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WO2002016600A2 - Novel proteins and nucleic acids encoding same - Google Patents

Novel proteins and nucleic acids encoding same Download PDF

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Publication number
WO2002016600A2
WO2002016600A2 PCT/US2001/026518 US0126518W WO0216600A2 WO 2002016600 A2 WO2002016600 A2 WO 2002016600A2 US 0126518 W US0126518 W US 0126518W WO 0216600 A2 WO0216600 A2 WO 0216600A2
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Prior art keywords
amino acid
polypeptide
nucleic acid
ofthe
seq
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PCT/US2001/026518
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French (fr)
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WO2002016600A3 (en
Inventor
Valerie Gerlach
John R. Macdougall
Glennda Smithson
David J. Stone
Karen Ellerman
Kimberly A. Spytek
Bryan D. Zerhusen
Luca Rastelli
Corine A. M. Vernet
Meera Patturajan
Velizar T. Tchernev
Muralidhara Padigaru
Raymond J. Taupier, Jr.
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Curagen Corporation
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Application filed by Curagen Corporation filed Critical Curagen Corporation
Priority to JP2002522273A priority Critical patent/JP2004516820A/en
Priority to EP01964417A priority patent/EP1332212A2/en
Priority to AU2001285271A priority patent/AU2001285271A1/en
Priority to CA002420538A priority patent/CA2420538A1/en
Publication of WO2002016600A2 publication Critical patent/WO2002016600A2/en
Publication of WO2002016600A3 publication Critical patent/WO2002016600A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the invention generally relates to nucleic acids and polypeptides encoded thereby. 5
  • the invention generally relates to nucleic acids and polypeptides encoded therefrom. More specifically, the invention relates to nucleic acids encodmg cytoplasmic, nuclear, membrane bound, and secreted polypeptides, as well as vectors, host cells, antibodies, and 10 recombinant methods for producing these nucleic acids and polypeptides.
  • the invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides.
  • novel nucleic acids and polypeptides are referred to herein as NOVX, i 5 or NOV1 , NOV2, NOV3 , NOV4, NOV5, NOV6, NOV7, and NOV8 nucleic acids and? polypeptides.
  • NOVX nucleic acid or polypeptide sequences.
  • the invention provides an isolated NOVX nucleic acid molecule
  • NOVX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a NOVX nucleic acid sequence.
  • .5 includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof.
  • the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37.
  • the nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid
  • oligonucleotide e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of aNOVX nucleic acid (e.g., SEQ JD NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36) or a complement of said oligonucleotide.
  • substantially purified NOVX polypeptides SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36.
  • the NOVX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human NOVX polypeptide.
  • the invention also features antibodies that immunoselectively bind to NOVX polypeptides, or fragments, homologs, analogs or derivatives thereof.
  • the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier.
  • the therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or an antibody specific for a NOVX polypeptide.
  • the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
  • the invention includes a method of producing a polypeptide by culturing a cell that includes a NOVX nucleic acid, under conditions allowing for expression ofthe NOVX polypeptide encoded by the DNA. If desired, the NOVX polypeptide can then be recovered.
  • the invention includes a method of detecting the presence of a NOVX polypeptide in a sample.
  • a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound.
  • the complex is detected, if present, thereby identifying the NOVX polypeptide within the sample.
  • the invention also includes methods to identify specific cell or tissue types based on their expression of a NOVX.
  • Also included in the invention is a method of detecting the presence of a NOVX nucleic acid molecule in a sample by contacting the sample with a NOVX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a NOVX nucleic acid molecule in the sample.
  • the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample that includes the NOVX polypeptide with a compound that binds to the NOVX polypeptide in an amount sufficient to modulate the activity of said polypeptide.
  • the compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
  • a small molecule such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
  • a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., Inflamation, Autoimmune disorders, scleroderma, transplantation, autoimmune disease, allergies, immunodeficiencies, systemic lupus erythematosus, lymphedema, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, graft versus host disease (GVHD), endometriosis, cystic fibrosis, pathological disorders involving spleen, thymus, lung, and peritoneal macrophages, Aging, Alzheimer's disease, stroke, Cancer, abnormal angiogenesis, like cancer and more specifically aggressive, metastatic cancer, more specifically tumor ofthe lung, kidney, brain, liver and colon, mastocytomas, prostate cancer, modulation of apoptosis, Oncogenesis such as lung, liver and ovarian cancers; cell growth and possibly differentiation, non-Hodgkin lympho
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding NOVX may be useful in gene therapy, and NOVX may be useful when administered to a subject in need thereof.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the method includes contacting a test compound with a NOVX polypeptide and determining if the test compound binds to said NOVX polypeptide. Binding ofthe test compound to the NOVX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
  • Also within the scope ofthe invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes.
  • the test animal expresses a recombinant polypeptide encoded by a NOVX nucleic acid. Expression or activity of NOVX polypeptide is then measured in the test animal, as is expression or activity ofthe protein in a control animal which recombinantly- expresses NOVX polypeptide and is not at increased risk for the disorder or syndrome.
  • the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX polypeptide, a NOVX nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount ofthe NOVX polypeptide in a test sample from the subject and comparing the amount ofthe polypeptide in the test sample to the amount ofthe NOVX polypeptide present in a control sample.
  • an alteration in the level ofthe NOVX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject.
  • the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the expression levels ofthe new polypeptides ofthe invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
  • the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subj ect a NOVX polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition.
  • the disorder includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
  • the invention can be used in a method to identity the cellular receptors and downstream effectors ofthe invention by any one of a number of techniques commonly employed in the art.
  • the present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences and their encoded polypeptides. The sequences are collectively referred to herein as “NOVX nucleic acids” or “NOVX polynucleotides” and the corresponding encoded polypeptides are referred to as “NOVX polypeptides” or “NOVX proteins.” Unless indicated otherwise, “NOVX” is meant to refer to any ofthe novel sequences disclosed herein. Table A provides a summary ofthe NOVX nucleic acids and their encoded polypeptides.
  • NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts.
  • the various NOVX nucleic acids and polypeptides according to the invention are useful as novel members ofthe protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members ofthe family to which the NOVX polypeptides belong. NO VI is homologous to a thioredoxin -like family of proteins.
  • NO VI nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; Inflamation, Autoimmune disorders, Aging and Cancer, and/or other pathologies/disorders.
  • NOV2a and 2b are homologous to the Retinoic acid responsive/STRA-6-like family of proteins.
  • NOV2 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, scleroderma, transplantation, autoimmune disease, allergies, immunodeficiencies, renal artery stenosis, intersti
  • NOV3a and 3b are homologous to a family of LRR/GPCR-like proteins.
  • the NOV3 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; abnormal angiogenesis, like cancer and more specifically aggressive, metastatic cancer, more specifically rumor ofthe lung, kidney, brain, liver and colon and/or other pathologies/disorders.
  • NOV4 is homologous to the Trypsin inhibitor-like family of proteins.
  • NOV4 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; chronic lung diseases, periodontitis, pancreatitis and/or other pathologies/disorders.
  • NOV5a, b, c, d, e, f, g, h and i are homologous to the apolipoprotein e-like family of proteins.
  • NOV5 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; atherosclerosis, dysbetalipoproteinemia, hyperlipoproteinemia type ILT, xanthomatosis, coronary and/or peripheral vascular disease, hypothyroidism, systemic lupus erythematosus, diabetic acidosis, hypercholesterolemia, planar and tendon xanthomas, dysbetalipoproteinemia, hypercholesterolemia, accelerated vascular disease, Alzheimer disease, familial amyloidotic polyneuropathy and/or other pathologies/disorders.
  • NOV6 is homologous to the mastocytoma protease precursor -like family of proteins.
  • NOV6 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; allergies, asthma and cystic fibrosis, cancers such as mastocytomas, vascular diseases and/or other pathologies/disorders.
  • NOV7 is homologous to members ofthe thymosin beta-4-like family of proteins.
  • the NOV7 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; prostate cancer, immunological and autoimmune disorders (ie hyperthyroidism), angiogenesis and wound healing, modulation of apoptosis, neurodegenerative and neuropsychiatric disorders, age-related disorders, pathological disorders involving spleen, thymus, lung, and peritoneal macrophages and Cancer and/or other pathologies/disorders.
  • NOV8 is homologous to the Protein-tyrosine-phosphatase -like family of proteins.
  • NOV8 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; hematopoiesic disorders; leukemogenesis; polycystic kidney disease; Oncogenesis such as lung, liver and ovarian cancers; apoptosis; type 2 diabetes and obesity; cell growth and possibly differentiation; non-Hodgkin lymphomas; musculoskeletal disorders; and CNS development disorders and/or other pathologies/disorders.
  • the NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., neurogenesis, cell differentiation, cell proliferation, hematopoiesis, wound healing and angiogenesis.
  • NOVX nucleic acids and polypeptides according to the invention are disclosed herein.
  • a disclosed NO VI nucleic acid of 620 nucleotides (also referred to as AC013554_dal) encoding a novel thioredoxin-like protein is shown in Table 1 A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 282-284 and ending with a TAA codon at nucleotides 618-620.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 1 A. The start and stop codons are in bold letters.
  • Table 1A NOV1 nucleotide sequence (SEQ ID NO:l).
  • the NO VI nucleic acid sequence, ocated on the p31 region of chromsome 9 has 232 of 331 bases (70%) identical to a thioredoxin mRNA from Ovis aries (GENBANK-ID: OATHIORD
  • Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
  • the "E-value” or “Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched.
  • the probability that the subject (“Sbjct”) retrieved from the NOV1 BLAST analysis, e.g., thioredoxin mRNA from Ovis aries, matched the Query NOV1 sequence purely by chance is 608e- .
  • the Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences.
  • the Expect value is used as a convenient way to create a significance threshold for reporting results.
  • the default value used for blasting is typically set to 0.0001.
  • the Expect value is also used instead ofthe P value (probability) to report the significance of matches.
  • an E value of one assigned to a hit can be interpreted as meaning that in a database ofthe current size one might expect to see one match with a similar score simply by chance.
  • An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education BLASTinfo/. Occasionally, a string of X's or N's will result from a BLAST search.
  • the disclosed NOVl polypeptide (SEQ ID NO:2) encoded by SEQ ID NO:l has 112 amino acid residues and is presented in Table IB using the one-letter amino acid code.
  • Signal P, Psort and/or Hydropathy results predict that NOVl has a signal peptide and is likely to be localized in the cytoplasm with a certainty of 0. 6500.
  • NOVl may also be localized to the mitochondrial matrix space with acertainty of 0.1000, or the lysosome (lumen) with a certainty of 0.1000.
  • the NOVl peptide is not likely to have a signal peptide.
  • Table IB Encoded NOVl protein sequence (SEQ ID NO:2).
  • the disclosed NOVl polypeptide has homology to the amino acid sequences shown in the BLASTP data listed in Table lC.
  • the "strong” group of conserved amino acid residues may be any one ofthe following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
  • Table IE lists the domain description from DOMAIN analysis results against NOVl. This indicates that the NOVl sequence has properties similar to those of other proteins known to contain this domain.
  • NOVl also has high homology to members ofthe patp database shown in Table IF.
  • Thioredoxin is an oxidoreductase enzyme of 12,000 daltons, containing a dithiol- disulfide active site. It is ubiquitous and found in many organisms from plants and bacteria to mammals. Multiple in vitro substrates for thioredoxin have been identified, including ribonuclease, choriogonadotropins, coagulation factors, glucocorticoid receptor, and insulin. Reduction of insulin is classically used as an activity test. Wollman et al. (1988) identified a full-length cDNA clone coding for human thioredoxin. The open reading frame coded for a protein of 104 amino acids (excluding the initial methionine).
  • This protein possessed the highly conserved enzymatic active site common to plant and bacterial thioredoxins: trp-cys- gly-pro-cys (amino acids 30-34). Tonissen and Wells (1991) determined that the TRX gene extends over 13 kb and consists of 5 exons encoding a 12-kD protein. Southern analysis demonstrated the presence of several TXN genes in the human genome. At least one of these is inactive, i.e., apseudogene. Kaghad et al. (1994) also cloned the TXN gene and demonstrated that the 5 exons are separated by 4 introns. They determined the +1 transcription start point by primer extension. The +1 site is located 22 bp downstream from a TATAA box and defines a 5-prime untranslated region of 74 bp. Southern hybridization of genomic DNAs from several donors detected only 1 active TXN gene.
  • Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter- and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin (TXN; 187700). (Arunachalam et al.; Nat. Acad. Sci. 97: 745- 750, 2000) described a lysosomal thiol reductase, which they called GILT, that was optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro.
  • TXN thioredoxin
  • the GILT gene encodes a predicted 261-amino acid protein composed of a 37- amino acid signal peptide and a 224-amino acid proform.
  • the enzyme was expressed constitutively in antigen-presenting cells and was induced by gamma-interferon (147570) in other cell types, suggesting a potentially important role in antigenprocessing.
  • T-cell thioredoxin reductase is a selenoenzyme that contains selenium in a conserved C-terminal region provides another example ofthe role of selenium in the major antioxidant enzyme system (i.e., thioredoxin-thioredoxin reductase), in addition to the well- known glutathione peroxidase enzyme system.
  • thioredoxin-thioredoxin reductase the major antioxidant enzyme system
  • glutathione peroxidase enzyme system i.e., glutathione peroxidase enzyme system.
  • the disclosed NOVl nucleic acid ofthe invention encoding a thioreductase-like protein includes the nucleic acid whose sequence is provided in Table 1 A, or a fragment thereof.
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 1 A while still encoding a protein that maintains its thioreductase-like activities and physiological functions, or a fragment of such a nucleic acid.
  • the invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any ofthe nucleic acids just described.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
  • modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 30 % percent ofthe bases may be so changed.
  • the disclosed NOVl protein ofthe invention includes the thioreductase-like protein whose sequence is provided in Table IB.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue.shown in Table 2 while still encoding a protein that maintains its thioreductase-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 38 % percent ofthe residues may be so changed.
  • the invention further encompasses antibodies and antibody fragments, such as F a b or (F a b)2, that bind immunospecifically to any ofthe proteins ofthe invention.
  • This thioreductase-like protein may function as a member of a "Thioredoxin family”. Therefore, the NOVl nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below.
  • the potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
  • NOVl nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in cancer including but not limited to Inflamation,
  • a cDNA encoding the thioreductase- like protein (NOVl) may be useful in gene therapy, and the thioreductase-like protein (NOVl) may be useful when administered to a subject in need thereof.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from Inflamation, Autoimmune disorders, Aging and Cancer.
  • the NOVl nucleic acid encoding thioreductase-like protein, and the thioreductase-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
  • NOVl nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOVl substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOVl protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOVl epitope is from about amino acids 1 to 10. In another embodiment, a NOVl epitope is from about amino acids 20 to 35.
  • NOVl epitopes are from about amino acids 45 to 50, from about amino acids 55 to 60, from about amino acid 65 to 75, and from about amino acids 90 to 115.
  • NOV2 NOV2 includes two novel retinoic acid/SRA6-like proteins disclosed below. The disclosed proteins have been named NOV2a and NOV2b. NOV2a
  • NOV2a nucleic acid of 2632 nucleotides also referred to as 3277789.0.83_dal
  • Table 2A An open reading frame was identified beginning with an ATG initiation codon at nucleotides 31-33 and ending with a TGA codon at nucleotides 2011-2013.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 2A, and the start and stop codons are in bold letters.
  • Table 2A NOV2a nucleotide sequence (SEQ ID NO:3).
  • the NOV2a nucleic acid was identified by TblastN using CuraGen Corporation's sequence file for Retinoic acid responsive/STRA-6 or homolog as run against the Genomic Daily Files made available by GenBank or from files downloaded from the individual sequencing centers.
  • the nucleic acid sequence was predicted from the genomic file Genbank by homology to a known Retinoic acid responsive/STRA-6 or homolog. Exons were predicted by homology and the intron/exon boundaries were determined using standard genetic rules. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlastN, BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein.
  • the disclosed NO V2a nucleic acid sequence, localized to chromsome 15, has 1511 of 2207 bases (68%) identical to a retinoic acid-responsive protein (Stra6) mRNA from Mus musculus (GENBANK-ID: AF062476
  • acc: AF062476 (GENBANK-ID: M77668)(E 1.2e " 189 ).
  • a NOV2a polypeptide (SEQ ID NO:4) encoded by SEQ ID NO:3 has 660 amino acid residues and is presented using the one-letter code in Table 2B.
  • Signal P, Psort and/or Hydropathy results predict that NOV2A contain a signal peptide and is likely to be localized in the plasmam membrane with a certainty of 0.6000.
  • NOV2a may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a NOV2A peptide is between amino acids 8 and 9, at: AGN-QT.
  • Table 2B Encoded NOV2a protein sequence (SEQ ID NO:4).
  • NOV2a also has high homology to members ofthe patp database shown in Table 2C.
  • a disclosedNOV2b nucleic acid of2663 nucleotides (also referred to as CG52276-07) encoding a novel STRA6-like protein is shown in Table 2D.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TGA codon at nucleotides 2068-2070.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 2D, and the start and stop codons are in bold letters.
  • a disclosed NOV2b polypeptide (SEQ ID NO:6) encoded by SEQ ID NO:5 has 689 amino acid residues and is presented using the one-letter code in Table 2E.
  • Signal P, Psort and/or Hydropathy results predict that NOV2b has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000.
  • NOV2b could be localized at the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or a microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a NOV2b peptide is between amino acids 8 and 9, at AGN-QT.
  • NOV2b maps to chromosome 15. This assignment was made using mapping information associated with genomic clones, public genes and ESTs sharing sequence identity with the disclosed sequence and CuraGen Corporation's Electronic Northern bioinformatic tool.
  • NOV2b is expressed in at least the following tissues: Brain, Cervix, Heart, Kidney, Lymph node, Lymphoid tissue, Ovary, Pituitary Gland, Placenta, Retina, Temporal Lobe, Thyroid, Uterus. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOV2b. NOV2a also homology to the amino acid sequences shown in the BLASTP data listed in Table 2F.
  • Carcinogenesis involves inactivation or subversion ofthe normal controls of proliferation, differentiation, and apoptosis (Hurst RE, et.al.; Adv Exp Med Biol
  • hRAR alpha nuclear receptor occupies a central position with respect to induction of gene transcription in that when bound to appropriate retinoid ligands, its homodimers and heterodimers with hRXR alpha regulate the transcription of a number of retinoid-responsive genes. These include genes in other signaling pathways, so that the whole forms a complex network.
  • a set of cultured bladder-derived cells representing different stages of bladder rumorigenesis formed a model system. It consisted of 2 immortalized bladder cell lines (HUC-BC and HUC-PC), one squamous cell carcinoma cell line (SCaBER), one papilloma line (RT4), and 4 transitional cell carcinomas (TCC-Sup, 5637, T24, J82) of varying stages and grades. This set of cells were used to model the range of behaviors of bladder cancers.
  • aTRA all-trans-retinoic acid
  • CRABP I was not expressed by J82, T24, 5637 and RT4, but was expressed at low levels that did not change in SCaBER and at moderate levels that decreased, increased, or decreased sharply in HUC-BC, TCC-Sup and HUC-PC, respectively.
  • telomeres The phenotypes for inhibition of proliferation showed no obvious relationship to the expression of any single gene, but cell lines that were inhibited by aTRA (HUC-BC and TCC-Sup) were not sensitive to 4-HPR, and vice versa.
  • One line (RT4) was insensitive to either retinoid.
  • Transfection showed very little retinoid- stimulated transfection ofthe CAT reporter gene with RT4 or HUC-PC.
  • About 2-fold enhancement transactivation was observed with SCaBER, HUC-BC, J82 and T24 cells and 3- 8 fold with 5637, TCC-Sup cells.
  • HUC-BC a G to T point mutation was found at position 606 ofthe hRAR alpha gene. This mutation would substitute tyrosine for asparagine in a highly conserved domain.
  • Relative RT-PCR analysis of a panel of retinoid-responsive and inducible genes demonstrated changes in expression levels of all the genes in response to retinoic acid treatment together with numerous aberrations dysregulations. They concluded that retinoid signaling may be a target for inactivation during tumorigenesis by uncoupling gene expression, proliferation and differentiation. Therefore retinoids are more likely to be effective for chemoprevention than for treatment of bladder carcinomas.
  • the proliferative effects of retinoids were examined in the MC-26 and LoVo colon adenocarcinoma cell lines (Stewart LV, et.al.; Exp Cell Res 1997 Jun 15;233(2):321-9).
  • the proliferation ofthe LoVo cell line was not altered in the presence ofthe retinoids all trans- retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA). Both retinoids, however, stimulated the growth, as measured by cell proliferation, of MC-26 cells. atRA and 9-cis-RA were equipotent in increasing MC-26 cell proliferation, suggesting that the growth stimulation is mediated by one or more retinoic acid receptors (RARs). To determine the RAR which might be responsible for this growth stimulatory effect, the authors characterized the RAR subtypes which were present in both cell lines. mRNA for the RAR alpha, RAR beta, and RAR gamma were detected in the MC-26 cell.
  • the RAR alpha does not mediate the growth stimulatory effects of retinoids, for a selective RAR alpha antagonist was unable to prevent the retinoid-induced increase in MC-26 cell growth.
  • RAR alpha, RAR beta, and RAR gamma mRNA are also expressed in the LoVo cell line; the lack of growth- stimulation by retinoids in LoVo cells, therefore, does not seem to be due to the absence of RARs.
  • the results obtained in these experiments demonstrate that the growth response elicited by retinoids can vary between colon cancer cells and that the differences in response may not be solely determined by the RAR subtypes which are expressed in a colon cancer cell line.
  • Retinoic acids well characterized regulators of proliferation and differentiation, partly re-differentiate follicular thyroid carcinoma cell lines (FTC-133, FTC-238, andHTC- TSHr) as well as SV40-transfected immortalized thyroid cell lines (ori3 and 7751) (Schmutzler C, et.al.;Exp Clin Endocrinol Diabetes 1996;104 Suppl 4:16-9). This is indicated by the stimulation of type 15'-deiodinase and other differentiation markers.
  • thyroid carcinoma cell lines express RA receptor rnRNAs and functional ligand- and DNA-binding receptor proteins able to mediate RA-dependent signal transduction. Together, these properties make these thyroid-derived cell lines useful in vitro models for studying the effects of an RA re-differentiation therapy of thyroid cancer.
  • RA chemotherapeutic agent retinoic acid
  • SCC head and neck squamous cell carcinoma
  • RAR retinoic acid receptors
  • RXR retinoid X receptors
  • CRABP cellular retinoic acid binding proteins I and II
  • clones expressing the antisense CRABP construct were less sensitive to RA mediated inhibition of proliferation. These clones were also less invasive in an in vitro invasion assay, likely due to downregulation of matrix metalloproteinase activity.
  • CRABP II affects the transcription of RA responsive genes which regulate proliferation and invasion of head and neck SCCs.
  • trans-RA trans-retinoic acid
  • RARs RA receptors
  • RXRs retinoid X receptors
  • Retinoic acid plays important roles in development, growth and differentiation by regulating the expression of target genes.
  • a new retinoic acid-inducible gene, Stra6 has been identified in PI 9 embryonal carcinoma cells using a subtractive hybridization cDNA cloning technique by Bouillet and coworkers (Mech Dev 1997;63:173-86).
  • Stra6 codes for a very hydrophobic membrane protein of a new type, which does not display similarities with previously characterized integral membrane proteins. Stra6, which exhibits a specific pattern of expression during development and in the adult, is strongly expressed at the level of blood- organ barriers.
  • Stra6 has a spermatogenic cycle-dependent expression which is lost in testes of RAR alpha null mutants where Stra6 is expressed in all tubules. This finding suggests that the Stra6 protein may be a component of an as yet unidentified transport machinery.
  • VHL Von Hippel-Lindau
  • Alzheimer's disease stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, scleroderma, transplantation, autoimmune disease, allergies, immunodeficiencies, renal artery stenosis, interstitial nephritis, glomeruloneph
  • VHL Von Hippel-Lindau
  • a cDNA encoding the Retinoic acid responsive/STRA-6-like protein may be useful in gene therapy, and the Retinoic acid responsive/STRA-6-like protein (NOV2) may be useful when administered to a subject in need thereof.
  • the compositions ofthe present invention will have efficacy for treatment of patients suffering from Inflamation, Autoimmune disorders, Aging or Cancer.
  • the NOV2A nucleic acid encoding Retinoic acid responsive/STRA-6-like protein, and the Retinoic acid responsive/STRA-6-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • NOV2 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV2 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV2a epitope is from about amino acids 20 to 30.
  • a NOV2a epitope is from about amino acids 40 to 60.
  • NOV2a epitopes are from about amino acids 75 to 80, from about 100 to 110, from about amino acids 180 to 190, from about amino acids 220 to 260, from about amino acids 310 to 330, from about amino acids 350 to 370, from about amino acids 380 to 400, from about amino acids 470 to 510, from about amino acids 550 to 570, and from about amino acids 590 to 650.
  • novel proteins can be used in assay systems for functional analysis of various human disorders, which are useful in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • a contemplated NOV2b epitope is from about amino acids 5 to 50. In another embodiment, a NOV2b epitope is from about amino acids 80 to 90. In additional embodiments, NOV2b epitopes are from about amino acids 140 to 160, from about 190 to 210, from about amino acids 230 to 290, from about amino acids 360 to 365, from about amino acids 390 to 440, from about amino acids 550 to 590, and from about amino acids 610 to 670. These novel proteins can be used in assay systems for functional analysis of various human disorders, which are useful in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • NOV3 NOV3 includes two novel LRR/GPCR-like proteins disclosed below. The disclosed proteins have been named NOV3a and NOV3b. NOV3a
  • a disclosed NOV3a nucleic acid of 7892 nucleotides (also referred to as 128900861ext) encoding a novel LRR/GPCR -like protein is shown in Table 3A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TAA codon at nucleotides 5728-5730.
  • a putative untranslated region downstream from the termination codon is underlined in Table 3A, and the start and stop codons are in bold letters.
  • a NOV3a polypeptide (SEQ ID NO:8) encoded by SEQ ID NO:7 is 1542 amino acid residues and is presented using the one-letter code in Table 3C.
  • Signal P, Psort and/or Hydropathy results predict that NOV3a contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.6400.
  • NOV3a may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3700, or the endoplasmic reticulum (lumen) with a certainty of 0.1000.
  • the most likely cleavage site for a NOV3a peptide is between amino acids 26 and 27, at: ARG-AP.
  • Table 3C Encoded NOV3a protein sequence (SEQ ID NO:8).
  • VFDELPAL WDLGTEFLTCDCHLR LLP AQNRSLQLSEHTLCAYPSALHAHALGSLQEAQLCCEGALEL HTHHLIPSLRQWFQGDRLPFQCSASYLGNDTRIR YHNRAPVEGDEQAGILLAESLIHDCTFITSELTLS HIGVWASGE ECTVSMAQGNASKKVEIWLETSASYCPAERVANNRGDFRWPRTLAGITAYQSCLQYPFTS
  • VPLGGGAPGTRASRRCDRAGR EPGDYSHCLYTNDITRVLYTFVLMPINASNALTLAHQLRVYTAEAASFS DMMD WAQMIQKFLGYvDQIKELVEVMVDMPSNLMLVDEHLLWLAQREDKACSRIVGAIERIGGAALSP HAQHISVNARNVALEAYLIKPHSYVGLTCTAFQRREGGVPGTRPGSPGQNPPPEPEPPADQQLRFRCTTGR PNVSLSSFHIKNSVALASIQLPPSLFSSLPAALAPPVPPDCTLQLLVFRNGRLFHSHSNTSRPGAAGPGKR
  • ptnr SPTREMBL-ACC : 094898 KIAA0806 PROTEIN - Homo sapiens . . . 240 1.4e-15 2 ptnr : SPTREMBL-ACC : P70193 MEMBRANE GLYCOPROTEIN - Mus muse . . . 234 1.4e-15 3 ptnr : SPTREMBL-ACC :088278 MEGF2 - Rattus norvegicus (Rat) , . . . 190 1.6e-15 6 ptnr : TREMBLNE -ACC :AAF61929 PROTOCADHERIN FLAMINGO 1 - Ho . . . 214 8 .2e-15 4
  • NOV3a also has high homology to members ofthe patp database shown in Table 3E.
  • NOV3a is expressed in at least the following tissues: Blood, Brain, Breast, Colon, Ear, Germ Cell, Heart, Kidney, Lung, Muscle, Pancreas, Placenta, Prostate, Skin, Stomach, Testis, Uterus, Whole embryo, colon, lung, ovary, uterus Eye, Prostate, Tonsil, placenta, and thymus..
  • sequence is predicted to be expressed in the following tissues based on the expression pattern of its homolog, SeqCalling assembly 128900861: fetal heart, fetal lung fetal retina fetal_liver_spleen, total fetal tissue and multiple tumor from colon, lung, geerm cells, kidney prostate.
  • NOV3b The cDNA coding for the a domain of NOV3a from residue 444 to 937 was targeted for "in-frame" cloning by PCR.
  • the PCR template is based on human cDNA(s).
  • oligonucleotide primers were used to clone the target cDNA sequence:
  • the forward primer includes an in-frame BamHI restriction site and the reverse primer contains an in-frame Xhol restriction site.
  • the pool is composed of 5 micrograms of each ofthe following human tissue cDNAs: adrenal gland, whole brain, amygdala, cerebellum, thalamus, bone marrow, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, liver, lymphoma, Burkitt's Raji cell line, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small Intestine, spleen, stomach, thyroid, trachea, uterus.
  • human tissue cDNAs adrenal gland, whole brain, amygdala, cerebellum, thalamus, bone marrow, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, liver, lymphoma, Burkitt's Raji cell line, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small Intestine
  • the second PCR was performed using the above primers and 0.5ng-l .0 ng of one of the following human tissue cDNAs: skeleton muscle, testis, mammary gland, adrenal gland, ovary, colon, normal cerebellum, normal adipose, normal skin, bone marrow, brain amygdala, brain hippocampus, brain substantia nigra, brain thalamus, thyroid, fetal lung, fetal liver, fetal brain, kidney, heart, spleen, uterus, pituitary gland, lymph node, salivary gland, small intestine, prostate, placenta, spinal cord, peripheral blood, trachea, stomach, pancreas, hypothalamus.
  • the reaction mixtures contained 2 microliters of each ofthe primers (original concentration: 5 pmol/ul), 1 microliter of lOmM dNTP (Clontech Laboratories, Palo Alto CA) and 1 microliter of 50xAdvantage-HF 2 polymerase (Clontech Laboratories) in 50 microliter- reaction volume.
  • PCR condition 1 a) 96°C 3 minutes b) 96°C 30 seconds denaturation c) 60°C 30 seconds, primer annealing d) 72°C 6 minutes extension Repeat steps b-d 15 times e) 96°C 15 seconds denaturation f) 60°C 30 seconds, primer annealing g) 72°C 6 minutes extension Repeat steps e-g 29 times e) 72°C 10 minutes final extension
  • PCR condition 2 a) 96°C 3 minutes b) 96°C 15 seconds denaturation c) 76°C 30 seconds, primer annealing, reducing the temperature by 1 °C per cycle d) 72°C 4 minutes extension Repeat steps b-d 34 times e) 72°C 10 minutes final extension
  • SF2 GTGCTGATGCCCATCAATGCCT (SEQ ID NO:94 SF3: CGCATCGTGGGTGCCATAG (SEQ ID NO:95)
  • SF4 ACCGGGAGGCCCAATGTTTCTCT (SEQ ID NO:96)
  • SR2 ACGAAGGTGTACAGCACCCTG (SEQ ID NO:99)
  • SR3 GGCTGCAGGCCTTGTCCTC (SEQ ID NO:100)
  • the insert assembly NOV3b was found to encode an open reading frame between residues 444 and 937 ofthe target sequence NOV3a.
  • the cloned insert differs from the original sequence at 8 nucleotide positions and 4 amino acid positions. It also differs by a 55 amino acid deletion between amino acid residues 491 and 546 ofthe original sequence.
  • the alignment with NOV3a is displayed in a ClustalW below. Note that differing amino acids have a white or grey background, and deleted/inserted amino acids can be detected by a dashed line in the sequence that does not code at that position.
  • a disclosed NOV3b nucleic acid of 1329 nucleotides (also referred to as 188822778) encoding a novel LRR/GPCR -like protein is shown in Table 3F.
  • An open reading frame was identified beginning with an GGA initiation codon at nucleotides 1-3 and ending with a GAG codon at nucleotides 1327-1329.
  • a putative untranslated region upstream from the initiation codon is underlined in Table 3F, and the start and stop codons are in bold letters.
  • This reading frame may be a portion of a larger reading frame, since it does not posess traditional start and stop codons.
  • the coding region of this nucleic acid may extend in both the 5' and 3' directions, and the sequence of its expressed protein may extend in the N- or C terminal directions.
  • a NOV3b polypeptide (SEQ ID NO:l 1 encoded by SEQ ID NO:9 is 443 amino acid residues and is presented using the one-letter code in Table 3H.
  • NOV3 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 31.
  • Novel NOV3 (SEQ ID NO: 8) 2) Novel NOV4 (SEQ ID N ⁇ :ll)
  • NOV3a APRL RLHSKCTLSMAGNQKNQGGSSSQEAASGLMPSRRLQRGEVEGLCTCLCTDLRNNI 300 NOV3b 1 Q9P1Z7 I Q9BYB8
  • NOV3a ISTVQPGAFLGLGELKRLDLSNNRIGCLTSETFQGLPRLLRLNISGNIFSSLQPGVFDEL 360 NOV3b X Q9P1Z7 Q9BYB8 I
  • NOV3a PALKVVDLGTEFLTCDCHLR LLP AQNRSLQLSEHTLCAYPSALHAHALGSLQEAQLCC 20 NOV3b 1 Q9P1Z7 Q9BYB8 MARPVRGGLGAPRRS- 15
  • NOV3 AV ⁇ QRWLPRaVCSCl ⁇ GVAAS LGLF(3FTHHCARRRDVRfi ⁇ RgCCPPA PAAPHAPPRA 1302 NOV3b :' 443 Q9P1Z7 AvSQRWLPR ⁇ CSCJ4gGVAAS ⁇ LGLF * FTHHCARRRDVRA[pRlCCPPA ⁇ PAAPHAPPRA 820 Q9BYB8 Vs lVLTSL3wVCl(rgQTR-0 ⁇ EEYs2 ⁇ NTDETvVPPDVp
  • Tables 3K through 3Q list the domain description from DOMAIN analysis results against NOV3. This indicates that the NOV3a sequence has properties similar to those of other proteins known to contain these domains.
  • ID NO: 75 51 residues, 96.1% aligned
  • CD-Length 51 residues, only 68.6% aligned
  • CD-Length 70 residues, only 67.1% aligned
  • CD-Length 49 residues, 93.9% aligned
  • CD-Length 49 residues, 93.9% aligned
  • tumor endothelial markers were expressed in a wide range of tumor types, as well as in normal vessels associated with wound healing and corpus luteum formation. These studies demonstrate that tumor and normal endothelium are distinct at the molecular level, a finding that may have significant implications for the development of antiangiogenic therapies.
  • Small leucine-rich proteoglycans belong to an expanding gene class whose distinctive feature is a structural motif, called the trypsin inhibitor, found in an increasing number of intracellular and extracellular proteins with diverse biological attributes (Iozzo RV Crit Rev Biochem Mol Biol 1997;32(2): 141-74).
  • Three-dimensional modeling of their prototype protein core proposes a flexible, arch-shaped binding surface suitable for strong and distinctive interactions with ligand proteins. Changes in the properties of individual proteoglycans derive from amino acid substitutions in the less conserved surface residues, changes in the number and length ofthe trypsin inhibitors, and/or variation in glycosylation.
  • proteoglycans are tissue organizers, orienting and ordering collagen fibrils during ontogeny and in pathological processes such as wound healing, tissue repair, and tumor stroma formation. These properties are rooted in their bifunctional character: the protein moiety binding collagen fibrils at strategic loci, the microscopic gaps between staggered fibrils, and the highly charged glycosaminoglycans extending out to regulate interfibrillar distances and thereby establishing the exact topology of fibrillar collagens in tissues. These proteoglycans also interact with soluble growth factors, modulate their functional activity, and bind to cell surface receptors.
  • Trypsin inhibitors are short sequence motifs present in a number of proteins with diverse functions and cellular locations (Kobe B et al., Trends Biochem Sci 1994 Oct;19(10):415-21). All proteins containing these repeats are thought to be involved in protein-protein interactions.
  • the crystal structure of ribonuclease inhibitor protein has revealed that trypsin inhibitors correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglobular shape.
  • Latrophilin is a brain-specific Ca2+-independent receptor of alpha-latrotoxin, a potent presynaptic neurotoxin. Matsushita H. et al. report the finding of two novel latrophilin homologues (Matsushita H. et al., FEBS Lett 1999 Jan 29;443(3):348-52). All three latrophilins are unusual G protein-coupled receptors. They exhibit strong similarities within their lectin, olfactomedin and transmembrane domains but possess variable C-termini.
  • Latrophilins have up to seven sites of alternative splicing; some splice variants contain an altered third cytoplasmic loop or a truncated cytoplasmic tail. Only latrophilin-1 binds alpha- latrotoxin; it is abundant in brain and is present in endocrine cells. Latrophilin-3 is also brain- specific, whereas latrophilin-2 is ubiquitous. Together, latrophilins form a novel family of heterogeneous G protein-coupled receptors with distinct tissue distribution and functions.
  • the protein similarity information, expression pattern, and map location for the LRR/GPCR-like protein and nucleic acid (NOV3) disclosed herein suggest that this LRR/GPCR-like protein may have important structural and/or physiological functions characteristic ofthe transient receptor potential-related protein family. Therefore, the NOV3 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from disorder linked to abnormal angiogenesis, like cancer and more specifically aggressive, metastatic cancer, more specifically tumor ofthe lung, kidney, brain, liver and colon.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from disorder linked to abnormal angiogenesis, like cancer and more specifically aggressive, metastatic cancer, more specifically tumor ofthe lung, kidney, brain, liver and colon.
  • These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods, specifically for the treatment of tumors, more specifically tumor ofthe lung, kidney, brain, liver and colon.
  • NOV3 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV3 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • the disclosed NOV3 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV3 epitope is from about amino acids 1 to 300. In another embodiment, a NOV3 epitope is from about amino acids 420 to 450.
  • NOV3 epitopes are from about amino acids 650 to 800, from about 700 to 800, from about amino acids 820 to 900, from about amino acids 1050 to 1150, from about amino acids 1200 to 1250, and from about amino acids 1300 to 1550.
  • NOV3 proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • NOV4 A disclosed NOV4 nucleic acid of 1123 nucleotides also referred to as
  • the disclosed NOV4 nucleic acid sequence has 1063 of its 1123 bases (95%) identical to an alpha- 1 -micro globulin mRNA from Homo sapiens (GENBANK-ID: HSHCR
  • acc:X04225) (E 3.3e -227 ).
  • a NOV4 polypeptide (SEQ ID NO: 13) encoded by SEQ ID NO: 12 is 372 amino acid residues and is presented using the one-letter amino acid code in Table 4B. Signal P, Psort and/or Hydropathy results predict that NOV4 has a signal peptide and is likely to be localized extracellularly with a certainty of 0.8200. In other embodiments, NOV4 may also be localized to the lysosome (lumen) with acertainty of 0.1900, the microbody (peroxisome) with a certainty of 0.1456, or the endoplasmic reticulum (membrane) with a certainty of 0.1000. The most likely cleavage site for a NOV4 peptide is between amino acids 19 and 20, at: VSA-GP.
  • the disclosed NOV4 amino acid sequence has
  • the disclosed NOV4 protein maps to chromosome 16 and is expressed in at least the following tissues: HEPG2 untreated (a hepatocellular carcinoma cell line); Fetal Liver; Pituitary Gland; pool of: Hypothalamus, Lymph Node, Fetal Liver, fetal_lung.
  • NOV4 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 4C.
  • AMBP_HUMAN h ambp protein precursor [contains: alpha-1-microglobulin (protein he) (complex-forming glycoprotein heterogeneous in charge) ; inter-alpha- trypsin inhibitor light chai (SEQ ID NO: 49)
  • AMBP_PIG sus scrofa (pig), ambp protein precursor [contains: alpha-1- microglobulin; inter-alpha- trypsin inhibitor light chain (iti-lc) (bikunin) (hi-30) (ei-14)] (fragment) (SEQ ID N ⁇ :50)
  • AMBP_MESAU mesocricetus auratus golden hamster
  • ambp protein precursor [contains: alpha-1-microglobulin; inter-alpha- trypsin inhibitor light chain (iti- lc) (bikunin) (hi-30)] (SEQ ID NO: 51)
  • AMBP_RAT rattus norvegicus rat
  • ambp protein precursor [contains: alpha-1- microglobulin; inter-alpha- trypsin inhibitor light chain (iti-lc) (bikunin) (hi- 30)]. 7/1999and (Pasted_No.1.18 :22-343) (SEQ ID N0:52)
  • AMBP_BOVIN b ambp protein precursor [contains: alpha-1-microglobulin; inter- alpha- trypsin inhibitor light chain (iti-lc) (bikunin) (hi-30) (bi-14) (cumulus extracellular matrix s (SEQ ID NO: 53)
  • Tables 4E-I list the domain description from DOMAIN analysis results against NOV4. This indicates that the NOV4 sequence has properties similar to those of other proteins known to contain this domain.
  • Table 4F Domain Analysis of NOV4 gnl I Smart
  • One member of the family is encoded by an alternatively-spliced form of Alzheimer's amyloid beta- protein (SEQ ID NO: 81)
  • NOV4 also has high homology to members ofthe patp database shown in Table 4J.
  • NOV4 is homologous in various ways to the following molecules whose functions are described below.
  • An imbalance between increased neutrophil elastase and a decreased antiprotease shield has been suggested as a factor contributing to the development of chronic lung disease (CLD).
  • CLD chronic lung disease
  • Stiskal JA et al. hypothesized that administration of alpha 1-proteinase inhibitor (A1PI), also known as alphal-antitrypsin, to premature neonates would prevent CLD.
  • A1PI alpha 1-proteinase inhibitor
  • alpha 1-PI alpha 1-proteinase inhibitor
  • the serum protein, alpha 1-proteinase inhibitor (alpha 1-PI) defends the host against serine proteinases, e.g. PMN elastase.
  • this protein in GCF was quantified from a standard curve constructed from dot-blot analysis and characterized by Western blot (Lee HM et al., J Periodontal Res 1997 Jan;32(l Pt 1):9-19).
  • GCF was collected on filter paper strips from healthy (H), gingivitis (G) and adult periodontitis (AP) patients, then extracted with Tris/NaCl/CaC12 buffer, pH 7.6. alpha 1-PI concentration increased with G and was highest in AP subjects.
  • alpha 1-PI degradation fragments were seen in 17% and 71% of GCF samples, respectively.
  • Both collagenase and alpha 1-PI-degrading activities in GCF increased with severity of inflammation (GCF flow).
  • the alpha 1-PI degrading (or serpinolytic) activity was characterized as a matrix metalloproteinase, probably collagenase, based on its in vitro response to a panel of different proteinase inhibitors including doxycycline.
  • protease- antiprotease balance was studied by measuring immunoreactive anionic trypsin (irAT), cationic trypsin (irCT), complexes between cationic trypsin and alpha 1 -protease inhibitor (irCT-alpha 1 PI), leukocyte elastase and neutrophil proteinase 4 (NP4), as well as the endogenous protease inhibitors, pancreatic secretory trypsin inhibitor (PSTI), alpha 2- macro globulin (alpha 2M), alpha 1 -protease inhibitor (alpha 1 PI), antichymotrypsin (ACHY), and secretory leukocyte protease inhibitor (SLPI).
  • irAT immunoreactive anionic trypsin
  • irCT cationic trypsin
  • irCT-alpha 1 PI complexes between cationic trypsin and alpha 1 -protease inhibitor
  • NP4 neutrophil proteinase 4
  • Intraperitoneal levels were studied before and after the lavage procedure, and plasma levels were followed for 21 d.
  • the control group had lower plasma levels of SLPI and analysis of peritoneal fluid showed the reduction of irCT- alpha 1 PI to be more pronounced in the aprotinin group. None ofthe other variables measured differed significantly between the two groups. All patients had very high levels of leukocyte elastase and NP4 both in peritoneal exudate and in plasma. Peritoneal levels of PSTI were higher after the lavage procedure in contrast to the other measured variables that all showed lower peritoneal levels after the lavage.
  • the protein similarity information, expression pattern, and map location for the trypsin inhibitor-like protein and nucleic acid (NOV4) disclosed herein suggest that this NOV4 protein may have important structural and/or physiological functions characteristic ofthe trypsin inhibitor family. Therefore, the NOV4 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
  • the NOV4 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from chronic lung diseases, periodontitis, pancreatitis.
  • the NOV4 nucleic acids, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
  • NOV4 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV4 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV4 epitope is from about amino acids 20 to 50.
  • a NOV4 epitope is from about amino acids 75 to 170.
  • NOV4 epitopes are from about amino acids 180 to 270, and from about amino acids 320 to 370.
  • NOV5 includes three novel apolipoprotein e-like proteins disclosed below.
  • the disclosed proteins have been named NOV5a, NOV5b, NOV5c, NOV5d, NOV5e, NOV5f, NOV5g, NOV5h, andNOV5i.
  • NOV5a A disclosed NOV5a nucleic acid of 2249 nucleotides (also referred to as sggc_draft_dj784al6_20000723_dal) encoding a novel apolipoprotein e-like protein is shown in Table 5A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 30-32 and ending with a TGA codon at nucleotides 2217-2219.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5A, and the start and stop codons are in bold letters.
  • the NOV5a sequence was identified by subjecting Ace. No. RP4-784A16 to an exon linking process.
  • PCR primers were designed to Ace. No. RP4-784A16 by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached.
  • suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species.
  • the resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5a sequence.
  • NOV5a nucleic acid was identified on chromosome 1 and has 2099 of 2118 bases (99%) identical to a 2549 bp apolipoprotein E receptor 2 mRNA from Homo sapiens (GENBANK-ID: HSZ75190
  • acc:Z75190 (E 0.0)
  • a disclosed NOV5a polypeptide (SEQ ID NO: 15) encoded by SEQ ID NO: 14 is 729 amino acid residues and is presented using the one-letter code in Table 5B.
  • Signal P, Psort and/or Hydropathy results predict that NOV5a has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000.
  • NOV5a may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum
  • the most likely cleavage site for a NOV5a peptide is between amino acids 59 and 60, at: AAA-AA.
  • NOV5b nucleic acid of 2348 nucleotides also referred to as sggc_draft_dj784al6_20000723_da2 encoding a novel apolipoprotein e-like protein is shown in Table 5C.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 30-32 and ending with a TGA codon at nucleotides 2316-2318.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5C, and the start and stop codons are in bold letters.
  • Table 5C NOV5b Nucleotide Sequence (SEQ ID NO:16)
  • the NOV5b sequence was identified through an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library contaimng a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5b sequence.
  • NOV5b nucleic acid was identified on chromosome 1 and has 1151 of 1206 bases (95%) identical to a a 2549 bp apolipoprotein E receptor 2 mRNA from Homo sapiens (GENBANK-ID: HSZ75190
  • acc:Z75190 (E 0.0)
  • a disclosed NOV5b polypeptide (SEQ ID NO: 17) encoded by SEQ ID NO: 16 is 762 amino acid residues and is presented using the one-letter code in Table 5D.
  • Signal P, Psort and/or Hydropathy results predict that NOV5b has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000.
  • NOV5b may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a NOV5b peptide is between amino acids 59 and 60, at: AAA-AA.
  • NOV5c nucleic acid of 2537 nucleotides also referred to as sggc_draft_dj784al6_20000723_da3 encoding a novel apolipoprotein e-like protein is shown in Table 5E.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 30-32 and ending with a TGA codon at nucleotides 2505-2507.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5E, and the start and stop codons are in bold letters.
  • the NOV5c sequence was identified through an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5c sequence.
  • NOV5c nucleic acid was identified on chromosome 1 and has 1626 of 1922 bases (84%) identical to a 4468 bp apolipoprotein E receptor 2 mRNA from Homo sapiens (GENBANK-ID:D50678
  • acc:D50678 ) (E ie '127 )
  • a disclosed NOV5c polypeptide (SEQ ID NO: 19) encoded by SEQ ID NO: 18 is 825 amino acid residues and is presented using the one-letter code in Table 5F.
  • Signal P, Psort and/or Hydropathy results predict that NOV5c has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000.
  • NOV5c may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a NOV5c peptide is between amino acids 44 and 45, at: AAA-DP.
  • a disclosed NOV5d nucleic acid was isolated by subjecting NOV5c to ⁇ & exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence ofthe target sequence, or by translated homology ofthe predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone manow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • the NOV5d sequence was identified through an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5d sequence.
  • the NOV5d nucleic acid was identified to the p34 region of human chromosome 1 and has 2508 of 2995 bases (83%) identical to a gb:GENBANK-ID:D50678
  • acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E 0.0)
  • a disclosed NOV5d polypeptide (SEQ ID NO:21) encoded by SEQ ID NO:20 is 1012 amino acid residues and is presented using the one-letter code in Table 5H. Signal P, Psort and/or Hydropathy results predict that NOV5d has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760.
  • NOV5d may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or the exfracellularly with a certainty of 0.1000.
  • the most likely cleavage site for a NOV5d peptide is between amino acids 32 and 33, at: AAA-AA.
  • the disclosed NOV5d amino acid sequence has 742 of 931 amino acid residues (79%) identical to, and 777 of 931 amino acid residues (83%) similar to, the 963 amino acid residue ptnr:SPTREMBL-ACC:Q14114 protein from Homo sapiens (Human) (APOLIPOPROTEIN E
  • NOV5d is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
  • sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:D50678
  • a disclosed NOV5e nucleic acid was isolated by subjecting NOV5c to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence ofthe target sequence, or by translated homology ofthe predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • the NOV5e sequence was identified through an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5e sequence.
  • the NOV5e nucleic acid was identified to the p34 region of human chromosome 1 and has 1359 of 1359 bases (100%) identical to a gb:GENBANK-ID:HSZ75190
  • acc:Z75190.1 mRNA from Homo sapiens (H.sapiens mRNA for apolipoprotein E receptor 2) (E 0.0)
  • a disclosed NOV5e polypeptide (SEQ ID NO:23) encoded by SEQ ID NO:22 is 695 amino acid residues and is presented using the one-letter code in Table 5 J. Signal P, Psort and/or Hydropathy results predict that NOV5e has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760. In other embodiments, NOV5e may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or the extracellularly with a certainty of 0.1000. The most likely cleavage site for a NOV5e peptide is between amino acids 32 and 33, at: AAA-AA.
  • NOV5e is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, and/or RACE sources.
  • sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-JD:HSZ75190
  • this protein contains the following protein domains (as defined by Interpro) at the indicated positions: low density lipoprotein receptor domains at amino acid positions 45 to 83, 84 to 165, 168 to 206; low density receptor repeat at amino acid ⁇ ositions333 to 378, 380 to 416, 418 to 460, 462 to 546; EGF-like domains at amino acid positions 86 tol22, 170 to 204, 211 to 245, 251 to 285, 556 to 600, 739 to 783; and trypsin inhibitor like cysteine rich domain at amino acid positions 195 to 251.
  • sequence ofthe invention has properties similar to those of other proteins known to contain this/these domain(s) and similar to the properties of these domains.
  • a disclosed NOV5f nucleic acid was isolated by subjecting NOV5c to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence ofthe target sequence, or by translated homology ofthe predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was. encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5f sequence.
  • the NOV5f nucleic acid was identified to the p34 region of human chromosome 1 and has 2047 of 2341 bases (87%) identical to a gb:GENBANK-JX>:D50678
  • acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E 0.0)
  • a disclosed NOV5f polypeptide (SEQ ID NO:25) encoded by SEQ ID NO:24 is 804 amino acid residues and is presented using the one-letter code in Table 5L. Signal P, Psort and/or Hydropathy results predict that NOV5f has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000.
  • NOV5f may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000.
  • the most likely cleavage site for a NOV5f peptide is between amino acids 44 and 45, at: AAA-DP.
  • NOV5f is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
  • sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:D50678
  • the presence of identifiable domains in NOV5f was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/).
  • this protein contains the following protein domains (as defined by Interpro) at the indicated positions: domain name low density lipoprotein receptor domains at amino acid positions 55 to 93, 96 to 177, 180 to 218; low density receptor repeat domains at amino acid positions 303 to 348, 350 to 391, 393 to 435, 437 to 521; EGF-like domains at amino acid positions 182 to 216, 223 to 257, 531 to 575; and fusion glycoprotein FO domain at amino acid positions 650 to 683.
  • domain name low density lipoprotein receptor domains at amino acid positions 55 to 93, 96 to 177, 180 to 218
  • low density receptor repeat domains at amino acid positions 303 to 348, 350 to 391, 393 to 435, 437 to 521
  • EGF-like domains at amino acid positions 182 to 216, 223 to 257, 531 to 575
  • fusion glycoprotein FO domain at amino acid positions 650 to 683.
  • NOV5g nucleic acid was isolated by The sequence of NOV5g was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence. cDNA fragments covering either the full length ofthe DNA sequence, or part ofthe sequence, or both, were cloned. In silico prediction was based on sequences available in Curagen's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof.
  • the novel NOV5g sequence of 4150 nucleotides also referred to as CG55256-05
  • Table 5M The novel NOV5g sequence of 4150 nucleotides (also referred to as CG55256-05) encoding a novel apolipoprotein e-like protein is shown in Table 5M.
  • the NOV5g sequence was identified through an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library contaimng a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5g sequence.
  • the NOV5g nucleic acid was identified to the p34 region of human chromosome 1 and has 3358 of 3361 bases (99%) identical to a gb:GENBANK-ID:D50678
  • acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E 0.0)
  • a disclosed NOV5g polypeptide (SEQ ID NO:27) encoded by SEQ ID NO:26 is 857 amino acid residues and is presented using the one-letter code in Table 5N. Signal P, Psort and/or Hydropathy results predict that NOV5g has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760.
  • NOV5g may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or exfracellularly with a certainty of 0.1000.
  • the most likely cleavage site for a NOV5g peptide is between amino acids 32 and 33, at: AAA-DP.
  • NOV5g is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus, Cerebral Medulla/Cerebral white matter, Colon, Lung, Lymphoid tissue, Parietal Lobe, Tonsils, Whole Organism. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOV5g.
  • IPR000033 YWTD repeat, from amino acid 356 to amino acid 401, from amino acid 403 to amino acid 444, from amino acid 446 to amino acid 488, from amino acid 490 to amino acid 533, from amino acid 534 to amino acid 574.
  • This domain is also known as the YWTD motif after the most conserved region ofthe repeat.
  • the YWTD repeat is found in multiple tandem repeats and has been predicted to form a beta-propeller structure. It is found in a variety of proteins that include, vitellogenin receptor from Drosophila, low-density lipoprotein (LDL) receptor, preproepidermal growth factor, nidogen (entactin) and others.
  • LDL Low density lipoprotein
  • LDLRA Low density lipoprotein-receptor class A domain
  • the receptor protein binds LDL and transports it into cells by endocytosis. In order to be internalised, the receptor-ligand complex must first cluster into clathrin-coated pits. Seven successive cysteine-rich repeats of about 40 amino acids are present in the N-terminal of this multidomain membrane protein.
  • the LDL- receptor class A domain contains 6 disulfide-bound cysteines and a highly conserved cluster of negatively charged amino acids, of which many are clustered on one face ofthe module.
  • the class A domains form the binding site for LDL and calcium.
  • the acidic residues between the fourth and sixth cysteines are important for high-affinity binding of positively charged sequences in LDLR's ligands.
  • the repeat has been shown to consist of a beta-hairpin structure followed by a series of beta turns. The binding of calcium seems to induce no significant conformational change. Following these repeats is a 350 residue domain that resembles part ofthe epidermal growth factor (EGF) precursor. Similar domains have been found in several extracellular and membrane proteins.
  • EGF epidermal growth factor
  • EGF-like domain from amino acid 234 to amino acid 268, from amino acid 274 to amino acid 308
  • a sequence of about thirty to forty amino-acid residues long found in the sequence of epidermal growth factor (EGF) has been shown to be present, in a more or less conserved form, in a large number of other, mostly animal proteins.
  • the list of proteins currently known to contain one or more copies of an EGF-like pattern is large and varied.
  • the functional significance of EGF domains in what appear to be unrelated proteins is not yet clear. However, a common feature is that these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G H synthase).
  • the EGF domain includes six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds.
  • the main structure is a two-stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet. Subdomains between the conserved cysteines vary in length.
  • NOV5h A disclosed NOV5h nucleic acid was isolated by The sequence of NOV5h was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence. cDNA fragments covering either the full length ofthe DNA sequence, or part ofthe sequence, or both, were cloned.
  • the NOV5h'sequence was identified through. an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5h sequence.
  • the NOV5h nucleic acid was identified to the p34 region of human chromosome 1 and has 3634 of 3851 bases (94%) identical to a gb:GENBANK-ID:D50678
  • acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E 0.0)
  • a disclosed NOV5h polypeptide (SEQ ID NO:29) encoded by SEQ ID NO:28 is 847 amino acid residues and is presented using the one-letter code in Table 5P. Signal P, Psort and/or Hydropathy results predict that NOV5h has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760.
  • NOV5h may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or exfracellularly with a certainty of 0.1000.
  • the most likely cleavage site for a NOV5h peptide is between amino acids 32 and 33, at: AAA-AA.
  • NOV5h is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus, Cerebral Medulla/Cerebral white matter, Colon, Lung, Lymphoid tissue, Parietal Lobe, Tonsils, Whole Organism. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOV5h
  • the sequence is predicted to be expressed in placenta because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:D50678
  • LDL Low density lipoprotein
  • LDLRA Low density lipoprotein-receptor class A domain
  • the receptor-ligand complex In order to be internalised, the receptor-ligand complex must first cluster into clathrin-coated pits. Seven successive cysteine-rich repeats of about 40 amino acids are present in the N-terminal of this multidomain membrane protein.
  • the LDL- receptor class A domain contains 6 disulfide-bound cysteines and a highly conserved cluster of negatively charged amino acids, of which many are clustered on one face ofthe module. In LDL-receptors the class A domains form the binding site for LDL and calcium.
  • the acidic residues between the fourth and sixth cysteines are important for high-affinity binding of positively charged sequences in LDLR's ligands.
  • the repeat has been shown to consist of a beta-hairpin structure followed by a series of beta turns. The binding of calcium seems to induce no significant confonnational change. Following these repeats is a 350 residue domain that resembles part ofthe epidermal growth factor (EGF) precursor. Similar domains have been found in several extracellular and membrane
  • TPR000033 YWTD repeat, from amino acid 346 to amino acid 391, from amino acid 393 to amino acid 434, from amino acid 436 to amino acid 478, from amino acid 480 to amino acid 523, from amino acid 524 to amino acid 564.
  • This domain is also known as the YWTD motif after the most conserved region ofthe repeat.
  • the YWTD repeat is found in multiple tandem repeats and has been predicted to form a beta-propeller structure. It is found in a variety of proteins that include, vitellogenin receptor from Drosophila, low-density lipoprotein (LDL) receptor, preproepidermal growth factor, nidogen (entactin) and others.
  • EGF-like domain from amino acid 224 to amino acid 258, from amino acid 264 to amino acid 298, from amino acid 574 to amino acid 618.
  • a sequence of about thirty to forty amino-acid residues long found in the sequence of epidermal growth factor (EGF) has been shown to be present, in a more or less conserved form, in a large number of other, mostly animal proteins.
  • the list of proteins currently known to contain one or more copies of an EGF-like pattern is large and varied.
  • the functional significance of EGF domains in what appear to be unrelated proteins is not yet clear. However, a common feature is that these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G/H synthase).
  • the EGF domain includes six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds.
  • the main structure is a two-stranded beta-sheet followed by a loop to a C-terminal short two- stranded sheet.
  • Subdomains between the conserved cysteines vary in length.
  • NOV5i A disclosed NOV5i nucleic acid was isolated by The sequence of NOV5i was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence. cDNA fragments covering either the full length ofthe DNA sequence, or part ofthe sequence, or both, were cloned. In silico prediction was based on sequences available in Curagen ' s proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof.
  • the novel NOV5i sequence of 4294 nucleotides (also referred to as CG55256-07) encoding a novel apolipoprotein e-like protein is shown in Table 5Q.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 103-105 and ending with a TGA codon at nucleotides 2818-2820.
  • a putative untranslated region upsfream from the initiation codon and downstream from the termination codon is underlined in Table 5Q, and the start and stop codons are in bold letters.
  • the NOV5i sequence was identified through an exon linking process.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5i sequence.
  • the NOV5i nucleic acid was identified to the p34 region of human chromosome 1 and has 3375 of 3389 bases (99%) identical to a gb:GENBANK-ID:D50678
  • acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E 0.0)
  • a disclosed NOV5i polypeptide (SEQ ID NO:31) encoded by SEQ ID NO:30 is 905 amino acid residues and is presented using the one-letter code in Table 5R. Signal P, Psort and/or Hydropathy results predict that NOV5i has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760.
  • NOV5i may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or extracellularly with a certainty of 0.1000.
  • the most likely cleavage site for a NOV5i peptide is between amino acids 32 and 33, at: AAA-AA. Table 5R. Encoded NOV5i protein sequence (SEQ ID NO:31)
  • NOV5i is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus, Cerebral Medulla/Cerebral white matter, Colon, Lung, Lymphoid tissue, Parietal Lobe, Tonsils, Whole Organism.
  • Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOV5L The sequence is predicted to be expressed in placenta because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:D50678
  • LDL Low density lipoprotein
  • LDLRA Low density lipoprotein-receptor class A domain
  • the receptor protein binds LDL and transports it into cells by endocytosis. In order to be internalised, the receptor-ligand complex must first cluster into clathrin-coated pits. Seven successive cysteine-rich repeats of about 40 amino acids are present in the N-terminal of this multidomain membrane protein.
  • the LDL- receptor class A domain contains 6 disulfide-bound cysteines and a highly conserved cluster of negatively charged amino acids, of which many are clustered on one face ofthe module.
  • the class A domains form the binding site for LDL and calcium.
  • the acidic residues between the fourth and sixth cysteines are important for high-affinity binding of positively charged sequences in LDLR's ligands.
  • the repeat has been shown to consist of a beta-hairpin structure followed by a series of beta turns. The binding of calcium seems to induce no significant conformational change. Following these repeats is a 350 residue domain that resembles part ofthe epidermal growth factor (EGF) precursor. Similar domains have been found in several extracellular and membrane proteins.
  • EGF epidermal growth factor
  • IPR000033 YWTD repeat, from amino acid 404 to amino acid 449, from amino acid 451 to amino acid 492, from amino acid 494 to amino acid 536, from amino acid 538 to amino acid 581, from amino acid 582 to amino acid 622.
  • This domain is also known as the YWTD motif after the most conserved region of the repeat.
  • the YWTD repeat is found in multiple tandem repeats and has been predicted to form a beta-propeller structure. It is found in a variety of proteins that include, vitellogenin receptor from Drosophila, low-density lipoprotein (LDL) receptor, preproepidermal growth factor, nidogen (entactin) and others.
  • EGF-like domain from amino acid 322 to amino acid 356, from amino acid 632 to amino acid 676.
  • a sequence of about thirty to forty amino-acid residues long found in the sequence of epidermal growth factor (EGF) has been shown to be present, in a more or less conserved form, in a large number of other, mostly animal proteins.
  • the list of proteins currently known to contain one or more copies of an EGF-like pattern is large and varied.
  • the functional significance of EGF domains in what appear to be unrelated proteins is not yet clear. However, a common feature is that these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G/H synthase).
  • the EGF domain includes six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds.
  • the main structure is a two-stranded beta- sheet followed by a loop to a C-terminal short two-stranded sheet. Subdomains between the conserved cysteines vary in length.
  • NOV5a also has homology to the amino acid sequences shown in the BLASTP data listed in Table 5S.
  • N0V5g SEQ ID NO: 7
  • NOV5h SEQ ID NO: 29
  • Tables 5U-Y list the domain description from DOMAIN analysis results against NOV5a. This indicates that the NOV5a sequence has properties similar to those of other proteins known to contain this domain.
  • Table 5U Domain Analysis of NOV5a qnl 1 Smart
  • LDL Low-density lipoprotein receptor domain class A
  • Cysteine-rich repeat in the low-density lipoprotein (LDL) receptor that plays a central role in mammalian cholesterol metabolism.
  • the N-terminal type A repeats in LDL receptor bind the lipoproteins.
  • Other homologous domains occur in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement . Mutations in the LDL receptor gene cause familial hypercholesterolemia. (SEQ ID NO:85)
  • NOV5a also has high homology to members ofthe patp database shown in Table 5Z.
  • NOV5c also has high homology to members ofthe patp database shown in Table 5AA.
  • Apolipoprotein E is a 34-kDa lipophilic protein that circulates in the plasma primarily as a major component of various lipoproteins including chylomicron remnants, intermediate density lipoprotein, very low density lipoprotein (VLDL), ⁇ -migrating VLDL ( ⁇ - VLDL), and high density lipoprotein (with apoE). It is a key molecule responsible for the cellular recognition and internalization of these lipoproteins. Biochemical and genetic studies have demonstrated that apoE is involved in the hepatic clearance of chylomicron remnants and VLDL remnants from the plasma. Apolipoprotein E receptor 2 binds apoE-rich ⁇ -VLDL with high affinity and internalizes it into the cells.
  • Apolipoprotein E receptor 2 is a receptor belonging to a family of low and very low density lipoprotein receptors (LDL-R). This family consists of cell-surface receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. The family members contain four major structural modules: the cysteine-rich complement-type repeats, epidermal growth factor precursor-like repeats, a transmembrane domain, and a cytoplasmic domain. Each structural module serves distinct and important functions. These receptors bind several structurally dissimilar ligands. It is proposed that instead of a primary sequence, positive electrostatic potential in different ligands constitutes a receptor bmding domain. This family of receptors plays crucial roles in various physiologic functions.
  • LDL-R low and very low density lipoprotein receptors
  • LDL-R plays an important role in cholesterol homeostasis. Mutations cause familial hypercholesterolemia and premature coronary artery disease. LDL-R-related protein plays an important role in the clearance of plasma-activated alpha 2-macroglobulin and apolipoprotein E-enriched lipoproteins. It is essential for fetal development and has been associated with Alzheimer's disease. The regulation of expression of these receptors occurs at the transcriptional level.
  • the NOV5 nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in atherosclerosis, dysbetalipoproteinemia, hyperlipoproteinemia type III, xanthomatosis, coronary and/or peripheral vascular disease, hypothyroidism, systemic lupus erythematosus, diabetic acidosis, hypercholesterolemia, planar and tendon xanthomas, dysbetalipoproteinemia, hypercholesterolemia, accelerated vascular disease, Alzheimer disease, familial amyloidotic polyneuropathy, as well as other diseases, disorders and conditions.
  • the LDL receptor related protein, the apolipoprotein E receptor 2 and the very low density lipoprotein (VLDL) receptor are preferentially expressed in the brain and they have been suspected to be involved in Alzheimer disease at various levels (Microsc Res Tech 2000 Aug 15;50(4):273-7)., and/or other pathologies and disorders.
  • a cDNA encoding the apolipoprotein e-like protein may be useful in gene therapy, and the apolipoprotein e-like protein may be useful when administered to a subject in need thereof.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from atherosclerosis, dysbetalipoproteinemia, hyperlipoproteinemia type III, xanthomatosis, coronary and or peripheral vascular disease, hypothyroidism, systemic lupus erythematosus, diabetic acidosis, hypercholesterolemia, planar and tendon xanthomas, dysbetalipoproteinemia, hypercholesterolemia, accelerated vascular disease, Alzheimer disease, familial amyloidotic polyneuropathy, as well as other diseases, disorders and conditions.
  • NOV5 nucleic acid, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
  • NOV5 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV5a, e, and f proteins have multiple hydrophilic regions, each of which can be used as an immunogen.
  • contemplated NOV5a, e, and f epitopes are from about amino acids 80 to 220.
  • NOV5a, e, and f epitopes are from about amino acids 250 to 340, from about amino acids 370 to 540, from about amino acids 590 to 650, or from about amino acids 680 to 720.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • the disclosed NOV5b protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV5b epitope is from about amino acids 80 to 280.
  • a NOV5b epitope is from about amino acids 290 to 350, from about amino acids 390 to 560, from about amino acids 600 to 670, or from about amino acids 690 to 750.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • the disclosed NOV5c protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV5c epitope is from about amino acids 50 to 220. In other embodiments, a NOV5c epitope is from about amino acids
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • the disclosed NOV5d protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV5d epitope is from about amino acids 50 to 180.
  • a NOV5d epitope is from about amino acids 190 to 230, from about amino acids 250 to 390, from about amino acids 420 to 550,from about amino acids 470 to 650, from about amino acids 780 to 850, from amino acids 870 to 900, from about amino acids 920 to 980, or from about amino acids 990 to 1050.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • contemplated NOV5g and h epitopes are from about amino acids 30 to 220. In other embodiments, NOV5g and h epitopes are from about amino acids 250 to 330, from about amino acids 350 to 540, from about amino acids 550 to 680, from about amino acids 690 to 720, from about amino acids 750 to 780, or from about amino acids 790 to 850.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • the disclosed NOV5i protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV5i epitope is from about amino acids 5 to 150.
  • a NOV5i epitope is from about amino acids 190 to 230, from about amino acids 170 to 320, or from about amino acids 360 to 450.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • a disclosed NOV6 nucleic acid of 843 nucleotides (also referred to as SC_129296119- A) encoding a novel mastocytoma protease precursor -like protein is shown in Table 6A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 26- 28 and ending with a TAG codon at nucleotides 836-838.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 6A, and the start and stop codons are in bold letters.
  • a disclosed NOV6 polypeptide (SEQ ID NO:33) encoded by SEQ ID NO:32 is 270 amino acid residues and is presented using the one-letter amino acid code in Table 6B.
  • Signal P, Psort and/or Hydropathy results predict that NOV6 contains a signal peptide and is likely to be localized in thecytosol with a certainty of 0.4500.
  • the most likely cleavage site for a NOV6 peptide is between amino acids 20 and 21, at: KVG-IT.
  • Table 6B Encoded NOV6 protein sequence (SEQ ID NO.33).
  • the global sequence homology (as defined by FASTA alignment with the full length sequence of NOV6) is 66.171 % amino acid homology and 61.338 % amino acid identity.
  • this protein contains the following protem domains (as defined by Interpro) at the indicated nucleotide positions: Trypsin domain (IPR001254) at amino acid positions 21 to 260.
  • NOV6 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 6F.
  • N0V6 SEQ ID NO: 33
  • TRYM_CANFA canis familiaris (dog), mastocytoma protease precursor (ec 3.4.21.-) (SEQ ID NO: 58)
  • MCT7_RAT rattus norvegicus rat
  • mast cell protease 7 precursor ec 3.4.21.-
  • rmcp-7 tryptase, skin
  • MCT6_MOUSE mus musculus (mouse), mast cell protease 6 precursor (ec 3.4.21.-) (mmcp-6) (tryptase) (SEQ ID NO: 61)
  • Table 6H lists the domain description from DOMAIN analysis results against NOV6.
  • Table 6H Domain Analysis of NOV6 gnl I Smart
  • CD-Length 217 residues, 100.0% aligned
  • Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation.
  • the tryptase sequence includes the essential residues ofthe catalytic triad and an aspartic acid at the base ofthe putative substrate binding pocket that confers PI Arg and Lys specificity on tryptic serine proteases.
  • the apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Mast cell activation in vivo is often associated with areas of oedema and connective- tissue degradation.
  • Tryptase and chymase are the major serine proteinases released by mast cells, but they appear to have little activity on most components ofthe extracellular matrix.
  • the matrix metalloproteinases (MMP) are purported to degrade almost all connective tissue elements and are secreted by cells in the form of inactive precursors. Since the mechanisms of MMP activation in vivo are poorly understood we have examined the potential of mast cell proteinases to activate the precursor forms of human collagenase (MMP-1), stromelysin (MMP-3), gelatinase A (MMP-2) and gelatinase B (MMP-9).
  • Mast cell proteinases prepared from purified dog mastocytoma cells were shown to process and activate purified precursor forms of both MMP-1 and MMP-3. Using antipain and chymostatin, inhibitors for tryptase and chymase, respectively, it was demonstrated that both pMMP-1 and pMMP-3 were effectively processed and activated by the chymase component. By contrast, tryptase activated only pMMP-3. The mast cell proteinases were unable to process or activate purified precursor forms of MMP-2 and MMP-9. However, MMP-3 previously activated by mast cell proteinases was shown to activate pMMP-9, but not pMMP-2. Since we have no evidence that mast cells express these four metalloenzymes, the release of mast cell serine proteinases following activation/degranulation could contribute to local metalloproteinase activation and subsequent matrix degradation.
  • the mast cell proteases tryptase and chymase have long been known to constitute one- fifth ofthe total protein in mast cells.
  • their biological functions have not been easy to study because ofthe difficulty in obtaining sufficient amounts ofthe enzymes to study their biological functions.
  • We have discovered unique and potent actions ofthe enzymes that may provide important insights into the pathogenesis of diseases such as asthma and cystic fibrosis. Important biological activities are also likely to exist in other tissues. Because of their structures, mast cell proteases are likely to act in proximity to their sites of release.
  • NOV6 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below.
  • the potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
  • nucleic acids and proteins of NOV6 are useful in potential therapeutic applications implicated in immune disorders and airway pathologies such as allergies, asthma and cystic fibrosis, cancers such as mastocytomas, vascular diseases, and/or other pathologies and disorders
  • a cDNA encodingNOV6 may be useful in gene therapy, and NOV6 may be useful when administered to a subject in need thereof.
  • NOV6 will have efficacy for treatment of patients suffering from immune disorders and airway pathologies such as allergies, asthma and cystic fibrosis, cancers such as mastocytomas, and vascular diseases.
  • novel NOV6 nucleic acid encoding NOV6 protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
  • NOV6 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOV6 substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders. NOV7
  • a disclosed NOV7 nucleic acid of 239 nucleotides (also referred to GM10d7_A) encoding a novel thymosin beta-4-like receptor protein is shown in Table 7A.
  • An open reading frame was identified beginning with a TCA initiation codon at nucleotides 40-42 and ending with a TGA codon at nucleotides 190-092.
  • the start and stop codons are in bold letters.
  • a disclosed NOV7 polypeptide (SEQ ID NO:35) encoded by SEQ ID NO:34 is 50 amino acid residues and is presented using the one-letter amino acid code in Table 7B. Signal P, Psort and/or Hydropathy results predict that NOV7 has no signal peptide and is likely to be localized in the cytoplasm with a certainty of 0.4500.
  • NOV7 maps to chromosome 5p35 and was found to be expressed in at least the following tissues: mammary gland, small intestine, colon, testis and leukocytes.
  • the disclosed NOV7 amino acid sequence has 26 of 43 amino acid residues (83 %) identical to, and 37 of 43 residues (86 %) positive with, the 50 amino acid residue
  • THYMOSIN BETA-4 protein ftom Mus musculus (ptnr:SPTREMBL-ACC: P20065) ( 2.2e " 12 ).
  • the global sequence homology (as defined by FASTA alignment with the full length sequence of this protein) is 72.000% amino acid homology and 72.000% amino acid identity.
  • NOV7 contains the following protein domains (as defined by Interpro) at the indicated nucleotide positions: Thymosin domain(JPR001152) at amino acid positions 8 to 48 NOV7 also has homology to the amino acid sequence shown in the BLASTP data listed in Table 7C.
  • TYB4_HUMAN homo sapiens human
  • bos taurus bos taurus
  • rattus norvegicus rat
  • equus caballus horse
  • thymosin beta-4 [contains: hematopoietic system regulatory peptide] (SEQ ID NO: 66)
  • TYB4_RABIT oryctolagus cuniculus (rabbit) .
  • thymosin beta-4 (SEQ ID NO: 67)
  • NOV7 also has high homology to members ofthe patp database shown in Table 7E.
  • Thymosin beta-4 is a small polypeptide whose exact physiological role is not yet known . It was first isolated as a thymic hormone that induces terminal deoxynucleotidyltransferase. It is found in high quantity in thymus and spleen but is widely distributed in many tissues. It has also been shown to bind to actin monomers and thus to inhibit actin polymerization .It has inhibitory activity on the proliferation of hematopoeitic pluripotent stem cells, and is localized in the cytosol of cells. It is induced by alpha interferon, nerve and fibroblast growth factors. It belongs to the thymosin beta family.
  • thymosin beta 4 is expressed in ROS 17/2.8 osteosarcoma cells in a regulated manner.
  • the differential expression of rnRNAs between the closely related rat osteosarcoma cell lines ROS 17/2.8 and ROS 25/1 was used to identify genes whose expression is associated with the osteoblast phenotype.
  • Thymosin beta 4 cDNA was cloned from an ROS 17/2.8 complimentary DAN library on the basis of its differential hybridization with radiolabeled cDNA prepared from ROS 17/2.8 and ROS 25/1 cells.
  • Northern blot analysis confirmed that thymosin beta 4, hitherto a putative immunodulatory hormone, was indeed differentially expressed.
  • Thymosin-beta(4) binds actin monomers stoichiometrically and maintains the bulk ofthe actin monomer pool in metazoan cells. Tbeta(4) binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylenediamine (AEDANS) conjugated to Cys(374) of actin monomers.
  • AEDANS N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylenediamine
  • the K(d) ofthe actin-Tbeta(4) complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation.
  • Tbeta(4) binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on subdomain 2 is >12 times faster when Tbeta(4) is bound. The C terminus of Tbeta(4) contacts actin near this cleavage site, at His(40). By tritium exchange, Tbeta(4) slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that Tbeta(4) changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of Tbeta(4) to sequester actin monomers and inhibit nucleotide exchange.
  • glucocorticoids upregulate the expression of anti-inflammatory mediators is an exciting prospect for therapy in inflammatory diseases, because these molecules could give the therapeutic benefits of steroids without toxic side effects.
  • Supernatants from monocytes and macrophages cultured in the presence of glucocorticoids increase the dispersion of neutrophils from a cell pellet in the capillary tube migration assay. This supernatant factor, unlike other neutrophil agonists, promotes dispersive locomotion of neutrophils at uniform concentration, lowers their adhesion to endothelial cells, inhibits their chemotactic response to fMLP and induces distinctive morphological changes.
  • thymosin beta4 sulfoxide is generated by monocytes in the presence of glucocorticoids and acts as a signal to inhibit an inflammatory response.
  • thymosin beta4 sulfoxide inhibited neutrophil chemotaxis, and in vivo, the oxidized peptide, but not the native form, was a potent inhibitor of carrageenin-induced edema in the mouse paw.
  • Thymosin beta4 is unique, because oxidation attenuates its intracellular G-actin sequestering activity, but greatly enhances its extracellular signaling properties. This description of methionine oxidation conferring extracellular function on a cytosolic protein may have far-reaching implications for future strategies of ai ti-inflammatory therapy.
  • Angiogenesis is an essential step in the repair process that occurs after injury.
  • Tbeta4 angiogenic thymic peptide thymosin beta4
  • Tbeta4 stimulated keratinocyte migration in the Boyden chamber assay. After 4-5 h, migration was stimulated 2-3-fold over migration with medium alone when as little as 10 pg of Tbeta4 was added to the assay.
  • NOV7 thymosin beta-4-like protein and nucleic acid
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
  • the NOV7 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from prostate cancer, immunological and autoimmune disorders (ie hyperthyroidism), angiogenesis and wound healing, modulation of apoptosis, neurodegenerative and neuropsychiatric disorders, age-related disorders, pathological disorders involving spleen, thymus, lung, and peritoneal macrophages and/or other pathologies and disorders.
  • immunological and autoimmune disorders ie hyperthyroidism
  • angiogenesis ie hyperthyroidism
  • wound healing modulation of apoptosis
  • neurodegenerative and neuropsychiatric disorders ie hyperthyroidism
  • age-related disorders e.g., chronic myethelial fibrosis
  • pathological disorders involving spleen thymus, lung, and peritoneal macrophages and/or other pathologies and disorders.
  • the NOV7 nucleic acid, or fragments thereof may further be useful in diagnostic applications, wherein the presence or
  • NOV7 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
  • These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV7 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • a disclosed NOV8 nucleic acid of 1348 nucleotides (also referred to as GMba550p23_A) encoding a novel Protein-tyrosine-phosphatase -like protein is shown in Table 8A.
  • An open reading frame was identified beginning with a ATG initiation codon at nucleotides 57-59 and ending with a TAA codon at nucleotides 1302-1304.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 8A, and the start and stop codons are in bold letters.
  • a disclosed NOV8 protein (SEQ ID NO:37) encoded by SEQ ID NO:36 has 415 amino acid residues, and is presented using the one-letter code in Table 8B.
  • Signal P, Psort and/or Hydropathy results predict that NOV8 is likely to be localized to the endoplasmic reticulum (membrane) with a certainty of 0.8500.
  • Table 8B Encoded NOV8 protein sequence (SEQ ID NO:37).
  • NOV8 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 8C.
  • PTN2_M0USE mus musculus (mouse) . protein-tyrosine phosphatase ptp-2 (ec 3.1.3.48) (mptp) (SEQ ID N ⁇ :69)
  • PTN2_RAT rattus norvegicus (rat) . protein-tyrosine phosphatase ptp-s (ec 3.1.3.48) (SEQ ID N ⁇ :70)
  • PTN1_CHICK gallus gallus (chicken) .
  • protein-tyrosine phosphatase lb (ec 3.1.3.48) (ptp-lb) (cptpl).
  • Pa (SEQ ID N0:71)
  • PTN1_M0USE mus musculus (mouse), protein-tyrosine phosphatase lb (ec 3.1.3.48) (ptp-lb) (protein- protein-tyrosine phosphatase ha2) (ptp-ha2) (SEQ ID NO: 72)
  • Tables 8E-8G list the domain description from DOMAIN analysis results against NOV8. This indicates that the NOV8 sequence has properties similar to those of other proteins known to contain this domain.
  • CD-Length 264 residues, 98.5% aligned
  • CD-Length 235 residues, 100.0% aligned
  • CD-Length 105 residues, 100.0% aligned
  • NOV8 also has high homology to members ofthe patp database shown in Table 8H.
  • T-cell PTPase was cloned by Cool et al. (1989) who showed that it shares 72% amino acid sequence identity with PTP1B in a 236- amino acid core region present in all PTPases.
  • Sakaguchi et al. (1991, 1992) used a human PTPT cDNA to assign the gene to human chromosome 18 and mouse chromosome 18 by Southern analysis of human/mouse and mouse/Chinese hamster somatic cell hybrids, respectively.
  • Johnson et al. (1993) mapped the PTPN2 gene to 18pll.3-pl 1.2 by fluorescence in situ hybridization.
  • PTPs Protein tyrosine phosphatases
  • NOV8 protein may have important structural and/or physiological functions characteristic of the protein-tyrosine-phosphatase F family. Therefore, the NOV8 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
  • the NOV8 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies.
  • compositions ofthe present invention will have efficacy for treatment of patients suffering from hematopoiesic disorders; leukemogenesis; polycystic kidney disease; Oncogenesis such as lung, liver and ovarian cancers; apoptosis; type 2 diabetes and obesity; cell growth and possibly differentiation; non-Hodgkin lymphomas; musculoskeletal disorders; and CNS development disorders.
  • NOV8 nucleic acid, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
  • NOV8 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV8 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g. , mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double- stranded DNA.
  • an NOVX nucleic acid can encode a mature NOVX polypeptide.
  • a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein.
  • the naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein.
  • the product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises.
  • Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage ofthe N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence.
  • a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine would have residues 2 through N remaining after removal ofthe N-terminal methionine.
  • a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved would have the residues from residue M+1 to residue N remaining.
  • a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event.
  • additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation.
  • a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
  • probes refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
  • isolated nucleic acid molecule is one, which is separated from other nucleic acid molecules which are present in the natural source ofthe nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3 '-termini ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived.
  • the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.).
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule ofthe invention e.g., a nucleic acid molecule having the nucleotide sequence SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et ⁇ l., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et ⁇ l., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
  • a nucleic acid ofthe invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g. , using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
  • an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or a complement thereof. Oligonucleotides maybe chemically synthesized and may also be used as probes.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an NOVX polypeptide).
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown SEQ JD NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19 is one that is sufficiently complementary to the nucleotide sequence shown SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains.
  • Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
  • Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
  • Derivatives and analogs maybe full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
  • nucleic acids or proteins ofthe invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins ofthe invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.
  • a “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above.
  • Homologous nucleotide sequences encode those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues ofthe same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • homologous nucleotide sequences include nucleotide sequences encoding for an NOVX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
  • homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations ofthe nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, as well as a polypeptide possessing NOVX biological activity. Various biological activities ofthe NOVX proteins are described below.
  • An NOVX polypeptide is encoded by the open reading frame ("ORF") of an NOVX nucleic acid.
  • An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide.
  • a stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon.
  • An ORF that represents the coding sequence for a full protein begins with an ATG "start” codon and terminates with one ofthe three “stop” codons, namely, TAA, TAG, or TGA.
  • an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both.
  • a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
  • the nucleotide sequences determined from the cloning ofthe human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX homologues from other vertebrates.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19; or an anti-sense strand nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19; or of a naturally occurring mutant of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36.
  • Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis- express an NOVX protein, such as by measuring a level of an NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX niRNA levels or determining whether a genomic NOVX gene has been mutated or deleted.
  • a polypeptide having a biologically-active portion of an NOVX polypeptide refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide ofthe invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
  • a nucleic acid fragment encoding a "biologically- active portion of NOVX” can be prepared by isolating a portion SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19, that encodes a polypeptide having an NOVX biological activity (the biological activities ofthe NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g. , by recombinant expression in vitro) and assessing the activity ofthe encoded portion of NOVX.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 due to degeneracy ofthe genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36.
  • an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g. , the human population).
  • Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an NOVX protein, preferably a vertebrate NOVX protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence ofthe NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity ofthe NOVX polypeptides, are intended to be within the scope ofthe invention.
  • nucleic acid molecules encoding NOVX proteins from other species and thus that have a nucleotide sequence that differs from the human SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 are intended to be within the scope ofthe invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNAs ofthe invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36.
  • the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length.
  • an isolated nucleic acid molecule ofthe mvention hybridizes to the coding region.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • Homologs i.e., nucleic acids encoding NOVX proteins derived from species other than human
  • other related sequences e.g., paralogs
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% ofthe probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% ofthe probes are occupied at equilibrium.
  • Tm thermal melting point
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al, (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.
  • a non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • An isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequences SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or fragments, analogs or derivatives thereof, under conditions of moderate stringency.
  • moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5%) SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C.
  • Other conditions of moderate stringency that maybe used are well-known within the art.
  • nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided.
  • low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C.
  • Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).
  • nucleotide sequences SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 can be introduced by mutation into the nucleotide sequences SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, thereby leading to changes in the amino acid sequences ofthe encoded NOVX proteins, without altering the functional ability of said NOVX proteins.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence SEQ ID NOS :2, 4, 6, 8, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31, 33, 35, or 37.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequences ofthe NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity.
  • amino acid residues that are conserved among the NOVX proteins ofthe invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
  • nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity.
  • NOVX proteins differ in amino acid sequence from SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37.
  • the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37; more preferably at least about 70% homologous SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37; still more preferably at least about 80% homologous to SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37; even more preferably at least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37; and most preferably at least about 95% homologous to SEQ ID NOS.2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
  • An isolated nucleic acid molecule encoding an NOVX protein homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity.
  • SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe protein can be determined.
  • the relatedness of amino acid families may also be determined based on side chain interactions.
  • Substituted amino acids may be fully conserved "strong” residues or fully conserved “weak” residues.
  • the "strong” group of conserved amino acid residues may be any one ofthe following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other.
  • the "weak” group of conserved residues may be any one ofthe following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLJJVI, HFY, wherein the letters within each group represent the single letter amino acid code.
  • a mutant NOVX protein can be assayed for (i) the ability to form proteimprotein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and an NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
  • a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
  • Another aspect ofthe invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or fragments, analogs or derivatives thereof.
  • An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence).
  • antisense nucleic acid molecules comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding an NOVX protein.
  • coding region refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding the NOVX protein.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of NOVX mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid ofthe invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
  • 2-methylthio-N6-isopentenyladenine 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules ofthe invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an NOVX protein to thereby inhibit expression of the protein (e.g. , by inhibiting transcription and/or translation).
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove ofthe double helix.
  • An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens).
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule ofthe invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other. See, e.g., Gaultier, et al, 1987. Nucl. Acids Res. 15: 6625-6641.
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (See, e.g., ⁇ noue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See, e.g., Inoue, et al, 1987. FEBS Lett. 215: 327-330.
  • Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
  • an antisense nucleic acid ofthe invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes as described in
  • Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA.
  • a ribozyme having specificity for an NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of an NOVX cDNA disclosed herein (i.e., SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in an NOVX-encoding mRNA.
  • NOVX-encoding mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Barrel et al, (1993) Science 261:1411-1418.
  • NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe NOVX nucleic acid (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe NOVX gene in target cells.
  • nucleotide sequences complementary to the regulatory region ofthe NOVX nucleic acid e.g., the NOVX promoter and/or enhancers
  • the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule.
  • the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al, 1996. Bioorg Med Chem 4: 5-23.
  • peptide nucleic acids refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al, 1996. supra; Perry-O'Keefe, et al, 1996. Proc. Natl Acad. Sci. USA 93: 14670-14675.
  • PNAs of NOVX can be used in therapeutic and diagnostic applications. For example,
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., Si nucleases (See, Hyrup, et al, 1996.supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al, 1996, supra; Perry-O'Keefe, et al, 1996. supra).
  • PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996. supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al, 1996. supra and Finn, et al, 1996. Nucl Acids Res 24: 3357-3363.
  • aDNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5 , -(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al, 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al, 1996. supra.
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al, 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al, 1987. Proc. Natl. Acad. Sci.
  • oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al, 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549).
  • the oligonucleotide maybe conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
  • a polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37 while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.
  • an NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues ofthe parent protein as well as the possibility of deleting one or more residues from the parent sequence.
  • Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
  • One aspect ofthe invention pertains to isolated NOVX proteins, and biologically- active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies.
  • native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques, hi another embodiment, NOVX proteins are produced by recombinant DNA techniques.
  • an NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of NOVX proteins in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly-produced.
  • the language "substantially free of cellular material” includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX proteins.
  • non-NOVX proteins also referred to herein as a "contaminating protein”
  • contaminating protein also preferably substantially free of non-NOVX proteins
  • the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% ofthe volume of the NOVX protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis ofthe protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.
  • Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences ofthe NOVX proteins (e.g., the amino acid sequence shown in SEQ JD NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of an NOVX protein.
  • biologically- active portions comprise a domain or motif with at least one activity ofthe NOVX protein.
  • a biologically-active portion of an NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
  • biologically-active portions in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native NOVX protein.
  • the NOVX protein has an amino acid sequence shown SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
  • the NOVX protein is substantially homologous to SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37, and retains the functional activity ofthe protein of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below.
  • the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37, and retains the functional activity of the NOVX proteins of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the nucleic acid sequence homology may be determined as the degree of identity between two sequences.
  • the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMol Biol 48: 443-453.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I, in the case of nucleic acids
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • the invention also provides NOVX chimeric or fusion proteins.
  • NOVX chimeric or fusion proteins.
  • NOVX "chimeric protein” or “fusion protein” comprises an NOVX polypeptide operatively- linked to a non-NOVX polypeptide.
  • An "NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to an NOVX protein SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37), whereas a “non-NOVX polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism.
  • an NOVX fusion protein comprises at least one biologically-active portion of an NOVX protein.
  • an NOVX fusion protein comprises at least two biologically- active portions of an NOVX protein.
  • an NOVX fusion protein comprises at least three biologically-active portions of an NOVX protein.
  • the term "operatively-linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another.
  • the non-NOVX polypeptide can be fused to the N-terminus or C-terminus ofthe NOVX polypeptide.
  • the fusion protein is a GST-NO VX fusion protein in which the
  • NOVX sequences are fused to the C-terminus ofthe GST (glutathione S -transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX polypeptides.
  • the fusion protein is an NOVX protein containing a heterologous signal sequence at its N-terminus.
  • NOVX a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence.
  • the fusion protein is an NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member ofthe immunoglobulin protein family.
  • the NOVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an NOVX ligand and an NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo.
  • the NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of an NOVX cognate ligand.
  • NOVX-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with an NOVX ligand.
  • An NOVX chimeric or fusion protein ofthe invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
  • the invention also pertains to variants ofthe NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists.
  • Variants ofthe NOVX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation ofthe NOVX protein).
  • An agonist ofthe NOVX protein can retain substantially the same, or a subset of, the biological activities ofthe naturally occurring form ofthe NOVX protein.
  • An antagonist of the NOVX protein can inhibit one or more ofthe activities ofthe naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein.
  • treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe NOVX proteins.
  • Variants ofthe NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g. , truncation mutants) of the NOVX proteins for NOVX protein agonist or antagonist activity.
  • a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein.
  • a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein.
  • methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • degenerate set of genes allows for the provision, in one mixture, of all ofthe sequences encoding the desired set of potential NOVX sequences.
  • Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al, 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al, 1984. Science 198: 1056; Ike, et al, 1983. Nucl. Acids Res. 11: 477.
  • libraries of fragments ofthe NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of an NOVX protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Si nuclease, and ligating the resulting fragment library into an expression vector.
  • expression libraries can be derived which encodes N-terminal and internal fragments of various sizes ofthe NOVX proteins.
  • Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al, 1993. Protein Engineering 6:327-331.
  • antibodies to NOVX proteins, or fragments of NOVX proteins.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin (Ig) molecules i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F a , F ab * and F (a ')2 fragments, and an F ab expression library.
  • an antibody molecule obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature ofthe heavy chain present in the molecule.
  • Certain classes have subclasses as well, such as IgGi, IgG 2 , and others.
  • the light chain may be a kappa chain or a lambda chain.
  • Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
  • An isolated NOVX-related protein ofthe invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments ofthe antigen for use as immunogens.
  • An antigenic peptide fragment comprises at least 6 amino acid residues ofthe amino acid sequence ofthe full length protein and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope.
  • the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions ofthe protein that are located on its surface; commonly these are hydrophilic regions.
  • At least one epitope encompassed by the antigenic peptide is a region of NOVX-related protein that is located on the surface ofthe protein, e.g., a hydrophilic region.
  • a hydrophobicity analysis ofthe human NOVX-related protein sequence will indicate which regions of a NOVX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78:
  • Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
  • a protein ofthe invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative ofthe foregoing.
  • An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.
  • the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by inimunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be immunized in vitro.
  • the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J.
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
  • antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells ofthe invention serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, N ⁇ twre 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody.
  • the antibodies directed against the protein antigens ofthe invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.
  • Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen- binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.
  • Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues ofthe human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin and all or substantially all ofthe framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al, 1988; and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)).
  • Fc immunoglobulin constant region
  • Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 hnmunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, hie, pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice ofthe present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825;
  • Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
  • WO94/02602 The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encodmg human heavy and light chain immunoglobulins are inserted into the host's genome.
  • the human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.
  • An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement ofthe modifications.
  • the preferred embodiment of such a nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
  • U.S. Patent No. 5,939,598 An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
  • a method for producing an antibody of interest such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • F a b Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein ofthe invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of F ab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F a fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.
  • Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F( a ' )2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F( a b ' ) 2 fragment; (iii) an F a b fragment generated by the treatment ofthe antibody molecule with papain and a reducing agent and (iv) F v fragments.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one ofthe binding specificities is for an antigenic protein ofthe invention.
  • the second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because ofthe random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, 1991 EMBO J, 10:3655-3659.
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one ofthe fusions.
  • CHI first heavy-chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part ofthe CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield ofthe heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One ofthe Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

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Abstract

Disclosed herein are nucleic acid sequences that encode novel polypeptides. Also disclosed are polypeptides encoded by these nucleic acid sequences, and antibodies, which immunospecifically-bind to the polypeptide, as well as derivatives, variants, mutants, or fragments of the aforementioned polypeptide, polynucleotide, or antibody. The invention further discloses therapeutic, diagnostic and research methods for diagnosis, treatment, and prevention of disorders involving any one of these novel human nucleic acids and proteins.

Description

NOVEL PROTEINS AND NUCLEIC ACIDS ENCODING SAME
FIELD OF THE INVENTION
The invention generally relates to nucleic acids and polypeptides encoded thereby. 5
BACKGROUND OF THE INVENTION
The invention generally relates to nucleic acids and polypeptides encoded therefrom. More specifically, the invention relates to nucleic acids encodmg cytoplasmic, nuclear, membrane bound, and secreted polypeptides, as well as vectors, host cells, antibodies, and 10 recombinant methods for producing these nucleic acids and polypeptides.
SUMMARY OF THE INVENTION
The invention is based in part upon the discovery of nucleic acid sequences encoding novel polypeptides. The novel nucleic acids and polypeptides are referred to herein as NOVX, i 5 or NOV1 , NOV2, NOV3 , NOV4, NOV5, NOV6, NOV7, and NOV8 nucleic acids and? polypeptides. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as "NOVX" nucleic acid or polypeptide sequences.
In one aspect, the invention provides an isolated NOVX nucleic acid molecule
.0 encoding a NOVX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36. In some embodiments, the NOVX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a NOVX nucleic acid sequence. The mvention also
.5 includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof. For example, the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37. The nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid
50 sequence of any of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36. Also included in the invention is an oligonucleotide, e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of aNOVX nucleic acid (e.g., SEQ JD NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36) or a complement of said oligonucleotide. Also included in the mvention are substantially purified NOVX polypeptides (SEQ ID
NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37). In certain embodiments, the NOVX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human NOVX polypeptide.
The invention also features antibodies that immunoselectively bind to NOVX polypeptides, or fragments, homologs, analogs or derivatives thereof.
In another aspect, the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier. The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or an antibody specific for a NOVX polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
In a further aspect, the invention includes a method of producing a polypeptide by culturing a cell that includes a NOVX nucleic acid, under conditions allowing for expression ofthe NOVX polypeptide encoded by the DNA. If desired, the NOVX polypeptide can then be recovered.
In another aspect, the invention includes a method of detecting the presence of a NOVX polypeptide in a sample. In the method, a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound. The complex is detected, if present, thereby identifying the NOVX polypeptide within the sample.
The invention also includes methods to identify specific cell or tissue types based on their expression of a NOVX.
Also included in the invention is a method of detecting the presence of a NOVX nucleic acid molecule in a sample by contacting the sample with a NOVX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a NOVX nucleic acid molecule in the sample. In a further aspect, the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample that includes the NOVX polypeptide with a compound that binds to the NOVX polypeptide in an amount sufficient to modulate the activity of said polypeptide. The compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
Also within the scope ofthe invention is the use of a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., Inflamation, Autoimmune disorders, scleroderma, transplantation, autoimmune disease, allergies, immunodeficiencies, systemic lupus erythematosus, lymphedema, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, graft versus host disease (GVHD), endometriosis, cystic fibrosis, pathological disorders involving spleen, thymus, lung, and peritoneal macrophages, Aging, Alzheimer's disease, stroke, Cancer, abnormal angiogenesis, like cancer and more specifically aggressive, metastatic cancer, more specifically tumor ofthe lung, kidney, brain, liver and colon, mastocytomas, prostate cancer, modulation of apoptosis, Oncogenesis such as lung, liver and ovarian cancers; cell growth and possibly differentiation, non-Hodgkin lymphomas, Von Hippel-Lindau (VHL) syndrome, Lesch-Nyhan syndrome, periodontitis, pancreatitis, musculoskeletal disorders, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, behavioral disorders, addiction, anxiety, pain, neurodegeneration, familial amyloidotic polyneuropathy, neurodegenerative and neuropsychiatric disorders, leukodystrophies, dysbetalipoproteinemia, hyperlipoproteinemia type III, xanthomatosis, coronary and/or peripheral vascular disease,ardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, accelerated vascular disease, angiogenesis and wound healing, leukemogenesis, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, renal tubular acidosis, IgA nephropathy, fertility, endocrine dysfunctions, diabetes, diabetic acidosis, hypercholesterolemia, planar and tendon xanthomas, obesity, growth and reproductive disorders, hyperthyroidism, hypothyroidism, type 2 diabetes, chronic lung diseases, asthma, tuberous sclerosis and/or other pathologies and disorders ofthe like. The therapeutic can be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or a NOVX- specific antibody, or biologically-active derivatives or fragments thereof.
For example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like. The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds. For example, a cDNA encoding NOVX may be useful in gene therapy, and NOVX may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like.
The invention further includes a method for screening for a modulator of disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like. The method includes contacting a test compound with a NOVX polypeptide and determining if the test compound binds to said NOVX polypeptide. Binding ofthe test compound to the NOVX polypeptide indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
Also within the scope ofthe invention is a method for screening for a modulator of activity, or of latency or predisposition to an disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes. The test animal expresses a recombinant polypeptide encoded by a NOVX nucleic acid. Expression or activity of NOVX polypeptide is then measured in the test animal, as is expression or activity ofthe protein in a control animal which recombinantly- expresses NOVX polypeptide and is not at increased risk for the disorder or syndrome. Next, the expression of NOVX polypeptide in both the test animal and the control animal is compared. A change in the activity of NOVX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency ofthe disorder or syndrome. In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX polypeptide, a NOVX nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount ofthe NOVX polypeptide in a test sample from the subject and comparing the amount ofthe polypeptide in the test sample to the amount ofthe NOVX polypeptide present in a control sample. An alteration in the level ofthe NOVX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject. Preferably, the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like. Also, the expression levels ofthe new polypeptides ofthe invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
In a further aspect, the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subj ect a NOVX polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition. In preferred embodiments, the disorder, includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders ofthe like. In yet another aspect, the invention can be used in a method to identity the cellular receptors and downstream effectors ofthe invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing ofthe present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages ofthe invention will be apparent from the following detailed description and claims. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences and their encoded polypeptides. The sequences are collectively referred to herein as "NOVX nucleic acids" or "NOVX polynucleotides" and the corresponding encoded polypeptides are referred to as "NOVX polypeptides" or "NOVX proteins." Unless indicated otherwise, "NOVX" is meant to refer to any ofthe novel sequences disclosed herein. Table A provides a summary ofthe NOVX nucleic acids and their encoded polypeptides.
TABLE A. Sequences and Corresponding SEQ ID Numbers
Figure imgf000008_0001
NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members ofthe protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members ofthe family to which the NOVX polypeptides belong. NO VI is homologous to a thioredoxin -like family of proteins. Thus, the NO VI nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; Inflamation, Autoimmune disorders, Aging and Cancer, and/or other pathologies/disorders. NOV2a and 2b are homologous to the Retinoic acid responsive/STRA-6-like family of proteins. Thus NOV2 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, scleroderma, transplantation, autoimmune disease, allergies, immunodeficiencies, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, Lesch-Nyhan syndrome, lymphedema, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, graft versus host disease (GVHD), endometriosis, fertility, endocrine dysfunctions, diabetes, obesity, growth and reproductive disorders, hyperthyroidism, hypothyroidism and/or other pathologies/disorders.
NOV3a and 3b are homologous to a family of LRR/GPCR-like proteins. Thus, the NOV3 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; abnormal angiogenesis, like cancer and more specifically aggressive, metastatic cancer, more specifically rumor ofthe lung, kidney, brain, liver and colon and/or other pathologies/disorders.
NOV4 is homologous to the Trypsin inhibitor-like family of proteins. Thus, NOV4 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; chronic lung diseases, periodontitis, pancreatitis and/or other pathologies/disorders.
NOV5a, b, c, d, e, f, g, h and i are homologous to the apolipoprotein e-like family of proteins. Thus NOV5 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; atherosclerosis, dysbetalipoproteinemia, hyperlipoproteinemia type ILT, xanthomatosis, coronary and/or peripheral vascular disease, hypothyroidism, systemic lupus erythematosus, diabetic acidosis, hypercholesterolemia, planar and tendon xanthomas, dysbetalipoproteinemia, hypercholesterolemia, accelerated vascular disease, Alzheimer disease, familial amyloidotic polyneuropathy and/or other pathologies/disorders.
NOV6 is homologous to the mastocytoma protease precursor -like family of proteins. Thus NOV6 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; allergies, asthma and cystic fibrosis, cancers such as mastocytomas, vascular diseases and/or other pathologies/disorders.
NOV7 is homologous to members ofthe thymosin beta-4-like family of proteins. Thus, the NOV7 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; prostate cancer, immunological and autoimmune disorders (ie hyperthyroidism), angiogenesis and wound healing, modulation of apoptosis, neurodegenerative and neuropsychiatric disorders, age-related disorders, pathological disorders involving spleen, thymus, lung, and peritoneal macrophages and Cancer and/or other pathologies/disorders. NOV8 is homologous to the Protein-tyrosine-phosphatase -like family of proteins. Thus, NOV8 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; hematopoiesic disorders; leukemogenesis; polycystic kidney disease; Oncogenesis such as lung, liver and ovarian cancers; apoptosis; type 2 diabetes and obesity; cell growth and possibly differentiation; non-Hodgkin lymphomas; musculoskeletal disorders; and CNS development disorders and/or other pathologies/disorders.
The NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., neurogenesis, cell differentiation, cell proliferation, hematopoiesis, wound healing and angiogenesis.
Additional utilities for the NOVX nucleic acids and polypeptides according to the invention are disclosed herein. NOV1
A disclosed NO VI nucleic acid of 620 nucleotides (also referred to as AC013554_dal) encoding a novel thioredoxin-like protein is shown in Table 1 A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 282-284 and ending with a TAA codon at nucleotides 618-620. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 1 A. The start and stop codons are in bold letters.
Table 1A. NOV1 nucleotide sequence (SEQ ID NO:l).
TGTAAAACAAGACCAGGCACAAGAAGGGTGACATCCCCAAGTCCCCAGAAGAAGCCATCCAGCACAAGGAGG GTGACATTCCCAAGTCTCCAAAACAAGCCATCCAGCCCAAGGAGGGTGACATTCCCAAGTCCCTAGAGGAAG CCATCCCACCCAAGGAGATTGACATCCCCAAGTCCCCAGAAGAAACCATCCAGCCCAAGGAGGATGACAGCC CCAAGTCCCTAGAAGAAGCCACCCCATCCAAGGAGGGTGACATCCTAAAGCCTGAAGAAGAAACAATGGAGT TCCCGGAGGGGGACAAGGTGAAAGTGATCCTGAGCAAGGAGGACTTTGAGACATCACTGAAGGAGGCCGGGG AGAGGCTGGTGGCTGTGGACTTCTCGGCCACGTGGTGTGGGCCCTGCAGGACCATCAGACCATTCTTCCATG CCCTGTCTGTGAAGCATGAGGATGTGGTGTTCCTGGAGGTGGACGCTGACAACTGTGAGGAGGTGGTGAGAG AGTGCGCCATCATGTGTGTCCCAACCTTTCAGTTTTATAAAAAAGAAGAAAAGGTGGATGAACTTTGCGGCG CCCTTAAGGAAAAA.CTTGAAGCAGTCATTGCAGAATTAAAGTAA
In a search of public sequence databases, the NO VI nucleic acid sequence, ocated on the p31 region of chromsome 9 has 232 of 331 bases (70%) identical to a thioredoxin mRNA from Ovis aries (GENBANK-ID: OATHIORD|acc:Z25864). Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
In all BLAST alignments herein, the "E-value" or "Expect" value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. For example, the probability that the subject ("Sbjct") retrieved from the NOV1 BLAST analysis, e.g., thioredoxin mRNA from Ovis aries, matched the Query NOV1 sequence purely by chance is 608e- . The Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences.
The Expect value is used as a convenient way to create a significance threshold for reporting results. The default value used for blasting is typically set to 0.0001. In BLAST 2.0, the Expect value is also used instead ofthe P value (probability) to report the significance of matches. For example, an E value of one assigned to a hit can be interpreted as meaning that in a database ofthe current size one might expect to see one match with a similar score simply by chance. An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education BLASTinfo/. Occasionally, a string of X's or N's will result from a BLAST search. This is a result of automatic filtering ofthe query for low- complexity sequence that is performed to prevent artifactual hits. The filter substitutes any low-complexity sequence that it finds with the letter "N" in nucleotide sequence (e.g., "NNNNNNNNNNNNN") or the letter "X" in protein sequences (e.g., "XXXXXXXXX"). Low-complexity regions can result in high scores that reflect compositional bias rather than significant position-by-position alignment. Wootton and Federhen, Methods Enzymol 266:554-571, 1996.
The disclosed NOVl polypeptide (SEQ ID NO:2) encoded by SEQ ID NO:l has 112 amino acid residues and is presented in Table IB using the one-letter amino acid code. Signal P, Psort and/or Hydropathy results predict that NOVl has a signal peptide and is likely to be localized in the cytoplasm with a certainty of 0. 6500. In other embodiments, NOVl may also be localized to the mitochondrial matrix space with acertainty of 0.1000, or the lysosome (lumen) with a certainty of 0.1000. The NOVl peptide is not likely to have a signal peptide.
Table IB. Encoded NOVl protein sequence (SEQ ID NO:2).
MEFPEGDKVKVILSKEDFETSLKEAGERLVAVDFSAT CGPCRTIRPFFHALSVKHEDWFLEVDADNCEEV VRECAIMCVPTFQFYKKEEKVDELCGAKEKLEAVIAELK
A search of public sequence databases reveals that the NOVl amino acid sequence has 65 of 103 amino acid residues (63%) identical to, and 80 of 103 amino acid residues (77%) similar to, the 105 amino acid residue Thioredoxin - protein from Equus caballus (SPTREMBL-ACC.O97508) (E = 3.2e-32). Public amino acid databases in elude the GenBank databases, SwissProt, PDB and PIR.
The disclosed NOVl polypeptide has homology to the amino acid sequences shown in the BLASTP data listed in Table lC.
Figure imgf000013_0002
The homology between these and other sequences is shown graphically in the ClustalW analysis shown in Table ID. In the ClustalW alignment ofthe NOVl protein, as well as all other ClustalW analyses herein, the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be altered to a much broader extent without altering protein structure or function.
Table ID. ClustalW Analysis of NOVl
1) Novel NOVl (SEQ ID NO: 2)
2) 097508 equus caballus (horse) . thioredoxin (SEQ ID NO:38)
3) THIO_SHEEP ovis aries (sheep) . thioredoxin (SEQ ID Nθ:39)
4) THIO_BOVIN bos taurus (bovine) . thioredoxin (SEQ ID NO.-40) 5) THIO_MACMU macaca mulatta (rhesus macaque) . thioredoxin (SEQ ID NO: 41)
6} THIO_RAT rattus norvegicus (rat) . thioredoxin (SEQ ID NO: 42)
Figure imgf000013_0001
Figure imgf000014_0001
The presence of identifiable domains in NOVl, as well as all other NOVX proteins, was determined by searches using software algorithms such as PROSITE, DOMAIN, Blocks, Pfam, ProDomain, and Prints, and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/ interpro). DOMAIN results for NOVl as disclosed in Tables IE, were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST analyses. This BLAST analysis software samples domains found in the Smart and Pfam collections. For Table IE and all successive DOMAIN sequence alignments, fully conserved single residues are indicated by black shading or by the sign (|) and "strong" semi-conserved residues are indicated by grey shading or by the sign (+). The "strong" group of conserved amino acid residues may be any one ofthe following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
Table IE lists the domain description from DOMAIN analysis results against NOVl. This indicates that the NOVl sequence has properties similar to those of other proteins known to contain this domain.
Table IE. Domain Analysis of NOVl gnl|Pfam[pfam00085, thiored, Thioredoxin. (SEQ ID NO: 73) CD-Length = 108 residues, 97.2% aligned Score = 89.4 bits (220), Expect = le-19
Query: 7 DKVKVILSKEDFETSItKEAGERLVAVDFSAT CGPCRTIRPFFHALSVKH-EDWFLEVD 65
+ I |+|+ |+|+ + +| ++ I III I IIIH+ + I + |+ ++ +|| I +||
Sbjct: 1 ESvVvVLTDENFDEEVLKASDKPVLvDFYAPWCGPCKMLAPEYEKLAEEYKDDVKFAKVD 60
Query: 66 ADNCEEWRECAIMCVPTFQFYKKEEKVDELCGALKEKLEAVIAE 110
II ++ I + II +1+1 +111 + 1 II I I I + Sbjct: 61 ADENPDLASEYGVRGFPTLKFFKNGKKVDYVGGRTKEALVAFIKK 105
NOVl also has high homology to members ofthe patp database shown in Table IF.
Table IF. PATP BLASTX results for NOVl
Gene Index/ Protein/ Length Identity Positives Expect Identifier Organism (aa) (%) (%)
Figure imgf000015_0001
Thioredoxin is an oxidoreductase enzyme of 12,000 daltons, containing a dithiol- disulfide active site. It is ubiquitous and found in many organisms from plants and bacteria to mammals. Multiple in vitro substrates for thioredoxin have been identified, including ribonuclease, choriogonadotropins, coagulation factors, glucocorticoid receptor, and insulin. Reduction of insulin is classically used as an activity test. Wollman et al. (1988) identified a full-length cDNA clone coding for human thioredoxin. The open reading frame coded for a protein of 104 amino acids (excluding the initial methionine). This protein possessed the highly conserved enzymatic active site common to plant and bacterial thioredoxins: trp-cys- gly-pro-cys (amino acids 30-34). Tonissen and Wells (1991) determined that the TRX gene extends over 13 kb and consists of 5 exons encoding a 12-kD protein. Southern analysis demonstrated the presence of several TXN genes in the human genome. At least one of these is inactive, i.e., apseudogene. Kaghad et al. (1994) also cloned the TXN gene and demonstrated that the 5 exons are separated by 4 introns. They determined the +1 transcription start point by primer extension. The +1 site is located 22 bp downstream from a TATAA box and defines a 5-prime untranslated region of 74 bp. Southern hybridization of genomic DNAs from several donors detected only 1 active TXN gene.
Using in situ chromosomal hybridization with a human TXN cDNA probe, Lafage- Pochitaloff-Huvale et al. (1989) localized the gene to 3pl2-pl 1. Taketo et al. (1994) found that the homologous gene in mouse is located on chromosome 4 and that there is a processed Txn pseudogene in the proximal region of mouse chromosome 1.
Heppell-Parton et al. (1995) concluded that the correct chromosomal localization ofthe transcribed thioredoxin gene is 9q31. They discovered this both by analysis of a somatic cell hybrid panel and by fluorescence in situ hybridization of a YAC encoding the transcribed gene. The localization to chromosome 9 was confirmed by PCR amplification from a human/hamster somatic cell hybrid containing chromosome 9 as its only human chromosome. No amplification signals were detected in any ofthe other monochromosome hybrid cells. The location ofthe mouse thioredoxin gene on chromosome 4 is noteworthy because part of that chromosome shares homology with human chromosome 9.
Proteins internalized into the endocytic pathway are usually degraded. Efficient proteolysis requires denaturation, induced by acidic conditions within lysosomes, and reduction of inter- and intrachain disulfide bonds. Cytosolic reduction is mediated enzymatically by thioredoxin (TXN; 187700). (Arunachalam et al.; Nat. Acad. Sci. 97: 745- 750, 2000) described a lysosomal thiol reductase, which they called GILT, that was optimally active at low pH and capable of catalyzing disulfide bond reduction both in vivo and in vitro. They found that the GILT gene encodes a predicted 261-amino acid protein composed of a 37- amino acid signal peptide and a 224-amino acid proform. The enzyme was expressed constitutively in antigen-presenting cells and was induced by gamma-interferon (147570) in other cell types, suggesting a potentially important role in antigenprocessing.
Selenium has been indirectly implicated in immunologic function and numerous nutritional studies over many years. Furthermore, HTV-infected persons are reported to have decreased levels of plasma selenium and selenium-containing glutathione peroxidase (e.g., 138321). For this reason, (Gladyshev et al.;Proc. Nat. Acad. Sci. 93: 6146-6151, 1996) initiated studies on selenium metabolism in human T cells. The authors identified one ofthe selenoproteins detected in T cells as thioredoxin reductase and demonstrated that the location of selenocysteme in this protein corresponds to a TGA codon in the cloned placental gene. The finding that T-cell thioredoxin reductase is a selenoenzyme that contains selenium in a conserved C-terminal region provides another example ofthe role of selenium in the major antioxidant enzyme system (i.e., thioredoxin-thioredoxin reductase), in addition to the well- known glutathione peroxidase enzyme system. (Gorlatov and Stadtman; Proc. Nat. Acad. Sci. 95: 8520-8525, 1998) demonstrated the essential role of selenocysteme in thioredoxin reductase isolated from HeLa cells by alkylation studies. Selective alkylation of selenocysteme in the protein inhibited enzyme activity, and reduction with NADPH influenced affinity to heparin.
The disclosed NOVl nucleic acid ofthe invention encoding a thioreductase-like protein includes the nucleic acid whose sequence is provided in Table 1 A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 1 A while still encoding a protein that maintains its thioreductase-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any ofthe nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 30 % percent ofthe bases may be so changed.
The disclosed NOVl protein ofthe invention includes the thioreductase-like protein whose sequence is provided in Table IB. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue.shown in Table 2 while still encoding a protein that maintains its thioreductase-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 38 % percent ofthe residues may be so changed.
The invention further encompasses antibodies and antibody fragments, such as Fab or (Fab)2, that bind immunospecifically to any ofthe proteins ofthe invention. The above defined information for this invention suggests that this thioreductase-like protein (NOVl) may function as a member of a "Thioredoxin family". Therefore, the NOVl nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below. The potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
The NOVl nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in cancer including but not limited to Inflamation,
Autoimmune disorders, Aging and Cancer. For example, a cDNA encoding the thioreductase- like protein (NOVl) may be useful in gene therapy, and the thioreductase-like protein (NOVl) may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from Inflamation, Autoimmune disorders, Aging and Cancer. The NOVl nucleic acid encoding thioreductase-like protein, and the thioreductase-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
NOVl nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOVl substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. The disclosed NOVl protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOVl epitope is from about amino acids 1 to 10. In another embodiment, a NOVl epitope is from about amino acids 20 to 35. In additional embodiments, NOVl epitopes are from about amino acids 45 to 50, from about amino acids 55 to 60, from about amino acid 65 to 75, and from about amino acids 90 to 115. These novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
NOV2 NOV2 includes two novel retinoic acid/SRA6-like proteins disclosed below. The disclosed proteins have been named NOV2a and NOV2b. NOV2a
A disclosed NOV2a nucleic acid of 2632 nucleotides (also referred to as 3277789.0.83_dal) encoding a novel Retinoic acid responsive/STRA-6-like protein is shown in Table 2A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 31-33 and ending with a TGA codon at nucleotides 2011-2013. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 2A, and the start and stop codons are in bold letters. Table 2A. NOV2a nucleotide sequence (SEQ ID NO:3).
CGCCTGGGGTCAAAGGAGAAGGGCCAGAGAATGTCGTCCCAGCCAGCAGGGAACCAGACCTCCCCCGGGGCC ACAGAGGACTACTCCTATGGCAGCTGGTACATCGATGAGCCCCAGGGGGGCGAGGAGCTCCAGCCAGAGGGG GTGCCCTCCTGCCACACCAGCATACCACCCGGCCTGTACCACGCCTGCCTGGCCTCGCTGTCATTTCAGATC CTTGTGCTGCTGCTCCTGGCCATGCTGGTGAGGCGCCGCCAGCTCTGGCCTCAGTGTGGACATGGCCTTAGC CCTGTGGATTTCTTGGCTGGGGACAGGCCCCGGGCAGTGCCTGCTGCTGTTTTCATGGTCCTCCTGAGCTCC CTGTGTTTGCTGCTCCCCGACGAGGACGCATTGCCCTTCCTGACTCTCGCCTCAGCACCCAGCGGGGCCTGG AAGATACTGGGACTGTTCTATTATGCTGCCCTCTACTACCCTCTGGCTGCCTGTGCCACGGCTGGCCACACA GCTGCACACCTGCTCGGCAGCACGCTGTCCTGGGCCCACCTTGGGGTCCAGGTCTGGCAGAGGGCAGAGTGT CCCCAGGTGCCCAAGATCTACAAGTACTACTCCCTGCTGGCCTCCCTGCCTCTCCTGCTGGGCCTCGGATTC CTGAGCCTTTGGTACCCTGTGCAGCTGGTGAGAAGCTTCAGCCGTAGGACAGGAGCAGGCTCCCAGGGGCTG CAGAGCAGCTACTCTGAGGAATATCTGAGGAACCTCCTTTGCAGGAAGAAGCTGGGAAGCTGCAGCTACCAC ACCTCCAAGCATGGCTTCCTGTCCTGGGCCCGCGTCTGCTTGAGACACTGCATCTACACTCCACAGCCAGGA TTCCATCTCCCGCTGAAGCTGGTGCTTTCAGCTACACTGACAGGGACGGCCATTTACCAGGTAGCCCTGCTG CTGCTGGTGGGCGTGGTACCCACTATCCAGAAGGTGAGGGCAGGGGTCACCACGGATGTCTCCTACCTGCTG GCCGGCTTTGGAATCGTGCTCTCCGAGGACAAGCAGGAGGTGGTGGAGCTGGTGAAGCACCATCTGTGGGCT CTGGAATGCTACATCTCAGCCTTGGTCTTGTCCTGCTTACTCACCTTCCTGGTCCTGATGCGCTCACTGGTG ACACACAGGACCAACCTTCGAGCTCTGCACCGAGGAGCTGCCCTGGACTTGAGTCCCTTGCATCGGAGTCCC CATCCCTCCCGCCAAGCCATATTCTGTTGGATGAGCTTCAGTGCCTACCAGACAGCCTTTATCTGCCTTCTC CCGCCCCTGGCAGGGCTCCTGGTGCAGCAGATCATCTTCTTCCTGGGAACCACGGCCCTGGCCTTCCTGGTG CTCATGCCTGTGCTCCATGGCAGGAACCTCCTGCTCTTCCGTTCCCTGGAGTCCTCGTGGCCCTTCTGGCTG ACTTTGGCCCTGGCTGTGATCCTGCAGAACATGGCAGCCCATTGGGTCTTCCTGGAGACTCATGATGGACAC CCACAGCTGACCAACCGGCGAGTGCTCTATGCAGCCACCTTTCTTCTCTTCCCCCTCAATGTGCTGGTGGGT GCCATGGTGGCCACCTGGCGAGTGCTCCTCTCTGCCCTCTACAACGCCATCCACCTTGGCCAGATGGACCTC AGCCTGCTGCCACCGAGAGCCGCCACTCTCGACCCCGGCTACTACACGTACCGAAACTTCTTGAAGATTGAA GTCAGCCAGTCGCATCCAGCCATGACAGCCTTCTGCTCCCTGCTCCTGCAAGCGCAGAGCCTCCTACCCAGG ACCATGGCAGCCCCCCAGGACAGCCTCAGACCAGGGGAGGAAGACGAAGGGATGCAGCTGCTACAGACAAAG GACTCCATGGCCAAGGGAGCTAGGCCCGGGGCCAGCCGCGGCAGGGCTCGCTGGGGTCTGGCCTACACGCTG CTGCACAACCCAACCCTGCAGGTCTTCCGCAAGACGGGCCTGTTGGGTGCCAATGGTGCCCAGCCCTGAGGG CAGGGAAGGTCAACCCACCTGCCCATCTGTGCTGAGGCATGTTCCTGCCTACCATCCTCCTCCCTCCCCGGC TCTCCTCCCAGCATCACACCAGCCATGCAGCCAGCAGGTCCTCCGGATCACCGTGGTTGGGTGGAGGTCTGT CTGCACTGGGAGCCTCAGGAGGGCTCTGCTCCACCCACTTGGCTATGGGAGAGCCAGCAGGGGTTGTGGAGA AAAAAACTGGTGGGTTAGGGCCTTGGTCCAGGAGCCAGTTGAGCCAGGGCAGCCACATCCAGGCGTCTCCCT ACCCTGGCTCTGCCATCAGCCTTGAAGGGCCTCGATGAAGCCTTCTCTGGAACCACTCCAGCCCAGGTCCAC CTCAGCCTTGGCCTTCACGCTGTGGAAGCAGCCAAGGCACTTCCTCACCCCCTCAGCGCCACGGACCTCTCT GGGGAGTGGCCGGAAAGCTCCCGGGCCTCTGGCCTGCAGGGCAGCCCAAGTCATGACTCAGACCAGGTCCCA CACTGAGCTGCCCACACTCGAGAGCCAGATATTTTTGTAGTTTTTATGCCTTTGGCTATTATGAAAGAGGTT AGTGTGTTCCCTGCAATAAACTTGGTCCTGAGAAAAAAAA
The NOV2a nucleic acid was identified by TblastN using CuraGen Corporation's sequence file for Retinoic acid responsive/STRA-6 or homolog as run against the Genomic Daily Files made available by GenBank or from files downloaded from the individual sequencing centers. The nucleic acid sequence was predicted from the genomic file Genbank by homology to a known Retinoic acid responsive/STRA-6 or homolog. Exons were predicted by homology and the intron/exon boundaries were determined using standard genetic rules. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlastN, BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein.
The disclosed NO V2a nucleic acid sequence, localized to chromsome 15, has 1511 of 2207 bases (68%) identical to a retinoic acid-responsive protein (Stra6) mRNA from Mus musculus (GENBANK-ID: AF062476|acc: AF062476 (GENBANK-ID: M77668)(E = 1.2e" 189).
A NOV2a polypeptide (SEQ ID NO:4) encoded by SEQ ID NO:3 has 660 amino acid residues and is presented using the one-letter code in Table 2B. Signal P, Psort and/or Hydropathy results predict that NOV2A contain a signal peptide and is likely to be localized in the plasmam membrane with a certainty of 0.6000. In other embodiments, NOV2a may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The most likely cleavage site for a NOV2A peptide is between amino acids 8 and 9, at: AGN-QT.
Table 2B. Encoded NOV2a protein sequence (SEQ ID NO:4).
MSSQPAGNQTSPGATEDYSYGS YIDEPQGGEELQPEGVPSCHTSIPPGLYHACLASLSFQILVLLLLAMLV RRRQL PQCGHGLSPVDFLAGDRPRAVPAAVFMVLLSSLCLLLPDEDALPFLTLASAPSGAWKILGLFYYAA LYYPLAACATAGHTAAHLLGSTLS AHLGVQV QRAECPQVPKIYKYYSLLASLPLLLGLGFLSL YPVQLV RSFSRRTGAGSQGLQSSYSEEYLRNLLCRKKLGSCSYHTSKHGFLS ARVCLRHCIYTPQPGFHLPLKLVLS ATLTGTAIYQVALLLLVGWPTiQKVRAGVTTDVSYLLAGFGIVLSED QEWELVKHHLWALECYISALVL SCLLTFLVLMRSLVTHRTNLRALHRGAALDLSPLHRSPHPSRQAIFC MSFSAYQTAFICLLPPLAGLLVQQ IIFFLGTTALAFLVLMPVLHGRNLLLFRSLESS PFWLTLALAVILQN AAHWVFLETHDGHPQLTNRRVLY AATFLLFPLNVLVGAMVAT RVLLSALYNAIHLGQMDLSLLPPRAATLDPGYYTYRNFLKIEVSQSHPAMTA FCSLLLQAQSLLPRTMAAPQDSLRPGEEDEGMQLLQTKDSMAKGARPGASRGRAR GLAYTLLHNPTLQVFR TALLGANGAQP
The disclosed NOV2a amino acid sequence has 497 of 670 amino acid residues (74%) identical to, and 556 of 670 amino acid residues (82%) similar to, the 670 amino acid residue Retinoic Acid-Responsive protein from Mus musculus (SPTREMBL-ACC.O70491) (E =
7.1e"257).
NOV2a also has high homology to members ofthe patp database shown in Table 2C.
Figure imgf000020_0001
Figure imgf000021_0001
NOV2b
A disclosedNOV2b nucleic acid of2663 nucleotides (also referred to as CG52276-07) encoding a novel STRA6-like protein is shown in Table 2D. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TGA codon at nucleotides 2068-2070. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 2D, and the start and stop codons are in bold letters.
Table 2D. NOV2b Nucleotide Sequence (SEQ ID NO:5)
ATGTCGTCCCAGCCAGCAGGGAACCAGACCTCCCCCGGGGCCACAGAGGACTACTCCTATGGCAGCTGGTA CATCGATGAGCCCCAGGGGGGCGAGGAGCTCCAGCCAGAGGGGGAAGTGCCCTCCTGCCACACCAGCATAC CACCCGGCCTGTACCACGCCTGCCTGGCCTCGCTGTCAATCCTTGTGCTGCTGCTCCTGGCCATGCTGGTG AGGCGCCGCCAGCTCTGGCCTGACTGTGTGCGTGGCAGGCCCGGCCTGCCCAGCTGGGAGGTAATGGGGGT GCAGGGGCTGAGCAACTCCCTCCTGCCCTGTCCTTGCCTCTGTGCTAGCCCTGTGGATTTCTTGGCTGGGG ACGGGCCCCGGGCAGTGCCTGCTGCTGTTTTCATGGTCCTCCTGAGCTCCCTGTGTTTGCTGCTCCCCGAC GAGGACGCATTGCCCTTCCTGACTCTCGCCTCAGCACCCAGCCAAGATGGGAAAACTGAGGCTCCAAGAGG GGCCTGGAAGATACTGGGACTGTTCTATTATGCTGCCCTCTACTACCCTCTGGCTGCCTGTGCCACGGCTG GCCACACAGCTGCACACCTGCTCGGCAGCACGCTGTCCTGGGCCCACCTTGGGGTCCAGGTCTGGCAGAGG GCAGAGTGTCCCCAGGTGCCCAAGATCTACAAGTACTACTCCCTGCTGGCCTCCCTGCCTCTCCTGCTGGG CCTCGGATTCCTGAGCCTTTGGTACCCTGTGCAGCTGGTGAGAAGCTTCAGCCGTAGGACAGGAGCAGGCT CCAAGGGGCTGCAGAGCAGCTACTCTGAGGAATATCTGAGGAACCTCCTTTGCAGGAAGAAGCTGGGAAGC AGCTACCACACCTCCAAGCATGGCTTCCTGTCCTGGGCCCGCGTCTGCTTGAGACACTGCATCTACACTCC ACAGCCAGGATTCCATCTCCCGCTGAAGCTGGTGCTTTCAGCTACACTGACAGGGACGGCCATTTACCAGG TGGCCCTGCTGCTGCTGGTGGGCGTGGTACCCACTATCCAGAAGGTGAGGGCAGGGGTCACCACGGATGTC TCCTACCTGCTGGCCGGCTTTGGAATCGTGCTCTCCGAGGACAAGCAGGAGGTGGTGGAGCTGGTGAAGCA CCATCTGTGGGCTCTGGAAGTGTGCTACATCTCAGCCTTGGTCTTGTCCTGCTTACTCACCTTCCTGGTCC TGATGCGCTCACTGGTGACACACAGGACCAACCTTCGAGCTCTGCACCGAGGAGCTGCCCTGGACTTGAGT CCCTTGCATCGGAGTCCCCATCCCTCCCGCCAAGCCATATTCTGTTGGATGAGCTTCAGTGCCTACCAGAC AGCCTTTATCTGCCTTGGGCTCCTGGTGCAGCAGATCATCTTCTTCCTGGGAACCACGGCCCTGGCCTTCC TGGTGCTCATGCCTGTGCTCCATGGCAGGAACCTCCTGCTCTTCCGTTCCCTGGAGTCCTCGTGGCCCTTC TGGCTGACTTTGGCCCTGGCTGTGATCCTGCAGAACATGGCAGCCCATTGGGTCTTCCTGGAGACTCATGA TGGACACCCACAGCTGACCAACCGGCGAGTGCTCTATGCAGCCACCTTTCTTCTCTTCCCCCTCAATGTGC TGGTGGGTGCCATGGTGGCCACCTGGCGAGTGCTCCTCTCTGCCCTCTACAACGCCATCCACCTTGGCCAG ATGGACCTCAGCCTGCTGCCACCGAGAGCCGCCACTCTCGACCCCGGCTACTACACGTACCGAAACTTCTT GAAGATTGAAGTCAGCCAGTCGCATCCAGCCATGACAGCCTTCTGCTCCCTGCTCCTGCAAGCGCAGAGCC TCCTACCCAGGACCATGGCAGCCCCCCAGGACAGCCTCAGACCAGGGGAGGAAGACGAAGGGATGCAGCTG CTACAGACAAAGGACTCCATGGCCAAGGGAGCTAGGCCCGGGGCCAGCCGCGGCAGGGCTCGCTGGGGTCT GGCCTACACGCTGCTGCACAACCCAACCCTGCAGGTCTTCCGCAAGACGGCCCTGTTGGGTGCCAATGGTG CCCAGCCCTGAGGGCAGGGAAGGTCAACCCACCTGCCGATCTGTGCTGAGGCATGTTCCTGCCTACCATCC TCCTCCCTCCCCGGCTCTCCTCCCAGCATCACACCAGCCATGCAGCCAGCAGGTCCTCCGGATCACTGTGG TTGGGTGGAGGTCTGTCTGCACTGGGAGCCTCAGGAGGGCTCTGCTCCACCCACTTGGCTATGGGAGAGCC AGCAGGGGTTCTGGAGAAAAAAAAGTGGTGGGTTAGGGCCTTGGTCCAGGAGCCAGTTGAGCCAGGGCAGC CACATCCAGGCGTCTCCCTACCCTGGCTCTGCCATCAGCCTTGAAGGGCCTCGATGAAGCCTTCTCTGGAA CCACTCCAGCCCAGCTCCACCTCAGCCTTGGCCTTCACGCTGTGGAAGCAGCCAAGGCACTTCCTCACCCC CTCAGCGCCACGGACCTCTCTGGGGAGTGGCCGGAAΆGCTCCCGGGCCTCTGGCCTGCAGGGCAGCCCAAG TCATGACTCAGACCAGGTCCCACACTGAGCTGCCCACACTCGAGAGCCAGATATTTTTGTAGTTTTTATGG CTTTGGCTATTATGAAAGAGGTTAGTGTGTTCCCTG The disclosed NOV2b nucleic acid sequence has 2395 of 2444 bases (97%) identical to a gb:GENBANK-ID:AF352728|acc:AF352728.1 mRNA from Homo sapiens (Homo sapiens STRA6 isoform 1 mRNA, complete eds, alternatively spliced) (E = 0.0). A disclosed NOV2b polypeptide (SEQ ID NO:6) encoded by SEQ ID NO:5 has 689 amino acid residues and is presented using the one-letter code in Table 2E. Signal P, Psort and/or Hydropathy results predict that NOV2b has a signal peptide and is likely to be localized at the plasma membrane with a certainty of 0.6000. In other embodiments, NOV2b could be localized at the Golgi body with a certainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or a microbody (peroxisome) with a certainty of 0.3000. The most likely cleavage site for a NOV2b peptide is between amino acids 8 and 9, at AGN-QT.
Table 2E. Encoded NOV2b protein sequence (SEQ ID NO:20). SSQPAGNQTSPGATEDYSYGSWYIDEPQGGEELQPEGEVPSCHTSIPPGLYHACLASLSILVLLLLAMLVR RRQL PDCVRGRPGLPSWEVMGVQGLSNSLLPCPCLCASPVDFLAGDGPRAVPAAVFMVLLSSLCLLLPDED ALPFLTLASAPSQDGKTEAPRGAWKILGLFYYAALYYPLAACATAGHTAAHLLGSTLSWAHLGVQV QRAEC PQVPKIYKYYSLLASLPLLLGLGFLSLWYPVQLVRSFSRRTGAGSKGLQSSYSEEYLRNLLCRKKLGSSYHT SKHGFLSWARVCLRHCIYTPQPGFHLPLKLVLSATLTGTAIYQVALLLLVGWPTIQKVRAGVTTDVSYLLA GFGIVLSEDKQEWELVKHHL ALEVCYISALVLSCLLTFLVLMRSLVTHRTNLRALHRGAALDLSPLHRSP HPSRQAIFCWMSFSAYQTAFICLGLLVQQIIFFLGTTALAFLVLMPVLHGRNLLLFRSLESS PF LTLALA VILQNMAAH VFLETHDGHPQLTNRRVLYAATFLLFPLNVLVGAMVAT RVLLSALYNAIHLGQMDLSLLPP RAATLDPGYYTYRNFLKIEVSQSHPAMTAFCSLLLQAQSLLPRTMAAPQDSLRPGEEDEGMQLLQTKDSMAK GARPGASRGRAR GLAYTLLHNPTLQVFRKTALLGANGAQP
The disclosed NOV2b amino acid sequence has 578 of 579 amino acid residues (99%) identical to, and 578 of 579 amino acid residues (99%) similar to, the 667 amino acid residue ptnr:SPTREMBL-ACC:Q9BX79 protein from Homo sapiens (Human) (STRA6 ISOFORM 1) (E = 0.0).
NOV2b maps to chromosome 15. This assignment was made using mapping information associated with genomic clones, public genes and ESTs sharing sequence identity with the disclosed sequence and CuraGen Corporation's Electronic Northern bioinformatic tool.
NOV2b is expressed in at least the following tissues: Brain, Cervix, Heart, Kidney, Lymph node, Lymphoid tissue, Ovary, Pituitary Gland, Placenta, Retina, Temporal Lobe, Thyroid, Uterus. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOV2b. NOV2a also homology to the amino acid sequences shown in the BLASTP data listed in Table 2F.
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 2G.
Table 2G. ClustalW Analysis of NOV2
1) NOV2a (SEQ ID NO: 4)
2) NOV2b (SEQ ID NO: 6)
2) Q9BX79 homo sapiens (human) stra6 isoform 1 (SEQ ID NO: 43)
3) Q9BX78 homo sapiens (human) stra6 isoform 2 (SEQ ID NO: 44)
4) Q9H9U8 homo sapiens (human) cdna flj 12541 fis, clone nt2rm4000433 , (SEQ ID NO: 45)
5) 070491 mus musculus (mouse) retinoic acid-responsive protein (SEQ ID NO: 46)
Figure imgf000023_0001
Figure imgf000024_0001
310 320 330 340 350 360
NOV2a ΛARVCLRHCIYTPQPGFHLPLKLVLSATLTGTAIYQVALLLLVG PTIQKVRAGVTTD\ 322
NOV2b WARVCLRHCIYTPQPGFHLPLKLVLSATLTGTAIYQVALLLLVGWPTIQKVRAGVTTD 355
Q9BX79 WARVCLRHCIYTPQPGFHLPLKLVLSATLTGTAIYQVALLLLVGWPTIQKVRAGVTTDV 333
Q9BX78 WARVCLRHCIYTPQPGFHLPLKLVLSATLTGTAIYQVALLLLVGWPTIQ VRAGVTTDV 324
Q9H9U8 WARVCLRHCIYTPQPGFHLPLKLVLSATLTGTAIYQVALLLLVGWPTIQKVRAGVTTDV 226
070491 RJ*SWAFSBj*3s.iailiaa3BH|Rp3lll'^ 334
370 380 390 400 410 420
NOV2a ■»Ain.tAunan«r«! !i*iAΛ».-tπi-r>->Aa?*i««ιuιifraiιM-uϋτ-Atι«L'ii4-4Atιι-ι÷ιriιuinrj 381
NOV2b LLAGFGIVLSEDKQEWELVKHHL ALEVCYISALVLSCLLTFLVLMRSLVTHRTNL 415
Q9BX79 YLLAGFGIVLSEDKQEWELVKHHL ALEVCYISALVLSCLLTFLVL RSLVTHRTNL 393
Q9BX78 YLLAGFGIVLSEDKQEWELVKHHL ALEVCYISALVLSCLLTFLVLMRSLVTHRTNL 384
Q9H9U8 YLLAGFGIVLSEDKQEWELVKHHL ALEVCY'ISALVLSCLLTFLVLMRSLVTHRTNL 286
O70491 BRMatfAl aHtfiai Bnai|Asrogw|Liiiiig3i 394
Figure imgf000024_0002
490 500 510 520 530 540
NOV2a LAFLVLMPVLHGRNLLLFRSLESSWPFWLTLALAVILQNMAAHWVFLETHDGHPQLTNRR
NOV2b LAFLVLMPVLHGRNLLLFRSLESS PFWLTLALAVILQNMAAH VFLETHDGHPQLTNRR
Q9BX79 LAFLVLMPVLHGRNLLLFRSLESSWPFWLTLALAVILQNMAAHWVFLETHDGHPQLTNRR
Q9BX78 LAFLVLMPVLHGRNLLLFRSLESS PF LTLALAVILQN AAHWVFLETHDGHPQLTNRR
Q9H9U8 LAFLVLMPVLHGRNLLLFRSLESS PF LTLALAVILQNMAAHVFLETHDGHPQLTNRR
O70491 LAFLV
Figure imgf000025_0001
610 620 630 640 650 660
I I ..I.. I I ..|.. ..|..
NOV2a FLKIEVSQSHPAMTAFCSLLLQAQSLLPRTMAAPQDSLRPGEEDEGMQLLQTKDSMAKGA 621
NOV2b FLKIEVSQSHPAMTAFCSLLLQAQSLLPRTMAAPQDSLRPGEEDEGMQLLQTKDSMAKGA 650
Q9BX79 FLKIEVSQSHPAMTAFCSLLLQAQSLLPRTMAAPQDSLRPGEEDEGMQLLQTKDSMAKGA 628
Q9BX78 FLKIEVSQSHPAMTAFCSLLLQAQSLLPRTMAAPQDSLRPGEEDEGMQLLQT DSMAKGA 619
Q9H9U8 FLKIEVSQSHPAMTAFCSLLLQAQSLLPRTMAAPQDSLRPGEEDEGMQLLQTKDSMAKGA 521
070491 ■jG^ι***Sgaj5^gp@PQJ^ppL|a^^5agAj**gi 629
670 680 690 700
NOV2a UU-n.AAitt.i! 660
NOV2b RPGASRGRAR GLAYTLLHNPTLQVFRKTALLG 689
Q9BX79 RPGASRGRAR GLAYTLLHNPTLQVFRKTALLG 667
Q9BX78 RPGASRGRARWGLAYTLLHNPTLQVFRKTALLGBANGAQP 658
Q9H9U8 560
O70491 670
Carcinogenesis involves inactivation or subversion ofthe normal controls of proliferation, differentiation, and apoptosis (Hurst RE, et.al.; Adv Exp Med Biol
1999;462:449-67). However, these controls are robust, redundant, and interlinked at the gene expression levels, regulation of mRNA lifetimes, transcription, and recycling of proteins. One ofthe central systems of control of proliferation, differentiation and apoptosis is retinoid signaling. The hRAR alpha nuclear receptor occupies a central position with respect to induction of gene transcription in that when bound to appropriate retinoid ligands, its homodimers and heterodimers with hRXR alpha regulate the transcription of a number of retinoid-responsive genes. These include genes in other signaling pathways, so that the whole forms a complex network. In this study the authors showed that simple, cause-effect interpretations in terms of hRAR alpha gene transcription being the central regulatory event would not describe the retinoid-responsive gene network. A set of cultured bladder-derived cells representing different stages of bladder rumorigenesis formed a model system. It consisted of 2 immortalized bladder cell lines (HUC-BC and HUC-PC), one squamous cell carcinoma cell line (SCaBER), one papilloma line (RT4), and 4 transitional cell carcinomas (TCC-Sup, 5637, T24, J82) of varying stages and grades. This set of cells were used to model the range of behaviors of bladder cancers. Relative gene expression before (constitutive) and after treatment with 10 microM all-trans-retinoic acid (aTRA) was measured for androgen and estrogen receptor; a set of genes involved with retinoid metabolism and action, liRAR alpha and beta, hRXR alpha and beta CRBP, CRABP I and II; and for signaling genes that are known to be sensitive to retinoic acid, EGFR, cytokine MK, ICAM I and transglutaminase. The phenotype for inhibition of proliferation and for apoptotic response to both aTRA and the synthetic retinoid 4-HPR was determined. Transfection with a CAT-containing plasmid containing an aTRA-sensitive promoter was used to determine if the common retinoic acid responsive element (RARE)-dependent pathway for retinoid regulation of gene expression was active. Each ofthe genes selected is known from previous studies to react to aTRA in a certain way, either by up- or down-regulation ofthe message and protein. A complex data set not readily interpretable by simple cause and effect was observed. While all cell lines expressed high levels ofthe mRNAs for hRXR alpha and beta that were not altered by treatment with exogenous aTRA, constitutive and stimulated responses ofthe other genes varied widely among the cell lines. For example, CRABP I was not expressed by J82, T24, 5637 and RT4, but was expressed at low levels that did not change in SCaBER and at moderate levels that decreased, increased, or decreased sharply in HUC-BC, TCC-Sup and HUC-PC, respectively. The expression of hRAR alpha, which governs the expression of many retinoid-sensitive genes, was expressed at moderate to high levels in all cell lines, but in some it was sharply upregulated (TCC-Sup, HUC-PC and J82), remained constant (5637 and HUC-BC), or was down-regulated (SCaBER, T24 and RT4). The phenotypes for inhibition of proliferation showed no obvious relationship to the expression of any single gene, but cell lines that were inhibited by aTRA (HUC-BC and TCC-Sup) were not sensitive to 4-HPR, and vice versa. One line (RT4) was insensitive to either retinoid. Transfection showed very little retinoid- stimulated transfection ofthe CAT reporter gene with RT4 or HUC-PC. About 2-fold enhancement transactivation was observed with SCaBER, HUC-BC, J82 and T24 cells and 3- 8 fold with 5637, TCC-Sup cells. In HUC-BC, a G to T point mutation was found at position 606 ofthe hRAR alpha gene. This mutation would substitute tyrosine for asparagine in a highly conserved domain. These data indicate that retinoid signaling is probably a frequent target of inactivation in bladder carcinogenesis.
Waliszewski P, et.al. ( Mol Cell Endocrinol 1999 Feb 25;148(l-2):55-65) investigated the presence and functionality of retinoid signaling pathways in human urinary bladder carcinoma and SV40-immortalized uroepithelial cell lines. Only two of eight cell lines were proliferation-inhibited by 10 microM of either all-trans or 13-cis-retinoic acid. Transactivation ofthe CAT gene under control of a retinoid-responsive element demonstrated functionality of the signaling pathway in both sensitive cell lines and four of six resistant cell lines. Relative RT-PCR analysis of a panel of retinoid-responsive and inducible genes demonstrated changes in expression levels of all the genes in response to retinoic acid treatment together with numerous aberrations dysregulations. They concluded that retinoid signaling may be a target for inactivation during tumorigenesis by uncoupling gene expression, proliferation and differentiation. Therefore retinoids are more likely to be effective for chemoprevention than for treatment of bladder carcinomas. The proliferative effects of retinoids were examined in the MC-26 and LoVo colon adenocarcinoma cell lines (Stewart LV, et.al.; Exp Cell Res 1997 Jun 15;233(2):321-9). The proliferation ofthe LoVo cell line was not altered in the presence ofthe retinoids all trans- retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA). Both retinoids, however, stimulated the growth, as measured by cell proliferation, of MC-26 cells. atRA and 9-cis-RA were equipotent in increasing MC-26 cell proliferation, suggesting that the growth stimulation is mediated by one or more retinoic acid receptors (RARs). To determine the RAR which might be responsible for this growth stimulatory effect, the authors characterized the RAR subtypes which were present in both cell lines. mRNA for the RAR alpha, RAR beta, and RAR gamma were detected in the MC-26 cell. Ofthe RARs present in MC-26 cells, the RAR alpha does not mediate the growth stimulatory effects of retinoids, for a selective RAR alpha antagonist was unable to prevent the retinoid-induced increase in MC-26 cell growth. RAR alpha, RAR beta, and RAR gamma mRNA are also expressed in the LoVo cell line; the lack of growth- stimulation by retinoids in LoVo cells, therefore, does not seem to be due to the absence of RARs. The results obtained in these experiments demonstrate that the growth response elicited by retinoids can vary between colon cancer cells and that the differences in response may not be solely determined by the RAR subtypes which are expressed in a colon cancer cell line. Retinoic acids (RAs), well characterized regulators of proliferation and differentiation, partly re-differentiate follicular thyroid carcinoma cell lines (FTC-133, FTC-238, andHTC- TSHr) as well as SV40-transfected immortalized thyroid cell lines (ori3 and 7751) (Schmutzler C, et.al.;Exp Clin Endocrinol Diabetes 1996;104 Suppl 4:16-9). This is indicated by the stimulation of type 15'-deiodinase and other differentiation markers. As demonstrated by RT-PCR, electrophoretic mobility shift, and [3H] -retinoic acid binding assays, thyroid carcinoma cell lines express RA receptor rnRNAs and functional ligand- and DNA-binding receptor proteins able to mediate RA-dependent signal transduction. Together, these properties make these thyroid-derived cell lines useful in vitro models for studying the effects of an RA re-differentiation therapy of thyroid cancer.
The chemotherapeutic agent retinoic acid (RA) inhibits the proliferation and invasion of many tumor types(Vo HP, et.al; Anticancer Res 1998 Jan-Feb;18(lA):217-24). RA chemotherapy in head and neck squamous cell carcinoma (SCC) patients reduces recurrence and induces regression of premalignant lesions. The effects of RA are mediated by both cytoplasmic and nuclear proteins. In the nucleus, a family of ligand-dependent transcription factors, the retinoic acid receptors (RAR) and the retinoid X receptors (RXR), regulate target gene response to RA. In the cytoplasm, the cellular retinoic acid binding proteins I and II (CRABP) regulate intracellular RA concentration, transport, and metabolism. Alterations in CRABP expression have been shown to affect target gene response and the phenotype of cancer cells. To elucidate the role of these proteins in mediating the RA response, we examined target gene expression and malignant phenotype in SCC25 cells expressing an antisense CRABP II construct. RA induced CRABP II mRNA levels 2 fold in SCC25 cells by transcriptional upregulation. Expression ofthe antisense construct reduced CRABP II expression to undetectable levels. Inhibition of CRABP II expression resulted in significant downregulation of RA responsive genes. These reductions were the result of decreased transcription from RA responsive promoters. Surprisingly, clones expressing the antisense CRABP construct were less sensitive to RA mediated inhibition of proliferation. These clones were also less invasive in an in vitro invasion assay, likely due to downregulation of matrix metalloproteinase activity. We conclude that CRABP II affects the transcription of RA responsive genes which regulate proliferation and invasion of head and neck SCCs.
All-trans-retinoic acid (trans-RA) and other retinoids exert anticancer effects through two types of retinoid receptors, the RA receptors (RARs) and retinoid X receptors (RXRs)(Wu Q, etal.; Mol Cell Biol 1997 Nov;17(ll):6598-608). Previous studies demonstrated that the growth-inhibitory effects of trans-RA and related retinoids are impaired in certain estrogen- independent breast cancer cell lines due to their lower levels of RAR alpha and RARbeta. In this study, we evaluated several synthetic retinoids for their ability to induce growth inhibition and apoptosis in both frans-RA-sensitive and trans-RA-resistant breast cancer cell lines. Our results demonstrate that RXR-selective retinoids, particularly in combination with RAR- selective retinoids, could significantly induce RARbeta and inhibit the growth and induce the apoptosis of trans-RA-resistant, RAR alpha-deficient MDA-MB-231 cells but had low activity against trans-RA-sensitive ZR-75-1 cells that express high levels of RAR alpha. Using gel retardation and transient transfection assays, we found that the effects of RXR-selective retinoids on MDA-MB-231 cells were most likely mediated by RXR-nur77 heterodimers that bound to the RA response element in the RARbeta promoter and activated the RARbeta promoter in response to RXR-selective retinoids. In contrast, growth inhibition by RAR- selective retinoids in trans-RA-sensitive, RAR alpha-expressing cells most probably occurred through RXR-RAR alpha heterodimers that also bound to and activated the RARbeta promoter. In MDA-MB-231 clones stably expressing RAR alpha, both RARbeta induction and growth inhibition by RXR-selective retinoids were suppressed, while the effects of RAR- selective retinoids were enhanced. Together, our results demonstrate that activation of RXR can inhibit the growth of trans-RA-resistant MDA-MB-231 breast cancer cells and suggest that low cellular RAR alpha may regulate the signaling switch from RAR-mediated to RXR- mediated growth inhibition in breast cancer cells.
Retinoic acid plays important roles in development, growth and differentiation by regulating the expression of target genes. A new retinoic acid-inducible gene, Stra6, has been identified in PI 9 embryonal carcinoma cells using a subtractive hybridization cDNA cloning technique by Bouillet and coworkers (Mech Dev 1997;63:173-86). Stra6 codes for a very hydrophobic membrane protein of a new type, which does not display similarities with previously characterized integral membrane proteins. Stra6, which exhibits a specific pattern of expression during development and in the adult, is strongly expressed at the level of blood- organ barriers. Interestingly, in testis Sertoli cells, Stra6 has a spermatogenic cycle-dependent expression which is lost in testes of RAR alpha null mutants where Stra6 is expressed in all tubules. This finding suggests that the Stra6 protein may be a component of an as yet unidentified transport machinery. The NOV2 nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, stroke, tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, cerebral palsy, epilepsy, Lesch-Nyhan syndrome, multiple sclerosis, ataxia-telangiectasia, leukodystrophies, behavioral disorders, addiction, anxiety, pain, neurodegeneration, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, scleroderma, transplantation, autoimmune disease, allergies, immunodeficiencies, renal artery stenosis, interstitial nephritis, glomerulonephritis, polycystic kidney disease, systemic lupus erythematosus, renal tubular acidosis, IgA nephropathy, Lesch-Nyhan syndrome, lymphedema, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, graft versus host disease (GVHD), endometriosis, fertility, endocrine dysfunctions, diabetes, obesity, growth and reproductive disorders, hyperthyroidism, hypothyroidism, and other diseases, disorders and conditions ofthe like. For example, a cDNA encoding the Retinoic acid responsive/STRA-6-like protein (NOV2) may be useful in gene therapy, and the Retinoic acid responsive/STRA-6-like protein (NOV2) may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from Inflamation, Autoimmune disorders, Aging or Cancer. The NOV2A nucleic acid encoding Retinoic acid responsive/STRA-6-like protein, and the Retinoic acid responsive/STRA-6-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
NOV2 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. The disclosed NOV2 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOV2a epitope is from about amino acids 20 to 30. In another embodiment, a NOV2a epitope is from about amino acids 40 to 60. In additional embodiments, NOV2a epitopes are from about amino acids 75 to 80, from about 100 to 110, from about amino acids 180 to 190, from about amino acids 220 to 260, from about amino acids 310 to 330, from about amino acids 350 to 370, from about amino acids 380 to 400, from about amino acids 470 to 510, from about amino acids 550 to 570, and from about amino acids 590 to 650. These novel proteins can be used in assay systems for functional analysis of various human disorders, which are useful in understanding of pathology ofthe disease and development of new drug targets for various disorders.
In another embodiment, a contemplated NOV2b epitope is from about amino acids 5 to 50. In another embodiment, a NOV2b epitope is from about amino acids 80 to 90. In additional embodiments, NOV2b epitopes are from about amino acids 140 to 160, from about 190 to 210, from about amino acids 230 to 290, from about amino acids 360 to 365, from about amino acids 390 to 440, from about amino acids 550 to 590, and from about amino acids 610 to 670. These novel proteins can be used in assay systems for functional analysis of various human disorders, which are useful in understanding of pathology ofthe disease and development of new drug targets for various disorders.
NOV3 NOV3 includes two novel LRR/GPCR-like proteins disclosed below. The disclosed proteins have been named NOV3a and NOV3b. NOV3a
A disclosed NOV3a nucleic acid of 7892 nucleotides (also referred to as 128900861ext) encoding a novel LRR/GPCR -like protein is shown in Table 3A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TAA codon at nucleotides 5728-5730. A putative untranslated region downstream from the termination codon is underlined in Table 3A, and the start and stop codons are in bold letters.
Table 3A. NOV3a Nucleotide Sequence (SEQ ID NO:7)
ATGCGGGGGGCGCCCGCGCGCCTGCTGCTGCCGCTGCTGCCGTGGCTCCTGCTGCTCCTGGCGCCCGAGG CTCGGGGCGCGCCCGGCTGCCCGCTATCCATCCGCAGCTGCAAGTGCTCGGGGGAGCGGCCCAAGGGGCT GAGCGGCGGCGTCCCTGGCCCGGCTCGGCGGAGGGTGGTGTGCAGCGGCGGGGACCTCCCGGAGCCTCCC GAGCCCGGCCTTCTGCCTAACGGCACCGTTACCCTTGGCAACCGTTTCTCCACTGCCCCTTGCCCGCAGT CGGGGCTGGCCCAGTCTTTTCGATTGTGTCCAGAACAATACAGTAACGCCTCTGGTTGGGTGAGTTCAGA GGCTGCCCGCTTCCTGACAGGACACAACTGGTCAGCCTCTGACGCTGGCCGCCCGGCCTGCATTTTCCCC GGCCCGGTCTCCTGGAGACAGGTACCAGCTTACAACGCCGGACAGGATCAGGGTAGCAAATATAGCTTCT ATGGAGGAAAGGTCAAAGATGAGTATCTGCGTAGGTCCCCGATCTGTCGCTCTGCTGCCGCCCTGCACAC TGACACCAGTGCCCGCAGCTGGTCTGAATTTAGGCTCTTGAGCAATAACAAGATCACGGGGCTCCGCAAT GGCTCCTTCCTGGGACTGTCACTGCTGGAGAAGCTTCTCATGAGAGCACATTTTTCTGATGAAGATGAAA AGACGGCCTGTCCTTCTCCAGCACCAAGGCTCTGGAGGCTGCATAGCAAGTGCACCCTGAGCATGGCAGG CAATCAGAAGAACCAGGGCGGCAGCAGCTCACAAGAGGCAGCATCTGGCTTGATGCCGAGTCGGCGGCTC CAGCGGGGAGAGGTTGAGGGCCTCTGCACTTGCCTCTGCACGGACCTGAGGAACAACATCATCAGCACAG TGCAGCCGGGCGCCTTCCTGGGCCTGGGGGAGCTGAAGCGTTTAGATCTCTCCAACAACCGGATTGGCTG TCTCACCTCCGAGACCTTCCAGGGCCTCCCCAGGCTTCTCCGACTAAACATATCTGGAAACATCTTCTCC AGTCTGCAACCTGGGGTCTTTGATGAGCTGCCAGCCCTTAAGGTTGTGGACTTGGGCACCGAGTTCCTGA CCTGTGACTGCCACCTGCGCTGGCTGCTGCCCTGGGCCCAGAATCGCTCCCTGCAGCTGTCGGAACACAC GCTCTGTGCTTACCCCAGTGCCCTGCATGCTCATGCCCTGGGCAGCCTCCAGGAGGCCCAGCTCTGCTGG GAGGGGGCCCTGGAGCTGCACACACACCACCTCATCCCGTCCCTACGCCAAGTGGTGTTCCAGGGGGATC GGCTGCCCTTCCAGTGCTCTGCCAGCTACCTGGGCAACGACACCCGCATCCGCTGGTACCACAACCGAGC CCCTGTGGAGGGTGATGAGCAGGCGGGCATCCTCCTGGCCGAGAGCCTCATCCACGACTGCACCTTCATC ACCAGTGAGCTGACGCTGTCTCACATCGGCGTGTGGGCCTCAGGCGAGTGGGAGTGCACCGTGTCCATGG CCCAAGGCAACGCCAGCAAGAAGGTGGAGATCGTGGTGCTGGAGACCTCTGCCTCCTACTGCCCCGCCGA GCGTGTTGCCAACAACCGCGGGGACTTCAGGTGGCCCCGAACTCTGGCTGGCATCACAGCCTACCAGTCC TGCCTGCAGTATCCCTTCACCTCAGTGCCCCTGGGCGGGGGTGCCCCGGGCACCCGAGCCTCCCGCCGGT GTGACCGTGCCGGCCGCTGGGAGCCAGGGGACTACTCCCACTGTCTCTACACCAACGACATCACCAGGGT GCTGTACACCTTCGTGCTGATGCCCATCAATGCCTCCAATGCGCTGACCCTGGCTCACCAGCTGCGCGTG TACACAGCCGAGGCCGCTAGCTTTTCAGACATGATGGATGTAGTGTATGTGGCTCAGATGATCCAGAAAT TTTTGGGTTATGTCGACCAGATCAAAGAGCTGGTAGAGGTGATGGTGGACATGCCCAGCAACTTGATGCT GGTGGACGAGCACCTGCTGTGGCTGGCCCAGCGCGAGGACAAGGCCTGCAGCCGCATCGTGGGTGCCATA GAGCGCATTGGGGGGGCCGCCCTCAGCCCCCATGCCCAGCACATCTCAGTGAATGCGAGGAACGTGGCAT TGGAGGCCTACCTCATCAAGCCGCACAGCTACGTGGGCCTGACCTGCACAGCCTTCCAGAGGAGGGAGGG AGGGGTGCCGGGCACACGGCCAGGAAGCCCTGGCCAGAACCCCCCACCTGAGCCCGAGCCCCCAGCTGAC CAGCAGCTCCGCTTCCGCTGCACCACCGGGAGGCCCAATGTTTCTCTGTCGTCCTTCCACATCAAGAACA GCGTGGCCCTGGCCTCCATCCAGCTGCCCCCGAGTCTATTCTCATCCCTTCCGGCTGCCCTGGCTCCCCC GGTGCCCCCAGACTGCACCCTGCAACTGCTCGTCTTCCGAAATGGCCGCCTCTTCCACAGCCACAGCAAC CCTCCCGCCCTGGAGCTGCTGGGCCTGGCAAGAGGCGTGGCGTGGCCACCCCCGTCATCTTCGCAGGAA CCAGTGGCTGTGGCGTGGGAAACCTGACAGAGCCAGTGGCCGTTTCGCTGCGGCACTGGGCTGAGGGAGC CGAACCTGTGGCCGCTTGGTGGAGCCAGGAGGGGCCCGGGGAGGCTGGGGGCTGGACCTCGGAGGGCTGC CAGCTCCGCTCCAGCCAGCCCAATGTCAGCGCCCTGCACTGCCAGCACTTGGGCAATGTGGCCGTGCTCA GGAGCTGAGCGCCTTTCCCAGGGAGGTGGGGGGCGCCGGGGGAGGGCTGCACCCCGTGGTATACCCCTG CACGGCCTTGCTGCTGCTCTGCCTCTTCGCCACCATCATCACCTACATCCTCAACCACAGCTCCATCCGT GTGTCCCGGAAAGGCTGGCACATGCTGCTGAACTTGTGCTTCCACATAGCCATGACCTCTGCTGTCTTTG CGGGGGGCATCACACTCACCAACTACCAGATGGTCTGCCAGGCGGTGGGCATCACCCTGCACTACTCCTC CCTATCCACGCTGCTCTGGATGGGCGTGAAGGCGCGAGTGCTCCATAAGGAGCTCACCTGGAGGGCACCC CCTCCGCAAGAAGGGGACCCCGCTCTGCCTACTCCCAGTCCTATGCTCCGGTACATACTTTCAATTCCAG CTTTGCAATTGGGGAGGGACTCCAACGCAGGCGTAGGAAACCTCCCAAGGTTCTATTTGATCGCTGGAGG GATTCCACTCATTATCTGTGGCATCACAGCTGCAGTCAACATCCACAACTACCGGGACCACAGCCCCTAC TGCTGGCTGGTGTGGCGTCCAAGCCTTGGCGCCTTCTACATCCCTGTGGCTTTGATTCTGCTCATCACCT GGATCTATTTCCTGTGCGCCGGGCTACGCTTACGGGGTCCTCTGGCACAGAACCCCAAGGCGGGCAACAG CAGGGCCTCCCTGGAGGCAGGGGAGGAGCTGAGGGGTTCCACCAGGCTCAGGGGCAGCGGCCCCCTCCTG AGTGACTCAGGTTCCCTTCTTGCTACTGGGAGCGCGCGAGTGGGGACGCCCGGGCCCCCGGAGGATGGTG ACAGCCTCTATTCTCCGGGAGTCCAGCTAGGGGCGCTGGTGACCACGCACTTCCTGTACTTGGCCATGTG GGCCTGCGGGGCTCTGGCAGTGTCCCAGCGCTGGCTGCCCCGGGTGGTGTGCAGCTGCTTGTACGGGGTG GCAGCCTCCGCCCTGGGCCTCTTCGTCTTCACTCACCACTGTGCCAGGCGGAGGGACGTGAGAGCCTCGT GGCGCGCCTGCTGCCCCCCTGCCTCTCCCGCGGCCCCCCATGCCCCGCCCCGGGCCCTGCCCGCCGCCGC AGAGGACGGTTCCCCGGTGTTCGGGGAGGGGCCCCCCTCCCTCAAGTCCTCCCCAAGCGGCAGCAGCGGC CATCCGCTGGCTCTGGGCCCCTGCAAGCTCACCAACCTGCAGCTGGCCCAGAGTCAGGTGTGCGAGGCGG GGGCGGCGGCCGGCGGGGAAGGAGAGCCGGAGCCGGCGGGCACCCGGGGAAACCTCGCCCACCGCCACCC CAACAACGTGCACCACGGGCGTCGGGCGCACAAGAGCCGGGCCAAGGGACACCGCGCGGGGGAGGCCTGC GGCAAGAACCGGCTCAAGGCCCTGCGCGGGGGCGCGGCGGGGGCGCTGGAACTGCTGTCCAGCGAGAGCG GTAGTCTGCACAACAGCCCCACCGACAGCTACCTGGGCAGCAGCCGCAACAGCCCGGGCGCCGGCCTGCA GCTGGAAGGCGAGCCCATGCTCACGCCGTCCGAGGGCAGCGACACCAGCGCCGCGCCGCTTTCTGAGGCG GGCCGGGCAGGCCAGCGCCGCAGCGCCAGCCGCGACAGTCTCAAGGGCGGCGGCGCGCTGGAGAAGGAGA GCCATCGCCGCTCGTACCCGCTCAACGCCGCCAGCCTAAACGGCGCCCCCAAGGGGGGCAAGTACGACGA CGTCACCCTGATGGGCGCGGAGGTAGCCAGCGGGGGCTGCATGAAGACCGGACTCTGGAAGAGCGAAACT ACCGTCTAAGGTGGGGCGGGCGACGCGGTAGACGGGTTGGCCACGCGGCTCGTTCCCCCGCTCCTCGGGG CCCTCCAAGGTGTCTCCGTAGTCAGCAGGTTGGAGGCAGAGGAGCCGATGGCTGGAGGAAGCCCACAGGC GGATGTTCCCCACTTGCCTAGAGGGCATCCCTCTGGGGTAGCGACAGACAATCCCAGAAACACGCATAAT ACATTTCCGTCCAGCCCGGGGCAGTCTGACTGTCGGTGCCCTCCCAGGAACGGGGAAGGCCTCCGTGTGT GTGAAAGGGCACAGCACATCCCAGGTGCACCCTCCCCAAGTACTCCCACCCCGCCTACTGTCCATGCGGC CTCACTGGGGGCCATCAGCCTCACCAGCAAAGCAGAGATGAGAGCGTGGGAACTGTGTTCTTTCCTCCCT GCCCTCTACTGATTTCAGCCCAGCCCCTGCCTAGATCCTAGGTCCCTTTTCCTCCCGAGTTTGGCTGGCA CGAGAGCTAGCCCAGCACATGAAGCAGGTGATGTTAAGTCACAAGGTGCTGCTTTTCAGATCCACTATGC AAGAGGGGAGGGTGGGGCCACGTGAAAGGCAGCTCTAGACATCAACCAGTCCTGGGGGAGGGGAGTGGGA ACCGGGCACAACTAGGAACAATGCCACCATTCCCACAGGAGTGGTACTTAAACCAGACAGCAGGGTTCAG AGGTGGCACACCGGGACAAAGCTGAGGCCCTGCACCTCAACAGCTGACTGCCAGGTGCCTGTGGGTGAAC TGAGGGGAGTAGAGGGAGAGGGCAGGTGGAACTGGGGCAGAATCTAGTCATGCCCTAAAGCTAGTCCTGT AAACAATGGTGCCCCAGAAAGCTGCAGGTGGTGTTTGGAGAAGCAGTTACTTTTCAGTTACAAGACCCAT CTCCCTAGTCTCAGCCTTACAACACCACGGGACTAAGGAAGAGCACTTCCTTGCCTCCGTAAGGCCAGAG GAAGAACCATCCCAATCATTTGATCTCCAGCTCCACAGTAGAGAGAAACCTACAAAATGTCAAACCAGCT TCCCGACTGCCAGGAGCTCAAGCCAAGCCCAGAGGCAGTGGCTGGGGTCCCTGCAGGTCATGAGGGGCCT ATGCCTTTACTCCTTTTAAACACCAGCACCCGTCTTTTCCCCAACCTAAAACCAACCACCAGCATTTCAC TACAGGACCAAATGGAAACCGAGGGAACCCTGGGTCTTGGGAAGAACAACAGGAAACCAAGGTCTGACCT AGGGTTCCCTCCCAGTCTTCACATCACTCTGGCCTCATCACCAAGGTGACAGAGGACACAGGGGAGGGGG AAAACCCACACACACTCCTTGGAATGGGTCCTGTTATTTATGCTTGCTGCACAGACATATTAGAAGAAAA AAAAAAAGCTTTGTATTATTCTTCCACATATGCTGGCTGCTGTTTACACACCCTGCCAATGCCTTAGCAC TGGAGAGCTTTTTGCAATATGCTGGGGAAAGGGGAGGGAGGGAATGAAAGTGCCAAAGAAAACATGTTTT TAAGAACTCGGGTTTTATACAATAGAATGTTTTCTAGCAGATGCCTCTTGTTTTAATATATTAAAATTTT GCAAAGCCCTTTGAGCTACTGCCTTAGTCTACCCACTGTCCTTTTGTTATGAGGTAGAGGATCTCATGAC ACCATACACACAAACCCATCATTGCCTGTGAATGCACGTAGGGCCAGAATTCCCCAGTTCCCGCTCCTCT GAGGGTTGATACTGCTGGGAATGCCAACCACTCCACAAGCAGAGGGAAGCCCCCTCAGGCCTGCAGGAGG AGCCGCftGC^GTGTGTCCAATTCAAACCAGCAGCAAAGAGCCTGACATTTTCCCATCCATCTATGAGGAA AGCCATCTCACAGAACATGGACATAGGCAACTTGCTCTCCCACACCAAGGGATGGGAATCTCTCCTACCT ATAGTCATCCCTGCACTCCTGACTTTACTCCAGGACGCAGGGTCCAACTAATGGCAGAGCCCCTCTTGGT TCCTTCAAACAAGAAAAGCAATACCTACGGACTGGTGTACACTTCCATCCTTGGTTATAACAGGAATGTT ATCAAGCTGTCAGAACAGGATGAAGTGCTCCCAGTGGATATCCATCAGGGAGGGTTAGGGACACTCGTGG CAGCCTGTCTAGCAGCCTGGGCTCTCTGAAAGTCCCTAACTTGCTGAGGGGTACGCAAATACTGTTCTAT TTCACTATCAGAAATGTTCTCATCTCCAGTGACAGTGGAGACAGGGGGTACAGGGCAGATCCGCTTCGGG GACTTCAACATGCAGGGTGGCAAGAGAAGGGCAGGACTGGCCGGCCGCTTCCCCTGGGGTAAACCTAAGG AATTATTTCCCACCTCCCCTTCTCCTTGCCCCTGTCCCCACCCCGGTGGCTCCTTCTCTCGGGTCTCCAC TTCTGCTGTCCCATCCCGAAAGGCAGAGCGGACCAGTGACTGGCGGTGCTGGAGAAGGTCACCGATGTGC TTCACCACAGACCGTTTGTCAAGTCTCAGAACTCGTAACCAGGCCAGCTGCTCAGCCATCCGCAGCAGCA CAGCCAGCAGCTCCTGCAGGCGGGAGGACGCCGGGTAGGGCAGGTCCACATTTGCCAATTTACAAAATCG GGCAAGGGAACATGAAAGCCGATCTGCAGGCTGCAGCGACTGCCAAGCCAGGAAAGTGGCAGCAGTGATG ACGGGCAAGGGATGCCTCCCGGTCACCAGCCACGTCTCATTTGCCAGCTCCACCAACTGCATTGTTCGAG ACAGCATCTTCTCTTTGTCTTCCACGTATTTGGCTGGCACAGAAGGTGAAGCTTGGAACAGTTTGAAGCT GAAATAACCAAAATGAGGGTTGGATCTTAATGATATAGGGGCTGCTCTCCCACAGTGAGGAAAGACAGCC CACTCAAGATGGGGAAGCTATTCTGCCCTCAGGAATAGTCAAGCTCACTGGGCAGCAAGTTAATAAAGGT AGTGAGAGAAAACAGGGCGTCTTCCGCTTGTTAGGGGAAGGTGGAGGGATGGAGGAGAGCACGAACATTT ATTGGGCGCCTCCCAATCACCATTATTCTGAGTGCTTTACAACGTTCTCATTTAATCTACGTGCACGTGC ACCATCTTATGTGCATGTATAGTTAAAAAACTTTCCCATAGTCATCCAGCCAGGCAGTAACCAAGCTTCA AATACAAGGCTATTTGACACCAACAGCCTCTACTTTCAACGTTATTTATCAG
The disclosed NOV3a nucleic acid sequence of this invention, localized to the pi 1.2 region of chromosome 8, has 2894 of 2908 bases (99% identity) with KIAA1531 mRNA from Homo sapiens (GENBANK-ID: AB040964) (E = 0.0).
Additional BLASTN results are shown in Table 3B.
Table 3B. BLASTN alignments of NOV3a
Sequences producing High- scoring Segment Pairs :
\
High Prob Reading Score P (N) Frame qb : GENBANK- ID : AB040964 | acc :AB040964 Homo sapiens mRNA for . . . 14396 0 . 0 2 qb : GENBANK- ID : HSM800904 | acc :AL110244 Homo sapiens mRNA; c . . . 13169 0 . 0 2 qb : GENBANK- ID : AP00050l | acc :AP000501 Homo sapiens qenomic . . . 22592 0 . 0 15 gb : GENBAN - ID :AK001914 | acc : AK001914 Homo sapiens cDNA FLJ . . . 6650 9 .4e- 295 1 qb : GENBANK- ID : HSM802368 | acc : AL137610 Homo sapiens mRNA; C . . . 5545 1 . 3e- 244 1
A NOV3a polypeptide (SEQ ID NO:8) encoded by SEQ ID NO:7 is 1542 amino acid residues and is presented using the one-letter code in Table 3C. Signal P, Psort and/or Hydropathy results predict that NOV3a contains a signal peptide and is likely to be localized to the plasma membrane with a certainty of 0.6400. In other embodiments, NOV3a may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3700, or the endoplasmic reticulum (lumen) with a certainty of 0.1000. The most likely cleavage site for a NOV3a peptide is between amino acids 26 and 27, at: ARG-AP.
Table 3C. Encoded NOV3a protein sequence (SEQ ID NO:8).
MRGAPARLLLPLLP LLLLLAPEARGAPGCPLSIRSCKCSGERPKGLSGGVPGPARRRWCSGGDLPEPPE PGLLPNGTVTLGNRFSTAPCPQSGLAQSFRLCPEQYSNASGVSSEAARFLTGHN SASDAGRPACIFPGP VSWRQVPAYNAGQDQGSKYSFYGGKVKDEYLRRSPICRSAAALHTDTSARSWSEFRLLSNNKITGLRNGSF LGLSLLEKLLMRAHFSDEDEKTACPSPAPRL RLHSKCTLS AGNQKNQGGSSSQEAASGLMPSRRLQRGE EGLCTCLCTDLRNNIISTVQPGAFLGLGELKRLDLSNNRIGCLTSETFQGLPRLLRLNISGNIFSSLQPG
VFDELPAL WDLGTEFLTCDCHLR LLP AQNRSLQLSEHTLCAYPSALHAHALGSLQEAQLCCEGALEL HTHHLIPSLRQWFQGDRLPFQCSASYLGNDTRIR YHNRAPVEGDEQAGILLAESLIHDCTFITSELTLS HIGVWASGE ECTVSMAQGNASKKVEIWLETSASYCPAERVANNRGDFRWPRTLAGITAYQSCLQYPFTS
VPLGGGAPGTRASRRCDRAGR EPGDYSHCLYTNDITRVLYTFVLMPINASNALTLAHQLRVYTAEAASFS DMMD WAQMIQKFLGYvDQIKELVEVMVDMPSNLMLVDEHLLWLAQREDKACSRIVGAIERIGGAALSP HAQHISVNARNVALEAYLIKPHSYVGLTCTAFQRREGGVPGTRPGSPGQNPPPEPEPPADQQLRFRCTTGR PNVSLSSFHIKNSVALASIQLPPSLFSSLPAALAPPVPPDCTLQLLVFRNGRLFHSHSNTSRPGAAGPGKR
RGVATPVIFAGTSGCGVGNLTEPVAVSLRHAEGAEPVAAWWSQEGPGEAGGWTSEGCQLRSSQPNVSALH CQHLGNVAVL ELSAFPREVGGAGAGLHPWYPCTALLLLCLFATIITYILNHSSIRVSRKGHMLLNLCF HIAMTSAVFAGGITLTNYQMVCQAVGITLHYSSLSTLLWMGVKARVLHKELTWRAPPPQΞGDPALPTPSPM LRYILSIPALQLGRDSNAGVGNLPRFYLIAGGIPLIICGITAAVNIHNYRDHSPYCWLV RPSLGAFYIPV ALILLIT IYFLCAGLRLRGPLAQNPKAGNSRASLEAGEELRGSTRLRGSGPLLSDSGSLLATGSARVGTP GPPEDGDSLYSPGVQLGALVTTHFLYLAMWACGALAVSQRWLPRWCSCLYGVAASALGLFVFTHHCARRR DVRASWRACCPPASPAAPHAPPRALPAAAEDGSPVFGEGPPSLKSSPSGSSGHPLALGPCKLTNLQLAQSQ VCEAGAAAGGEGEPEPAGTRGNLAHRHPNNVHHGRRAHKSRAGHRAGEACGKNRLKALRGGAAGALELLS SESGSLHNSPTDSYLGSSRNSPGAGLQLEGEPMLTPSEGSDTSAAPLSEAGRAGQRRSASRDSLKGGGALE KESHRRSYPLNAASLNGAPKGGKYDDVTLMGAEVASGGCMKTGLWKSETTV
The disclosedNOV3a amino acid sequence has 638 of647 amino acidresidues (98%) identical to, and 643 of647 amino acid residues (99%) similar to, the KIAAl531 PROTEIN of 1060 amino acid residue LRR/GPCR-like protein from Homo sapiens (BAA96055) (E = 0.0). Additional BLASTP results forNOV3a are shown in Table 3D
Table 3D BLASTP alignments ofNOV3a
Sequences producing High-scoring Segment Pairs: \
High Prob Reading Score P(N) Frame ptnr:TREMBLNEW-ACC:BAA96055 KIAA1531 PROTEIN - Homo sapie. 3381 0.0 2 ptnr:SPTREMBL-ACC:Q9UFY4 HYPOTHETICAL 34.9 KDA PROTEIN - . 1804 7.1e-186 1 ptnr:SPTREMBL-ACC:Q9VYFl CG15744 PROTEIN - Drosophila mel . 273 ,le-60 6 ptnr:SPTRE BL-ACC:035161 SEVEN-PASS TRANSMEMBRANE RECEPTO. 217 ,le-19 3 ptnr:REMTREMBL-ACC:E1260970 SEQUENCE 1 FROM PATENT O9707. 213 ,9e-17 3 ptnr: SPTREMBL-ACC :075092 MEGF2 - Homo sapiens (Human), 13. 219 .9e-17 4 ptnr:REMTREMBL-ACC:E1260972 SEQUENCE 3 FROM PATENT W09707. 213 3e-16 3 ptnr:SPTREMBL-ACC:095722 DJ1163J1.1 (ORTHOLOG OF MOUSE TR. 203 .3e-16 4 ptnr:TREMBLNE -ACC:AAF61930 PROTOCADHERIN FLAMINGO 2 - Ho. 203 .2e-15 4 ptnr : SPTREMBL-ACC : 094898 KIAA0806 PROTEIN - Homo sapiens . . . 240 1.4e-15 2 ptnr : SPTREMBL-ACC : P70193 MEMBRANE GLYCOPROTEIN - Mus muse . . . 234 1.4e-15 3 ptnr : SPTREMBL-ACC :088278 MEGF2 - Rattus norvegicus (Rat) , . . . 190 1.6e-15 6 ptnr : TREMBLNE -ACC :AAF61929 PROTOCADHERIN FLAMINGO 1 - Ho . . . 214 8 .2e-15 4
NOV3a also has high homology to members ofthe patp database shown in Table 3E.
Table 3E PATP BLASTX results for NOV3a
Gene Index/ Protein/ Length Identity Positives Expect Identifier Organism (aa) (%) (%) patp :AAB71863 Human hl5571 1338 779/811 785/ 811 0 . 0 GPCR - Homo ( 96%) ( 96%) sapiens patp :AAB71863 Human hl5571 1338 453/453 453/453 0 . 0 GPCR - Homo ( 100%) (100%) sapiens
NOV3a is expressed in at least the following tissues: Blood, Brain, Breast, Colon, Ear, Germ Cell, Heart, Kidney, Lung, Muscle, Pancreas, Placenta, Prostate, Skin, Stomach, Testis, Uterus, Whole embryo, colon, lung, ovary, uterus Eye, Prostate, Tonsil, placenta, and thymus..
In addition, the sequence is predicted to be expressed in the following tissues based on the expression pattern of its homolog, SeqCalling assembly 128900861: fetal heart, fetal lung fetal retina fetal_liver_spleen, total fetal tissue and multiple tumor from colon, lung, geerm cells, kidney prostate.
Sage analysis ofthe 2 Unigene clusters (Example 4) indicates expression in several brain, ovarian and prostate tumors.
NOV3b The cDNA coding for the a domain of NOV3a from residue 444 to 937 was targeted for "in-frame" cloning by PCR. The PCR template is based on human cDNA(s).
The following oligonucleotide primers were used to clone the target cDNA sequence:
F2 5'-GGATCC CGGCTGCCCTTCCAGTGCTCTGCCAGCTACCTGG-3 ' (SEQ ID NO: 135) Rl 5'-CTCGAG GCTCAGCTCCATGAGCACGGCCACATTGCC-3 ' (SEQ ID NO: 136)
For downstream cloning purposes, the forward primer includes an in-frame BamHI restriction site and the reverse primer contains an in-frame Xhol restriction site.
Two parallel PCR reactions were set up using a total of 0.5-1.0 ng human pooled cDNAs as template for each reaction. The pool is composed of 5 micrograms of each ofthe following human tissue cDNAs: adrenal gland, whole brain, amygdala, cerebellum, thalamus, bone marrow, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, liver, lymphoma, Burkitt's Raji cell line, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small Intestine, spleen, stomach, thyroid, trachea, uterus.
When the tissue of expression is known and available, the second PCR was performed using the above primers and 0.5ng-l .0 ng of one of the following human tissue cDNAs: skeleton muscle, testis, mammary gland, adrenal gland, ovary, colon, normal cerebellum, normal adipose, normal skin, bone marrow, brain amygdala, brain hippocampus, brain substantia nigra, brain thalamus, thyroid, fetal lung, fetal liver, fetal brain, kidney, heart, spleen, uterus, pituitary gland, lymph node, salivary gland, small intestine, prostate, placenta, spinal cord, peripheral blood, trachea, stomach, pancreas, hypothalamus.
The reaction mixtures contained 2 microliters of each ofthe primers (original concentration: 5 pmol/ul), 1 microliter of lOmM dNTP (Clontech Laboratories, Palo Alto CA) and 1 microliter of 50xAdvantage-HF 2 polymerase (Clontech Laboratories) in 50 microliter- reaction volume. The following reaction conditions were used: PCR condition 1: a) 96°C 3 minutes b) 96°C 30 seconds denaturation c) 60°C 30 seconds, primer annealing d) 72°C 6 minutes extension Repeat steps b-d 15 times e) 96°C 15 seconds denaturation f) 60°C 30 seconds, primer annealing g) 72°C 6 minutes extension Repeat steps e-g 29 times e) 72°C 10 minutes final extension
PCR condition 2: a) 96°C 3 minutes b) 96°C 15 seconds denaturation c) 76°C 30 seconds, primer annealing, reducing the temperature by 1 °C per cycle d) 72°C 4 minutes extension Repeat steps b-d 34 times e) 72°C 10 minutes final extension
An amplified product was detected by agarose gel electrophoresis. The fragment was gel-purified and ligated into the pCR2.1 vector (Invitrogen, Carlsbad, CA) following the manufacturer's recommendation. Twelve clones per PCR reaction were picked and sequenced. The inserts were sequenced using vector-specific Ml 3 Forward and Ml 3 Reverse primers and the following gene-specific primers:
SF1 : CCTCTGCCTCCTACTGCCCC (SEQ ID NO:93)
SF2: GTGCTGATGCCCATCAATGCCT (SEQ ID NO:94 SF3: CGCATCGTGGGTGCCATAG (SEQ ID NO:95)
SF4: ACCGGGAGGCCCAATGTTTCTCT (SEQ ID NO:96)
SF5: CAGCCACAGCAACACCTCCC (SEQ ID NO:97)
SRI : GTCTCCAGCACCACGATCTCC (SEQ ID NO:98)
SR2: ACGAAGGTGTACAGCACCCTG (SEQ ID NO:99) SR3: GGCTGCAGGCCTTGTCCTC (SEQ ID NO:100)
SR4: GAAGCGGAGCTGCTGGTCA (SEQ ID NO:101)
SR5: TGTTGCTGTGGCTGTGGAAGA (SEQ ID NO:102)
The insert assembly NOV3b was found to encode an open reading frame between residues 444 and 937 ofthe target sequence NOV3a. The cloned insert differs from the original sequence at 8 nucleotide positions and 4 amino acid positions. It also differs by a 55 amino acid deletion between amino acid residues 491 and 546 ofthe original sequence. The alignment with NOV3a is displayed in a ClustalW below. Note that differing amino acids have a white or grey background, and deleted/inserted amino acids can be detected by a dashed line in the sequence that does not code at that position. A disclosed NOV3b nucleic acid of 1329 nucleotides (also referred to as 188822778) encoding a novel LRR/GPCR -like protein is shown in Table 3F. An open reading frame was identified beginning with an GGA initiation codon at nucleotides 1-3 and ending with a GAG codon at nucleotides 1327-1329. A putative untranslated region upstream from the initiation codon is underlined in Table 3F, and the start and stop codons are in bold letters. This reading frame may be a portion of a larger reading frame, since it does not posess traditional start and stop codons. The coding region of this nucleic acid may extend in both the 5' and 3' directions, and the sequence of its expressed protein may extend in the N- or C terminal directions.
Table 3F. NOV3b Nucleotide Sequence (SEQ ID NO:9)
GGATCCCGGCTGCCCTTCCAGTGCTCTGCCAGCTACCTGGGCAACGACACCCGCATCCGCTGGTACCACA ACCGAGCCCCTGTGGAGGGTGATGAGCAGGCGGGCATCCTCCTGGCCGAGAGCCTCATCCACGACTGCAC CTTCATCACCAGGTGGCCCCGAACTCTGGCTGGCATCACAGCCTACCAGTCCTGCCTGCAGTATCCCTTC ACCTCAGTGCGCCTGGGCGGGGGTGCCCCGGGCACCCGAGCCTCCCGCCGGTGTGACCGTGCCGGCCGCT GGGAGCCAGGGGACTACTCCCACTGTCTCTACACCAACGACATCACCAGGGTGCTGTACACCTTCGTGCT GATGCCCATCAATGCCTCCAATGCGCTGACCCTGGCTCACCAGCTGCGCGTGTACACAGCCGAGGCCGCT AGCTTTTCAGACATGATGGATGTGGTCTATGTGGCTCAGATGATCCAGAAATTTTTGGGTTATGTCGACC AGATCAAAGAGCTGGTAGGGGTGATGGTGGACATGGCCAGCAACCTGATGCTGGTGGACGAGCACCTGCT GTGGCTGGCCCAGCGCGAGGACAAGGCCTGCAGCCGCATCGTGGGTGCCCTGGAGCGCATTGGGGGGGCC GCCCTCAGCCCCCATGCCCAGCACATCTCAGTGAATGCGAGGAACGTGGCATTGGAGGCCTACCTCATCA AGCCGCACAGCTACGTGGGCCTGACCTGCACAGCCTTCCAGAGGAGGGAGGGAGGGGTGCCGGGCACACG GCCAGGAAGCCCTGGCCAGAACCCCCCACCTGAGCCCGAGCCCCCAGCTGACCAGCAGCTCCGCTTCCGC TGCACCACCGGGAGGCCCAATGTTTCTCTGTCGTCCTTCCACATCAAGAACAGCGTGGCCCTGGCCTCCA TCCAGCTGCCCCCGAGTCTATTCTCATCCCTTCCGGCTGCCCTGGCTCCCCCGGTGCCCCCAGACTGCAC CCTGCAACTGCTCGTCTTCCGAAATGGCCGCCTCTTCCACAGCCACAGCAACGCCTCCCGCCCTGGAGCT GCTGGGCCTGGCAAGAGGCGTGGCGTGGCCACCCCCGTCATCTTCGCAGGAACCAGTGGCTGTGGCGTGG GAAACCTGACAGAGTCAGTGGCCGTTTCGCTGCGGCACTGGGCTGAGGGAGCCGAACCTGTGGCCGCTTG GTGGAGCCAGGAGGGGCCCGGGGAGGCTGGGGGCTGGACCTCGGAGGGCTGCCAGCTCCGCTCCAGCCAG CCCAATGTCAGCGCCCTGCACTGCCAGCACTTGGGCAATGTGGCCGTGCTCATGGAGCTGAGCCTCGAG
The reverse complement of NOV3b nucleic acid is shown in Table 3G
Table 3G. NOV3b Nucleotide Sequence (SEQ ID NO:10)
CTCGAGGCTCAGCTCCATGAGCACGGCCACATTGCCCAAGTGCTGGCAGTGCAGGGCGCTGACATTGGGC TGGCTGGAGCGGAGCTGGCAGCCCTCCGAGGTCCAGCCCCCAGCCTCCCCGGGCCCCTCCTGGCTCCACC AAGCGGCCACAGGTTCGGCTCCCTCAGCCCAGTGCCGCAGCGAAACGGCCACTGACTCTGTCAGGTTTCC CACGCCACAGCCACTGGTTCCTGCGAAGATGACGGGGGTGGCCACGCCACGCCTCTTGCCAGGCCCAGCA GCTCCAGGGCGGGAGGCGTTGCTGTGGCTGTGGAAGAGGCGGCCATTTCGGAAGACGAGCAGTTGCAGGG TGCAGTCTGGGGGCACCGGGGGAGCCAGGGCAGCGGGAAGGGATGAGAATAGACTCGGGGGCAGCTGGAT GGAGGCCAGGGCCACGCTGTTCTTGATGTGGAAGGACGACAGAGAAACATTGGGGCTCCCGGTGGTGCAG CGGAAGGGGAGCTGCTGGTCAGCTGGGGGCTCGGGCTCAGGTGGGGGGTTCTGGCCAGGGCTTCCTGGCC GTGTGCCCGGCACCCCTCCCTCCCTCCTCTGGAAGGCTGTGCAGGTCAGGCGCACGTAGCTGTGCGGCTT GATGAGGTAGGCCTCCAATGCCACGTTCCTCGCATTCACTGAGATGTGCTGGGCATGGGGGCTGAGGGCG GCCCCCCCAATGCGCTCCAGGGCACCCACGATGCGGCTGCAGGCCTTGTCCTCGCGCTGGGCCAGCCACA GCAGGTGCTCGTCCACCAGCATCAGGTTGCTGGCCATGTCCACCATCACCCCTACCAGCTCTTTGATCTG GTCGACATAACCCAAAAATTTCTGGATCATCTGAGCCACATAGACCACATCCATCATGTCTGAAAAGCTA GCGGCCTCGGCTGTGTACACGCGCAGCTGGTGAGCCAGGGTCAGCGCATTGGAGGCATTGATGGGCATCA GCACGAAGGTGTACAGCACCCTGGTGATGTCGTTGGTGTAGAGAGAGTGGGAGTAGTCCCCTGGCTCCCA GCGGCCGGCACGGTCACACCGGCGGGAGGCTCGGGTGCCCGGGGCACCCCCGCCCAGGGGCACTGAGGTG AAGGGATACTGCAGGCAGGACTGGTAGGCTGTGATGCCAGCCAGAGTTCGGGGCCACCTGGTGATGAAGG TGCAGTCGTGGATGAGGCTCTCGGCCAGGAGGATGCCCGCCTGCTCATCACCCTCCACAGGGGCTCGGTT GTGGTACCAGCGGATGCGGGTGTCGTTGCCCAGGTAGCTGGCAGAGCACTGGAAGGGCAGCCGGGATCC
A NOV3b polypeptide (SEQ ID NO:l 1 encoded by SEQ ID NO:9 is 443 amino acid residues and is presented using the one-letter code in Table 3H.
Table 3H. Encoded NOV3b protein sequence (SEQ ID NO:ll).
GSRLPFQCSASYLGNDTRIR YHNRAPVEGDEQAGILLAESLIHDCTFITR PRTLAGITAYQSCLQYPFT SVPLGGGAPGTRASRRCDRAGR EPGDYSHCLYTNDITRVLYTFVLMPINASNALTLAHQLRVYTAEAASF SDMMDVVYVAQMIQKFLGYVDQIKELVGVMVDMASNLMLVDEHLL LAQREDKACSRIVGALERIGGAALS PHAQHISVNARNVALEAYLIKPHSYVGLTCTAFQRREGGVPGTRPGSPGQNPPPEPEPPADQQLRFRCTTG RPNVSLSSFHIKNSVALASIQLPPSLFSSLPAALAPPVPPDCTLQLLVFRNGRLFHSHSNASRPGAAGPGK RRGVATPVIFAGTSGCGVGNLTESVAVSLRH AEGAEPVAAWWSQEGPGEAGG TSEGCQLRSSQPNVSAL HCQHLGNVAVLMELSLE
NOV3 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 31.
Figure imgf000039_0001
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 3J.
Table 3J. ClustalW Analysis of NOV3
1) Novel NOV3 (SEQ ID NO: 8) 2) Novel NOV4 (SEQ ID Nθ:ll)
2) Q9P1Z7 homo sapiens (human) kiaal531 protein (fragment) (SEQ ID Nθ:47)
3) Q9BYB8 homo sapiens (human) membrane glycoprotein lig-1 (SEQ ID NO: 8)
10 20 30 40 50 60 ....|....|....|....|....|....|....|....|....|....|....|....| NOV3a MRGAPARLLLPLLP LLLLLAPEARGAPGCPLSIRSCKCSGERPKGLSGGVPGPARRRW 60
NOV3b ;- 1
Q9P1Z7 1
Q9BYB8 1
70 80 90 100 110 120 ....|....|....|....|....|....|....|....|....|....|....|....| NOV3a CSGGDLPEPPEPGLLPNGTVTLGNRFSTAPGPQSGLAQSFRLCPEQYSNASG VSSEAAR 120
NOV3b 1
Q9P1Z7 1
Q9BYB8 1
130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....|
NOV3a FLTGHNWSASDAGRPACIFPGPVSWRQVPAYNAGQDQGSKYSFYGGKVKDEYLRRSPICR 180
NOV3b 1
Q9P1Z7 1
Q9BYB8 1 190 200 210 220 230 240
NOV3a SAAALHTDTSARSWSEFRLLSNNKITGLRNGSFLGLSLLEKLLMRAHFSDEDEKTACPSP 240 NOV3b X Q9P1Z7 1 Q9BYB8 X
250 260 270 280 290 300
NOV3a APRL RLHSKCTLSMAGNQKNQGGSSSQEAASGLMPSRRLQRGEVEGLCTCLCTDLRNNI 300 NOV3b 1 Q9P1Z7 I Q9BYB8
310 320 330 340 350 360
NOV3a ISTVQPGAFLGLGELKRLDLSNNRIGCLTSETFQGLPRLLRLNISGNIFSSLQPGVFDEL 360 NOV3b X Q9P1Z7 Q9BYB8 I
370 380 390 400 410 420
NOV3a PALKVVDLGTEFLTCDCHLR LLP AQNRSLQLSEHTLCAYPSALHAHALGSLQEAQLCC 20 NOV3b 1 Q9P1Z7 Q9BYB8 MARPVRGGLGAPRRS- 15
Figure imgf000040_0001
730 740 750 760 770 780 NOV3a fVALSPHAQHISVNARNVALEAYLIKPHSYVGLTCTAFQRREGGVPGTRPGSPGQNPPPEP 765 NOV3b ?^ALSPHAQHISVNARNVALEAYLIKPHSYVGLTCTAFQRREGGVPGTRPGSPGQNPPPEP 269 Q9P1Z7 ΛALSPHAQHISVNARNVALEAYLIKPHSYVGLTCTAFQRREGGVPGTRPGSPGQNPPPEP 336
Q9BYB8 ELyLSFNNLTRLDEEpJ^EL^SJjSVLRLSHNSISHIA GAFKl^RSLj^LDLDHiJiEISG- 369 790 8 80000 810 820 830 840
I ..|.. I I I ..I
NOV3a EPPADQQLRFRCTTGRPNVSLSSFHIKNSVALASIQLPPSLFSSLPAALAPPVPPDCTLQI 325 NOV3b EPPADQQLRFRCTTGRPNVSLSSFHIKNSVALASIQLPPSLFSSLPAALAPPVPPDCTLQI 329 Q9P1Z7 EPPADQQLRFRCTTGRPNVSLSSFHIKNSVALASIQLPPSLFSSLPAALAPPVPPDCTLQI 396 Q9BYB8 TIEjgTSGAgSGLDSLSKLΪjJFGNK^S A-O^FSGJJEGLEHL^jjGGNAIRSgQFigAFjK 428
850 860 8 87700 8 88800 890 900
Figure imgf000041_0001
910 992200 993300 940 950 960 ■ ■■■]■■..|....1....|....|....|. I....].... I NOV3a WAEGAEPVAAWWSQEGPGEAGGWTSEGCQLRSSQPNVSALHCQHLGNVAVLMELSΘ "FIPRE 942 NOV3b WAEGAEPVAAWWSQEGPGEAGGWTSEGCQLRSSQPNVSALHCQHLGI AVLMELSg*"} - 443 Q9P1Z7 WAEGAEPVAAWWSQEGPGEAGGWTSEGCQLRSS'QPNVSALHCQHLGNVAVLMELSgF PRE 513 Q9BYB8 CDpFLKgQIITQP TTMAMVgKDIRFTgS. SS |SS^PMTFAWjK DNEgLTNAD^ENHVHV 548
970 980 990 1000 1010 1020
NOV3a GG-^GfbHP YPCTALfLLCLFATIITYg f*^SSIR §R^^ 1002
NOV3b *- It--- 4 3
Q9P1Z7 VGGA^Gf&P VYPCTALt LCLFATIITYnLraSSIRVBRraG HMjlL LCFi ΑMΦSAV 573
Q9BYB8 HAQDgEV||EYTTILHLRQ TFGHEGRYQCvBτ|SFGSTγ HgAR--3τ---Vg3LPSFTK 603
2
Figure imgf000041_0002
1090 1100 1110 1120 1130 1140
NOV3a SPcLRYILSIPALQLGRDSNAGVGNLPRFYLIAGGIPLIICGITAAVNIHNYRDHSPYCW 1122
NOV3b ■ 443
Q9P1Z7 SPMLR CW 640
Q9BYB8 VKJEDD G VYSCTAQNSAGS 680
1150 1160 1170 1180 1190 1200
Figure imgf000041_0003
1210 1220 1230 1240 1250 1260
NOV3a |RGSGP[gSDSGSpLATGSARVGTPGPPEDG3SL'gPGVQJjGgLVTTHFLYLAMWACG[i|C 1 42 NOV3b Q9P1Z7 RGSGPJT"" " .. . _ _ Q9BYB8 TPDNQΠ
1270 1280 1290 1300 1310 1320
NOV3 AV^QRWLPRaVCSCl^GVAAS LGLF(3FTHHCARRRDVRfi{^RgCCPPA PAAPHAPPRA 1302 NOV3b :' 443 Q9P1Z7 AvSQRWLPR^CSCJ4gGVAAS^LGLF* FTHHCARRRDVRA[pRlCCPPA{ PAAPHAPPRA 820 Q9BYB8 Vs lVLTSL3wVCl(rgQTR-0§EEYs2τNTDETvVPPDVp|γLgSQGTLgDRQETVVRTE 860
1330 1340 1350 1360 1370 1380 ....| . | ....| ....| ....| ....| ....| ....| ....| ....| ....| .... | NOV3a LPAAAEI^SPVFGEGPgSLK(|SPSGSSG[*jjP^LGPC[*g3TNLQLAQSΩVCtegGAAgGgEGE 1362 NOV3b 443
Q9P1Z7 LPAAAE^SPVFGEGpgSL HsPSGSSGgPjfflLGPC'jjgTNLQLAQS^VCpgGAAgGgEGE 880 Q9BYB8 GAGPQASJ iESNGVC@RDAgHFPEPDTgS^CRQP^CAGSAYHKJ3PWKgIEKgEgTPG 920
1390 1400 1410 1420 1430 1440
Figure imgf000042_0001
1450 1460 1470 1480 1490 1500
NOV3a S •G•H***jL*j*j»VSP*rø* 1.!j3Y*L*^*j*@S****^***1 ****1^*1 ****1^**1 ****1 ***^**** 1 1480
NOV3b ---" ' 443
Q9P1Z7 SG L[j^SPTD^YL*fflSRNSP* AGLQLEGfeP**graPS"|GSDT !AAPLSEAG--RAG|RRSAS 998
Q9BYB8 EHgP^QCβJ feAΑ^CPECQgS YPSNiφj j^A ^KKPMA^LDG GDSS TLAiLYHPD 1033
1510 1520 1530 1540 1550 1560
Figure imgf000042_0002
N0V3a §V 1542 N0V3b -- 443
Q9P1Z7 J?V 1060
Q9BYB8 fe- 1094
Tables 3K through 3Q list the domain description from DOMAIN analysis results against NOV3. This indicates that the NOV3a sequence has properties similar to those of other proteins known to contain these domains.
Table 3K. Domain Analysis of NOV3a
gnl I Pfam| pfam00002 , 7tm_2, 7 transmembrane receptor (Secretin family) (SEQ ID NO: 74)
CD-Length = 249 residues, only 56.2% aligned Score = 58.5 bits (140), Expect = 3e-09
Query: 978 SIRVSRKGWHMLLNLCFHIAMTSAVFAGGITLTNYQMVCQAVGITLHYSSLSTLLWMGVK 1037
+1 +1 1+ I I + I + + I + ++ 1+ I + III 1+ 11 1+
Sbjct: 31 KLRCTRNYIHINLFLSLILRALSFLIGDAVLLNSVELGCKWAVFLHYFFLANFFWMLVE 90 Query: 1038 ARVLHKELTWRAPPPQEGDPALPTPSPMLRYILSIPALQLGRDSNAGVGNLPRFYLIAGG 1097 1+ I +
Sbjct: 91 GLYLYTLLVETFFSER- -KRLLWYLLIGWG 118
Query: 1098 IPLIICGITAAVNIHNYRDHSPYCWL-VWRPSLGAFYIPVALILLITWIYFLC 1149
+1 ++ II I I I + 11+ I 1+ I++I+ +1+1+
Sbjct: 119 VPAVWGIWALVRPKGYGNPG-CCWISNEAPFWWIIRGPILLVILVNFIFFIN 170
Table 3L. Domain Analysis of NOV3a
gnl I Pfam| pfam01463 , LRRCT, Leucine rich repeat C-terminal domain. (SEQ
ID NO: 75) = 51 residues, 96.1% aligned
Score = 43.9 bits (102), Expect = 6e-05
Query: 373 LTCDCHLRWLLP A-QNRSLQLSEHTLCAYPSALHAHALGSLQEAQLCCE 421
III Mill I I I I II I +1 I I + I
Sbjct: 3 FICDCELRWLLRWLRAPRRLPDPEDLRCASPESL-RGPLLDLLPSDFSCP 51
Table 3M. Domain Analysis of NO 3a
gnl I Pfam| pf m01463 , LRRCT, Leucine rich repeat C-terminal domain. (SEQ
ID NO: 75)
CD-Length = 51 residues, only 68.6% aligned
Score = 42.7 bits (99), Expect = le-04
Query: 373 LTCDCHLRWLLPWA-QNRSLQLSEHTLCAYPSALH 406
III Nil! I II II II I +1
Sbjct: 3 FICDCELRWLLRWLEANRHLQDPVDLRCASPESLR 37
Table 3N. Domain Analysis of NOV3a gnl I Smart | smart00008, HormR, Domain present in hormone receptors. (SEQ
ID NO: 76
CD-Length = 70 residues, only 67.1% aligned
Score = 39.7 bits (91), Expect = 0.001
Query: 548 WPRTLAGITAYQSCLQYPFTSVPLGGGAPGTRASRRCDRAGRWEPG--DYSHC 598
II I II I I I + III I I I I +11+1
Sbj ct : 15 WPATPAGELVEVPCPDY FSGFSNKEGASRNCTEDGGWSPPFPNYSNC 61
Table 3O. Domain Analysis of NO 3 gnl I Smart | smart00303, GPS, G-protein-coupled receptor proteolytic site domain; Present in latrophilin/CL-1, sea urchin REJ and polycystin
(SEQ ID NO: 77)
CD-Length = 49 residues, 93.9% aligned
Score = 37 . 7 bits ( 86) , Expect = 0 . 005
Query: 888 EPVAAWWSQEGPGEAGGWTSEGCQLRSSQPNVSALHCQHLGNVAVLMELS 937
1 + + 1 ++ I I ++ l l + l + + 1 1 1 + I I I I ++
Sbjct: 2 NPICVFWD ESSGEWSTRGCELLETSSTHTTCSCNHLTSFAVLMDVP 47 Table 3P. Domain Analysis of NO 3a gnl I Pfam| pfam01825 , GPS, Latrophilin/CL-1-like GPS domain. (SEQ ID
NO: 78)
CD-Length = 49 residues, 93.9% aligned
Score = 37.0 bits (84), Expect = 0.008
Query: 889 PVAAWWSQEGPGEAGGWTSEGCQLRSSQPNVSALHCQHLGNVAVLMELSA 93£
1 + + 1 + I I ++ l l + l + + 1 1 1 + I I M ++
Sbjct: 3 PICVFWDESS GVWSTRGGKLLELSKSHTTCSCNHLTSFAVLMDVPP 48
Table 3Q. Domain Analysis of NO 3a gnl I Pfam| pfam02793 , HRM, Hormone receptor domain. (SEQ ID NO: 79 Score = 37.0 bits (84), Expect = 0.008
Query: 533 YCPAERVANNRGDFRWPRTLAGITAYQSCLQYPFTSVPLGGGAPGTRASRRCDRAGRWEP 592
MM I INI M I I I IN I I
Sbjct: 4 YCPAT WDGGVGWPRTPAGTLVEVPCPDYF SGFNYDNASRNCLSNGGWAS 52
Query: 593 G DYSHC 598
||++|
Sbj ct : 53 NWPWPDYTNC 62
Many sttudies have been performed showing the function ofthe sequences that are in varying ways homologous to NOV3. To gain a molecular understanding of tumor angiogenesis, the gene expression patterns of endothelial cells derived from blood vessels of normal and malignant colorectal tissues were compared (St. Croix et al.. Science 2000 August 18; 289:1197-1202). Of over 170 transcripts predominantly expressed in the endothelium, 79 were differentially expressed, including 46 that were specifically elevated in tumor-associated endothelium. Several of these genes encoded extracellular matrix proteins, but most were of unknown function. Most of these tumor endothelial markers were expressed in a wide range of tumor types, as well as in normal vessels associated with wound healing and corpus luteum formation. These studies demonstrate that tumor and normal endothelium are distinct at the molecular level, a finding that may have significant implications for the development of antiangiogenic therapies.
To provide information regarding the coding sequences of unidentified human genes, studies were conducted on a sequencing project of human cDNAs which encode large proteins (Nagase T et al. DNA Res 2000 Apr 28;7(2): 143-50). Therein the researchers present the entire sequences of 100 cDNA clones of unknown human genes, named KIAAl 444 to KIAAl 543, from two sets of size-fractionated human adult and fetal brain cDNA libraries. The average sizes ofthe inserts and corresponding open reading frames of cDNA clones analyzed were 4.4 kb and 2.6 kb (856 amino acid residues), respectively. Database searches of the predicted amino acid sequences classified 53 predicted gene products into the following five functional categories: cell signaling/communication, nucleic acid management, cell structure/motility, protein management and metabolism. It was also revealed that homologues for 32 KIAA gene products were detected in the databases, which were similar in sequence through almost their entire regions. Additionally, the chromosomal loci ofthe genes were determined by using human-rodent hybrid panels unless their chromosomal loci were already assigned in the public databases. The expression levels ofthe genes were monitored in spinal cord, fetal brain and fetal liver, as well as in 10 human tissues and 8 brain regions, by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.
Small leucine-rich proteoglycans belong to an expanding gene class whose distinctive feature is a structural motif, called the trypsin inhibitor, found in an increasing number of intracellular and extracellular proteins with diverse biological attributes (Iozzo RV Crit Rev Biochem Mol Biol 1997;32(2): 141-74). Three-dimensional modeling of their prototype protein core proposes a flexible, arch-shaped binding surface suitable for strong and distinctive interactions with ligand proteins. Changes in the properties of individual proteoglycans derive from amino acid substitutions in the less conserved surface residues, changes in the number and length ofthe trypsin inhibitors, and/or variation in glycosylation. These proteoglycans are tissue organizers, orienting and ordering collagen fibrils during ontogeny and in pathological processes such as wound healing, tissue repair, and tumor stroma formation. These properties are rooted in their bifunctional character: the protein moiety binding collagen fibrils at strategic loci, the microscopic gaps between staggered fibrils, and the highly charged glycosaminoglycans extending out to regulate interfibrillar distances and thereby establishing the exact topology of fibrillar collagens in tissues. These proteoglycans also interact with soluble growth factors, modulate their functional activity, and bind to cell surface receptors. The latter interaction affects cell cycle progression in a variety of cellular systems and could explain the purported changes in the expression of these gene products around the invasive neoplastic cells and in regenerating tissues. Trypsin inhibitors are short sequence motifs present in a number of proteins with diverse functions and cellular locations (Kobe B et al., Trends Biochem Sci 1994 Oct;19(10):415-21). All proteins containing these repeats are thought to be involved in protein-protein interactions. The crystal structure of ribonuclease inhibitor protein has revealed that trypsin inhibitors correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglobular shape. These two features may be responsible for the protein-binding functions of proteins containing trypsin inhibitors.
Latrophilin is a brain-specific Ca2+-independent receptor of alpha-latrotoxin, a potent presynaptic neurotoxin. Matsushita H. et al. report the finding of two novel latrophilin homologues (Matsushita H. et al., FEBS Lett 1999 Jan 29;443(3):348-52). All three latrophilins are unusual G protein-coupled receptors. They exhibit strong similarities within their lectin, olfactomedin and transmembrane domains but possess variable C-termini. Latrophilins have up to seven sites of alternative splicing; some splice variants contain an altered third cytoplasmic loop or a truncated cytoplasmic tail. Only latrophilin-1 binds alpha- latrotoxin; it is abundant in brain and is present in endocrine cells. Latrophilin-3 is also brain- specific, whereas latrophilin-2 is ubiquitous. Together, latrophilins form a novel family of heterogeneous G protein-coupled receptors with distinct tissue distribution and functions. The protein similarity information, expression pattern, and map location for the LRR/GPCR-like protein and nucleic acid (NOV3) disclosed herein suggest that this LRR/GPCR-like protein may have important structural and/or physiological functions characteristic ofthe transient receptor potential-related protein family. Therefore, the NOV3 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
NOV3 nucleic acids and proteins are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from disorder linked to abnormal angiogenesis, like cancer and more specifically aggressive, metastatic cancer, more specifically tumor ofthe lung, kidney, brain, liver and colon. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods, specifically for the treatment of tumors, more specifically tumor ofthe lung, kidney, brain, liver and colon.
NOV3 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. The disclosed NOV3 protein has multiple hydrophilic regions, each of which can be used as an immunogen. The disclosed NOV3 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOV3 epitope is from about amino acids 1 to 300. In another embodiment, a NOV3 epitope is from about amino acids 420 to 450. In additional embodiments, NOV3 epitopes are from about amino acids 650 to 800, from about 700 to 800, from about amino acids 820 to 900, from about amino acids 1050 to 1150, from about amino acids 1200 to 1250, and from about amino acids 1300 to 1550. These NOV3 proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
NOV4 A disclosed NOV4 nucleic acid of 1123 nucleotides (also referred to as
AL03171 l_dal) encoding a novel trypsin inhibitor -like protein is shown in Table 4A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TGA codon at nucleotides 1117-1119. A putative untranslated region downstream from the termination codon is underlined in Table 4A, and the start and stop codons are in bold letters. Table 4A. NOV4 Nucleotide Sequence (SEQ ID NO:12)
ATGAGGAGCCTCGGGGCCCTGCTCTTGCTGCTGAGCGCCTGCCTGGCGGTGAGCGCTGGCCCTGTGCCAA CGCCGCCCGACAACATCCAAGTGCAGGAAAACTTCAATATCTCTCGGATCTATGGGAAGTGGTACAACCT GGCCATCGGTTCCACCTGCCCCTGGCTGAAGAAGATCATGGACAGGATGACAGTGAGCACGCTGGTGCTG GGAGAGGGCGCTACAGAGGCGGAGATCAGCATGACCAGCACTCGTTGGCGGAAAGGTGTCTGTGAGGAGA CGTCTGGAGCTTATGAGAAAACAGATACTGATGGGAAGTTTCTCTATCACAAATCCAAATGGAACATAAC CATGGAGTCCTATGTGGTCCACACCAACTATGATGAGTATGCCATTTTCCTGACCAAGAAATTCAGCCGC CATCATGGACCCACCATTACTGCCAAGCTCTACGGGCGGGCGCCGCAGCTGAGGGAAACTCTCCTGCAGG ACTTCAGAGTGGTTGCCCAGGGTGTGGGCATCCCTGAGGACTCCATCTTCACCATGGCTGACCGAGGTGA ATGTGTCCCTGGGGAGCAGGAACCAGAGCCCATCTTAATCCCGAGAGTCCGGAGGGCTGTGCTACCCCAA GAAGAGGAAGGATCAGGGGGTGGGCAACTGGTAACTGAAGTCACCAAGAAAGAAGATTCCTGCCAGCTGG GCTACTCGGCCGGTCCCTGCATGGGAATGACCAGCAGGTATTTCTATAATGGTACATCCATGGCCTGTGA GACTTTCCAGTACGGCGGCTGCATGGGCAACGGTAACAACTTCGTCACAGAAAAGGAGTGTCTGCAGACC TGCCGAACTGTGGCGGCCTGCAATCTCCCCATAGTCCGGGGCCCCTGCCGAGCCTTCATCCAGCTCTGGG CATTTGATGCTGTCAAGGGGAAGTGCGTCCTCTTCCCCTACGGGGGCTGCCAGGGCAACGGGAACAAGTT CTACTCAGAGAAGGAGTGCAGAGAGTACTGCGGTGTCCCTGGTGATGGTAGGCAGCCCTGCAGGGTGCCA GGCTGGGCCGAGGGGCTGTTGGAGTCGAGGTGGGAGAGCCCTGGGAGGAGTAGGGGTCTGGAGAGCTGAG CGT
The disclosed NOV4 nucleic acid sequence has 1063 of its 1123 bases (95%) identical to an alpha- 1 -micro globulin mRNA from Homo sapiens (GENBANK-ID: HSHCR|acc:X04225) (E = 3.3e-227).
A NOV4 polypeptide (SEQ ID NO: 13) encoded by SEQ ID NO: 12 is 372 amino acid residues and is presented using the one-letter amino acid code in Table 4B. Signal P, Psort and/or Hydropathy results predict that NOV4 has a signal peptide and is likely to be localized extracellularly with a certainty of 0.8200. In other embodiments, NOV4 may also be localized to the lysosome (lumen) with acertainty of 0.1900, the microbody (peroxisome) with a certainty of 0.1456, or the endoplasmic reticulum (membrane) with a certainty of 0.1000. The most likely cleavage site for a NOV4 peptide is between amino acids 19 and 20, at: VSA-GP.
Table 4B. NOV4 protem sequence (SEQ ID NO:13)
MRSLGALLLLLSACLAVSAGPVPTPPDNIQVQENFNISRIYGKWrøLAIGSTCPWLKKIMDR TVST LVLGEGATEAEISMTSTR RKGVCEETSGAYEKTDTDGKFLYHKSKWNITMESYVVHTNYDEYAIFL TKKFSRHHGPTITAKLYGRAPQLRETLLQDFRWAQGVGIPEDSIFTMADRGECVPGEQEPEPILIP RVRRAVLPQEEEGSGGGQLVTEVTKKEDSCQLGYSAGPC GMTSRYFYNGTSMACETFQYGGCMGNG NNFVTEKECLQTCRTVAACNLPIVRGPCRAFIQL AFDAVKGKCVLFPYGGCQGNGNKFYSEKECRE YCGVPGDGRQPCRVPGWAEGLLESRWESPGRSRGLES
The disclosed NOV4 amino acid sequence has |acc:X04225) (Fig. 3A). The full amino acid sequence of the protein of the invention was found to have 343 of its 372 amino acid residues (92%) identical to and positive with, the 352 amino acid residue AMBP PROTEIN PRECURSOR protein from Homo sapiens (SWISSPROT-ACC:P02760 ) (E = 1.3e 191). The disclosed NOV4 protein maps to chromosome 16 and is expressed in at least the following tissues: HEPG2 untreated (a hepatocellular carcinoma cell line); Fetal Liver; Pituitary Gland; pool of: Hypothalamus, Lymph Node, Fetal Liver, fetal_lung.
NOV4 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 4C.
Figure imgf000049_0001
AMBP_RAT rattus norvegicus 349 250/322 281/322, le-155 (rat) . ambp (78%) (87%) protein precursor [contains: alpha- 1-microglobulin ; inter-alpha- trypsin inhibitor flight chain (iti- lc) (bikunin) (hi- 30)] . 7/1999 and
(Pasted_No.l.l8:22 -343)
AMBP_BOVIN b ambp protein 352 248/321 280/321, le-155 precursor (77%) (87%) [contains : alpha- 1-microglobulin; inter-alpha- trypsin inhibitor light chain (iti-lc) (bikunin) (hi-30) (bi-14) (cumulus extracellular matrix s
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 4D.
Table 4D ClustalW Analysis of NOV4 1) NOV4 (SEQ ID NO: 13)
2) AMBP_HUMAN h ambp protein precursor [contains: alpha-1-microglobulin (protein he) (complex-forming glycoprotein heterogeneous in charge) ; inter-alpha- trypsin inhibitor light chai (SEQ ID NO: 49)
3) AMBP_PIG sus scrofa (pig), ambp protein precursor [contains: alpha-1- microglobulin; inter-alpha- trypsin inhibitor light chain (iti-lc) (bikunin) (hi-30) (ei-14)] (fragment) (SEQ ID Nθ:50)
4) AMBP_MESAU mesocricetus auratus (golden hamster) . ambp protein precursor [contains: alpha-1-microglobulin; inter-alpha- trypsin inhibitor light chain (iti- lc) (bikunin) (hi-30)] (SEQ ID NO: 51) 5) AMBP_RAT rattus norvegicus (rat) . ambp protein precursor [contains: alpha-1- microglobulin; inter-alpha- trypsin inhibitor light chain (iti-lc) (bikunin) (hi- 30)]. 7/1999and (Pasted_No.1.18 :22-343) (SEQ ID N0:52)
6) AMBP_BOVIN b ambp protein precursor [contains: alpha-1-microglobulin; inter- alpha- trypsin inhibitor light chain (iti-lc) (bikunin) (hi-30) (bi-14) (cumulus extracellular matrix s (SEQ ID NO: 53)
Figure imgf000050_0001
Figure imgf000051_0001
370
NOV4 EgPGRSRG ES 372
AMBP_HUMAN 352
AMBP_PIG 337
AMBP_MESAU 349
AMBP_RAT 349
AMBP BOVIN 35
Tables 4E-I list the domain description from DOMAIN analysis results against NOV4. This indicates that the NOV4 sequence has properties similar to those of other proteins known to contain this domain.
Table 4E. Domain Analysis of NOV4 gnl I Pfam|pfam00061, lipocalin, Lipocalin / cytosolic fatty-acid binding protein family (SEQ ID NO: 80) CD-Length = 145 residues, 100.0% aligned Score = 101 bits (252) , Expect = 6e-23
Query : 39 RIYG WYNLAIGSTCPWLKKIMDRMTVSTLVLGEGATEAEISMTSTR RKGVCEETSGAY 98
+ m i +| + i | |+ + + + + i + + + i + i i n i
Sbjct: 1 KFAGKWYLVASANFDPELKEELGVLEATRK ITPL-KEGNLEIVFDGDKNGICEETFGKL 59 Query: 99 EKTDTDGKFLYHKSKWNITMESYVVHTNYDEYAIFLTKKFSRHHGPTITAKLYGRAPQLR 158
III I + + I |+ i +n I + +| + l + HII l+ l Sbjct: 60 EKTKKLGVEFDYYTGDN RFWLDTDYDNYLLVCVQKGDGG-ETSRVAELYGRTPELS 115
Query: 159 ETLLQDFRWAQGVGIPEDSIFTMADRGEC 188
1+ II + +IIIII++ I
Sbjct: 116 PEALELFRTLTEELGIPEDNWCTRQTERC 145
Table 4F. Domain Analysis of NOV4 gnl I Smart | smart00131, KU, BPTI/Kunitz family of serine protease inhibitors.; Serine protease inhibitors. One member of the family is encoded by an alternatively-spliced form of Alzheimer's amyloid beta- protein (SEQ ID NO: 81)
CD-Length = 54 residues, 100.0% aligned Score = 73.6 bits (179), Expect = 2e-14
Query: 229 DSCQLGYSAGPCMGMTSRYFYNGTSMACETFQYGGCMGNGNNFVTEKECLQTCR 282
I I I III i U+I+ + II i nn i in +++11 +11
Sbjct: 1 DVCSLPPDTGPCGGSIPRYYYDPETGTCEPFTYGGCGGNANNFESKEECERTCG 54
Figure imgf000052_0001
Query: 287 CNLPIVRGPCRAFIQLWAFDAVKGKCVLFPYGGCQGNGNKFYSEKECREYCG 338 l + ll III I + +1 I I I MM II I I I++I II
Sbjct: 3 CSLPPDTGPCGGSIPRYYYDPETGTCEPFTYGGCGGNANNFESKEECERTCG 54
Table 4H. Domain Analysis of NO 4 gnl I Pfam|pfam00014, Kunitz_BPTI; Kunitz/Bovine pancreatic trypsin inhibitor domain (SEQ ID NO: 82) CD-Length = 53 residues, 100.0% aligned Score = 70.1 bits (170), Expect = 2e-13
Query 287 CNLPIVRGPCRAFIQLWA--FDAVKGKCVLFPYGGCQGNGNKFYSEKECREYC 337 l + ll III I ++I +1+ I I I MM II I I +++II I Sbjct: 1 CSLPPDTGPCTGFPRIWRWYYDSSTGTCEPFTYGGCGGNANNFETKEECESTC 53 Table 41. Domain Analysis of NOV4 gnl |Pfam|pfam00014, Kunitz_BPTI, Kunitz/Bovine pancreatic trypsin inhibitor domain (SEQ ID NO: 82) CD-Length = 53 residues, 100.0% aligned Score = 67.0 bits (162), Expect = 2e-12
Query: 231 CQLGYSAGPCMGMTS--RYFYNGTSMACETFQYGGCMGNGNNFVTEKECLQTC 281
I I III I I++I+ + II I MM II III I++II II
Sbj ct : 1 CSLPPDTGPCTGFPRI RWYYDSSTGTCEPFTYGGCGGNANNFETKEECESTC 53
NOV4 also has high homology to members ofthe patp database shown in Table 4J.
Figure imgf000053_0001
NOV4 is homologous in various ways to the following molecules whose functions are described below. An imbalance between increased neutrophil elastase and a decreased antiprotease shield has been suggested as a factor contributing to the development of chronic lung disease (CLD). Stiskal JA et al. hypothesized that administration of alpha 1-proteinase inhibitor (A1PI), also known as alphal-antitrypsin, to premature neonates would prevent CLD. Design (Stiskal JA et al., Pediatrics 1998 Jan l;101(l):89-94). In this study a randomized, placebo-controlled, prospective study of A1PI supplementation was performed. Neonates <24 hours of age with birth weights 600- 1000 g on respiratory support, and 1001 - 1250 g with respiratory distress syndrome (RDS) were eligible. Intravenous A1PI (60 mg/kg) or placebo was infused on days 0, 4, 7, and 14. Primary outcome was CLD in survivors, defined as the need for supplemental oxygen on day 28. Results. A total of 106 patients were recruited. There were no significant differences between groups in birth weight or incidence of RDS. The incidence of CLD in survivors was lower in the treated group, but the difference did not reach statistical significance (relative risk [RR], 0.79; confidence interval [Cl], 0.60-1.02). This beneficial trend persisted at 36 weeks corrected gestational age (RR, 0.48; Cl, 0.23-1.00). The incidence of pulmonary hemorrhage was lower in the treated group (RR, 0.22; Cl, 0.05-0.98). Other complications were not significantly different between groups. In this, the first trial of a protease inhibitor for the prevention of CLD in premature infants, the infusions were well- tolerated. A1PI therapy may impede the development of CLD and appears to reduce the incidence of pulmonary hemorrhage in some neonates born prematurely.
The serum protein, alpha 1-proteinase inhibitor (alpha 1-PI), defends the host against serine proteinases, e.g. PMN elastase. Using a rabbit anti-serum against human alpha 1-PI, this protein in GCF was quantified from a standard curve constructed from dot-blot analysis and characterized by Western blot (Lee HM et al., J Periodontal Res 1997 Jan;32(l Pt 1):9-19).. GCF was collected on filter paper strips from healthy (H), gingivitis (G) and adult periodontitis (AP) patients, then extracted with Tris/NaCl/CaC12 buffer, pH 7.6. alpha 1-PI concentration increased with G and was highest in AP subjects. H sites only showed intact alpha 1-PI (52 kDa); no degradation fragments (48 kDa) were detected. In G and AP subjects, alpha 1-PI degradation fragments were seen in 17% and 71% of GCF samples, respectively. Both collagenase and alpha 1-PI-degrading activities in GCF increased with severity of inflammation (GCF flow). Moreover, the alpha 1-PI degrading (or serpinolytic) activity was characterized as a matrix metalloproteinase, probably collagenase, based on its in vitro response to a panel of different proteinase inhibitors including doxycycline. Lee et al. propose: (1) that collagenase promotes periodontal breakdown not only by degrading collagen, but also by depleting alpha 1-PI regulation of elastase and other serine-proteinases, thereby favoring a broader attack on extracellular matrix (ECM) constituents, and (2) based on a recent longitudinal double-blind study using the techniques described above for alpha 1-PI analysis, that low-dose doxycycline administration to humans with adult periodontitis can inhibit this broad cascade of ECM degradation.
Although high-dose aprotinin given intraperitoneally to patients with severe acute pancreatitis seems to inhibit activated trypsin in the peritoneal cavity, the treatment has little effect on the balance between proteases and antiproteases. (Berling R et al., Int J Pancreatol 1998 Aug;24(l):9-17). Plasma levels of leukocyte proteases were high in all the patients, indicating leukocyte activation to be an important feature ofthe pathophysiology of severe acute pancreatitis. A surprise finding was that the patients had higher peritoneal levels of pancreatic secretory trypsin inhibitor (PSTI) after the lavage procedure. Although most studies have shown protease inhibitor therapy to have little or no effect on acute pancreatitis, in an earlier study Berling et al. found that very high doses ofthe protease inhibitor aprotinin given intraperitoneally to patients with severe acute pancreatitis seemed to reduce the need of surgical treatment for pancreatic necrosis. In the present study they have further analyzed plasma and peritoneal samples from the same patients to ascertain whether the aprotinin treatment affects the balance between proteases and endogenous antiproteases. In a prospective double-blind randomized multicenter trial by Berling et al., 48 patients with severe acute pancreatitis were treated with intraperitoneal lavage. One group (aprotinin group, n = 22) was also treated with high doses (20 million KTU given over 30 h) of aprotinin intraperitoneally. The remaining 26 patients made up the control group. The protease- antiprotease balance was studied by measuring immunoreactive anionic trypsin (irAT), cationic trypsin (irCT), complexes between cationic trypsin and alpha 1 -protease inhibitor (irCT-alpha 1 PI), leukocyte elastase and neutrophil proteinase 4 (NP4), as well as the endogenous protease inhibitors, pancreatic secretory trypsin inhibitor (PSTI), alpha 2- macro globulin (alpha 2M), alpha 1 -protease inhibitor (alpha 1 PI), antichymotrypsin (ACHY), and secretory leukocyte protease inhibitor (SLPI). Intraperitoneal levels were studied before and after the lavage procedure, and plasma levels were followed for 21 d. The control group had lower plasma levels of SLPI and analysis of peritoneal fluid showed the reduction of irCT- alpha 1 PI to be more pronounced in the aprotinin group. None ofthe other variables measured differed significantly between the two groups. All patients had very high levels of leukocyte elastase and NP4 both in peritoneal exudate and in plasma. Peritoneal levels of PSTI were higher after the lavage procedure in contrast to the other measured variables that all showed lower peritoneal levels after the lavage.
The protein similarity information, expression pattern, and map location for the trypsin inhibitor-like protein and nucleic acid (NOV4) disclosed herein suggest that this NOV4 protein may have important structural and/or physiological functions characteristic ofthe trypsin inhibitor family. Therefore, the NOV4 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo. The NOV4 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from chronic lung diseases, periodontitis, pancreatitis. The NOV4 nucleic acids, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
NOV4 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. For example, the disclosed NOV4 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOV4 epitope is from about amino acids 20 to 50. In another embodiment, a NOV4 epitope is from about amino acids 75 to 170. In additional embodiments, NOV4 epitopes are from about amino acids 180 to 270, and from about amino acids 320 to 370. These novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
NOV5
NOV5 includes three novel apolipoprotein e-like proteins disclosed below. The disclosed proteins have been named NOV5a, NOV5b, NOV5c, NOV5d, NOV5e, NOV5f, NOV5g, NOV5h, andNOV5i. NOV5a A disclosed NOV5a nucleic acid of 2249 nucleotides (also referred to as sggc_draft_dj784al6_20000723_dal) encoding a novel apolipoprotein e-like protein is shown in Table 5A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 30-32 and ending with a TGA codon at nucleotides 2217-2219. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5A, and the start and stop codons are in bold letters.
Table 5A. NO 5a Nucleotide Sequence (SEQ ID NO:14)
TGTTTTTTTTCTTCAGTACTTTAAa.TGTCATGCCACTCTCCTGGCCTGTAAGCTTTCCACAGA GAAGCCTGCTGCCAGACCCATTGGAGCTTCTTTGTGTGTTATTTGTTTCTTTTTTCTTGCTGC TTTTACCTCTCCGGCTTCTGGCACTGTTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCAGCTCC AGCATCTTGCGGCGGCAGCGGCTGATCCGCTGCTCGGCGGCCAAGGGCCGGCCAAGGATTGCG AAAAGGACCAATTCCAGTGCCGGAACGAGCGCTGCATCCCCTCTGTGTGGAGATGCGACGAGG ACGATGACTGCTTAGACCACAGCGACGAGGACGACTGCCCTGCAGCCAAGAAGACCTGTGCAG ACAGTGACTTCACCTGTGACAACGGCCACTGCATCCACGAACGGTGGAAGTGTGACGGCGAGG AGGAGTGTCCTGATGGCTCCGATGAGTCCGAGGCCACTTGCACCAAGCAGGTGTGTCCTGCAG AGAAGCTGAGCTGTGGACCCACCAGCCACAAGTGTGTACCTGCCTCGTGGCGCTGCGACGGGG AGAAGGACTGCGAGGGTGGAGCGGATGAGGCCGGCTGTGCTACCTCACTGGGCACCTGCCGTG GGGACGAGTTCCAGTGTGGGGATGGGACATGTGTCCTTGCAATCAAGCACTGCAACCAGGAGC AGGACTGTCCAGATGGGAGTGATGAAGCTGGCTGCCTACAGGGGCTGAACGAGTGTCTGCACA ACAATGGCGGCTGCTCACACATCTGCACTGACCTCAAGATTGGCTTTGAATGCACGTGCCCAG CAGGCTTCCAGCTCCTGGACCAGAAGACCTGTGGCGACATTGATGAGTGCAAGGACCCAGATG CCTGCAGCCAGATCTGTGTCAATTACAAGGGCTATTTTAAGTGTGAGTGCTACCCTGGCTACG AGATGGACCTACTGACCAAGAACTGCAAGGCTGCTGCTGGCAAGAGCCCATCCCTAATCTTCA CCAACCGGCACGAGGTGCGGAGGATCGACCTGGTGAAGCGGAACTATTCACGCCTCATCCCCA TGCTCAAGAATGTCGTGGCACTAGATGTGGAAGTTGCCACCAATCGCATCTACTGGTGTGACC TCTCCTACCGTAAGATCTATAGCGCCTACATTGGACAAGGCCAGGACCCGAAAGAGCAGGAGG TCCTCATTGACGAGCAGTTGCACTCTCCAGAGGGCCTGGCAGTGGACTGGGTCCACAAGCACA TCTACTGGACTGACTCGGGCAATAAGACCATCTCAGTGGCCACAGTTGATGGTGGCCGCCGAC GCACTCTCTTCAGCCGTAACCTCAGTGAACCCCGGGCCATCGCTGTTGACCCCCTGCGAGGGT TCATGTATTGGTCTGACTGGGGGGACCAGGCCAAGATTGAGAAATCTGGGCTCAACGGTGTGG ACCGGCAAACACTGGTGTCAGACAATATTGAATGGCCCAACGGAATCACCCTGGATCTGCTGA GCCAGCGCTTGTACTGGGTAGACTCCAAGCTACACCAACTGTCCAGCATTGACTTCAGTGGAG GCAACAGAAAGACGCTGATCTCCTCCACTGACTTCCTGAGCCACCCTTTTGGGATAGCTGTGT TTGAGGACAAGGTGTTCTGGACAGACCTGGAGAACGAGGCCATTTTCAGTGCAAATCGGCTCA ATGGCCTGGAAATCTCCATCCTGGCTGAGAACCTGAACAACCCACATGACATTGTCATCTTCC ATGAGCTGAAGCAGCCAAGAGCTCCAGATGCCTGTGAGCTGAGTGTCCAGCCTAATGGAGGCT GTGAATACCTGTGCCTTCCTGCTCCTCAGATCTCCAGCCACTCTCCCAAGTACACATGTGCCT GTCCTGACACAATGTGGCTGGGTCCAGACATGAAGAGGTGCTACCGAGATGCAAATGAAGACA GTAAGATGGGCTCAACAGTCACTGCCGCTGTTATCGGGATCATCGTGCCCATAGTGGTGATAG CCCTCCTGTGCATGAGTGGATACCTGATCTGGAGAAACTGGAAGCGGAAGAACACCAAAAGCA TGAATTTTGACAACCCAGTCTACAGGAAAACAACAGAAGAAGAAGACGAAGATGAGCTCCATA TAGGGAGAACTGCTCAGATTGGCCATGTCTATCCTGCACGAGTGGCATTAAGCCTTGAAGATG ATGGACTACCCTGAGGATGGGATCACCCCCTTCGTGCCTCATGG
The NOV5a sequence was identified by subjecting Ace. No. RP4-784A16 to an exon linking process. PCR primers were designed to Ace. No. RP4-784A16 by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5a sequence.
The NOV5a nucleic acid was identified on chromosome 1 and has 2099 of 2118 bases (99%) identical to a 2549 bp apolipoprotein E receptor 2 mRNA from Homo sapiens (GENBANK-ID: HSZ75190|acc:Z75190 (E = 0.0)
A disclosed NOV5a polypeptide (SEQ ID NO: 15) encoded by SEQ ID NO: 14 is 729 amino acid residues and is presented using the one-letter code in Table 5B. Signal P, Psort and/or Hydropathy results predict that NOV5a has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000. In other embodiments, NOV5a may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum
(membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The most likely cleavage site for a NOV5a peptide is between amino acids 59 and 60, at: AAA-AA.
Table 5B. Encoded NOV5a protein sequence (SEQ ID NO:15)
MPLSWPVSFPQRSLLPDPLELLCVLFVSFFLLLLPLRLLALLLLLLLLLLLQLQHLAAAAADPLLGGQGPAK DCEKDQFQCRNERCIPSVWRCDEDDDCLDHSDEDDCPAAKKTCADSDFTCDNGHCIHER KCDGEEECPDGS DESEATCTKQVCPAEKLSCGPTSHKCVPASWRCDGEKDCEGGADEAGCATSLGTCRGDEFQCGDGTCVLAIK HCNQEQDCPDGSDEAGCLQGLNECLHNNGGCSHICTDLKIGFECTCPAGFQLLDQKTCGDIDECKDPDACSQ ICVNYKGYFKCECYPGYEMDLLTKNCKAAAGKSPSLIFTNRHEVRRIDLVKRNYSRLIPMLKNWALDVEVA TNRIYWCDLSYRKIYSAYIGQGQDPKEQEVLIDEQLHSPEGLAVDWVHKHIYWTDSGNKTISVATVDGGRRR TLFSRNLSEPRAIAVDPLRGFMYWSDWGDQAKIEKSGLNGVDRQTLVSDNIEWPNGITLDLLSQRLYWVDSK LHQLSSIDFSGGNRKTLISSTDFLSHPFGIAVFEDKVFWTDLENEAIFSANRLNGLEISILAENLNNPHDIV IFHELKQPRAPDACELSVQPNGGCEYLCLPAPQISSHSPKYTCACPDTMWLGPDMKRCYRDANEDSKMGSTV TAAVIGIIVPIWIALLCMSGYLIWRNWKRKNTKSMNFDNPVYRKTTEEEDEDELHIGRTAQIGHVYPARVA LSLEDDGLP
The disclosed NOV5a amino acid sequence has 676 of 695 amino acid residues (97%) identical to, and 681 of 695 amino acid residues (98%) similar to, the 699 amino acid residue APOLIPOPROTEIN E RECEPTOR 2 906 protein from Homo sapiens (Human) (SPTREMBL-ACC: Q99876) (E = 0.0). NOV5b
A disclosed NOV5b nucleic acid of 2348 nucleotides (also referred to as sggc_draft_dj784al6_20000723_da2) encoding a novel apolipoprotein e-like protein is shown in Table 5C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 30-32 and ending with a TGA codon at nucleotides 2316-2318. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5C, and the start and stop codons are in bold letters. Table 5C. NOV5b Nucleotide Sequence (SEQ ID NO:16)
TGTTTTTTTTCTTCAGTACTTTAAATGTCATGCCACTCTCCTGGCCTGTAAGCTTTCCACAGA GAAGCCTGCTGCCAGACCCATTGGAGCTTCTTTGTGTGTTATTTGTTTCTTTTTTCTTGCTGC TTTTACCTCTCCGGCTTCTGGCACTGTTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCAGCTCC AGCATCTTGCGGCGGCAGCGGCTGATCCGCTGCTCGGCGGCCAAGGGCCGGCCAAGGATTGCG AAAAGGACCAATTCCAGTGCCGGAACGAGCGCTGCATCCCCTCTGTGTGGAGATGCGACGAGG ACGATGACTGCTTAGACCACAGCGACGAGGACGACTGCCCTGCAGCCAAGAAGACCTGTGCAG ACAGTGACTTCACCTGTGACAACGGCCACTGCATCCACGAACGGTGGAAGTGTGACGGCGAGG AGGAGTGTCCTGATGGCTCCGATGAGTCCGAGGCCACTTGCACCAAGCAGGTGTGTCCTGCAG AGAAGCTGAGCTGTGGACCCACCAGCCACAAGTGTGTACCTGCCTCGTGGCGCTGCGACGGGG AGAAGGACTGCGAGGGTGGAGCGGATGAGGCCGGCTGTGCTACCTCACTGGGCACCTGCCGTG GGGACGAGTTCCAGTGTGGGGATGGGACATGTGTCCTTGCAATCAAGCACTGCAACCAGGAGC AGGACTGTCCAGATGGGAGTGATGAAGCTGGCTGCCTACAGGTTCCGCCAACATTCCTGGGAA ACAGGAGGAGGCCCAGGGGGCTGAACGAGTGTCTGCACAACAATGGCGGCTGCTCACACATCT GCACTGACCTCAAGATTGGCTTTGAATGCACGTGCCCAGCAGGCTTCCAGCTCCTGGACCAGA AGACCTGTGGCGACATTGATGAGTGCAAGGACCCAGATGCCTGCAGCCAGATCTGTGTCAATT ACAAGGGCTATTTTAAGTGTGAGTGCTACCCTGGCTACGAGATGGACCTACTGACCAAGAACT GCAAGGCTGCTGCTGGAAAGAGCCCATCCCTAATCTTCACCAACCGGCACGAGGTGCGGAGGA TCGACCTGGTGAAGCGGAACTATTCACGCCTCATCCCCATGCTCAAGAATGTCGTGGCACTAG ATGTGGAAGTTCCACCAATCGCATCTACTGGTGTGACCTCTCCTACCGTAAGATCTATAGCGC CTACATGGGACAGGCACCCCTCACCCCCACTCTTCATGCCTTGCTGCAGAGGATATGGCCGCT CACCTAGACAAGGCCAGGACCCGAAAGAGCAGGAGGTCCTCATTGACGAGCAGTTGCACTCTC CAGAGGGCCTGGCAGTGGACTGGGTCCACAAGCACATCTACTGGACTGACTCGGGCAATAAGA CCATCTCAGTGGCCACAGTTGATGGTGGCCGCCGACGCACTCTCTTCAGCCGTAACCTCAGTG AACCCCGGGCCATCGCTGTTGACCCCCTGCGAGGGTTCATGTATTGGTCTGACTGGGGGGACC AGGCCAAGATTGAGAAATCTGGGCTCAACGGTGTGGACCGGCAAACACTGGTGTCAGACAATA TTGAATGGCCCAACGGAATCACCCTGGATCTGCTGAGCCAGCGCTTGTACTGGGTAGACTCCA AGCTACACCAACTGTCCAGCATTGACTTCAGTGGAGGCAACAGAAAGACGCTGATCTCCTCCA CTGACTTCCTGAGCCACCCTTTTGGGATAGCTGTGTTTGAGGACAAGGTGTTCTGGACAGACC TGGAGAACGAGGCCATTTTCAGTGCAAATCGGCTCAATGGCCTGGAAATCTCCATCCTGGCTG AGAACCTGAACAACCCACATGACATTGTCATCTTCCATGAGCTGAAGCAGCCAAGAGCTCCAG ATGCCTGTGAGCTGAGTGTCCAGCCTAATGGAGGCTGTGAATACCTGTGCCTTCCTGCTCCTC AGATCTCCAGCCACTCTCCCAAGTACACATGTGCCTGTCCTGACACAATGTGGCTGGGTCCAG ACATGAAGAGGTGCTACCGAGATGCAAATGAAGACAGTAAGATGGGCTCAACAGTCACTGCCG CTGTTATCGGGATCATCGTGCCCATAGTGGTGATAGCCCTCCTGTGCATGAGTGGATACCTGA TCTGGAGAAACTGGAAGCGGAAGAACACCAAAAGCATGAATTTTGACAACCCAGTCTACAGGA AAACAACAGAAGAAGAAGACGAAGATGAGCTCCATATAGGGAGAACTGCTCAGATTGGCCATG TCTATCCTGCACGAGTGGCATTAAGCCTTGAAGATGATGGACTACCCTGAGGATGGGATCACC CCCTTCGTGCCTCATGG
The NOV5b sequence was identified through an exon linking process. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library contaimng a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5b sequence. The NOV5b nucleic acid was identified on chromosome 1 and has 1151 of 1206 bases (95%) identical to a a 2549 bp apolipoprotein E receptor 2 mRNA from Homo sapiens (GENBANK-ID: HSZ75190|acc:Z75190 (E = 0.0)
A disclosed NOV5b polypeptide (SEQ ID NO: 17) encoded by SEQ ID NO: 16 is 762 amino acid residues and is presented using the one-letter code in Table 5D. Signal P, Psort and/or Hydropathy results predict that NOV5b has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000. In other embodiments, NOV5b may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The most likely cleavage site for a NOV5b peptide is between amino acids 59 and 60, at: AAA-AA.
Table 5D. Encoded NOV5b protein sequence (SEQ ID NO:17)
MPLSWPVSFPQRSLLPDPLELLCVLFVSFFLLLLPLRLLALLLLLLLLLLLQLQHLAAAAADPLLGGQGPAK DCEKDQFQCRNERCIPSVWRCDEDDDCLDHSDEDDCPAAKKTCADSDFTCDNGHCIHERWKCDGEEECPDGS DESEATCTKQVCPAEKLSCGPTSHKCVPASWRCDGEKDCEGGADEAGCATSLGTCRGDΞFQCGDGTCVLAIK HCNQEQDCPDGSDEAGCLQVPPTFLGNRRRPRGLNECLHNNGGCSHICTDLKIGFECTCPAGFQLLDQKTCG DIDECKDPDACSQICVNYKGYFKCECYPGYEMDLLTKNCKAAAGKSPSLIFTNRHEVRRIDLVKRNYSRLIP MLKNWALDVEVPPIASTGVTSPTVRSIAPTWDRHPSPPLFMPCCRGYGRSPRQGQDPKEQEVLIDEQLHSP EGLAVDWVHKHIYWTDSGNKTISVATVDGGRRRTLFSRNLSEPRAIAVDPLRGFMYWSDWGDQAKIEKSGLN GVDRQTLVSDNIEWPNGITLDLLSQRLYWVDSKLHQLSSIDFSGGNRKTLISSTDFLSHPFGIAVFEDKVFW TDLENEAIFSANRLNGLEISILAENLNNPHDIVIFHELKQPRAPDACELSVQPNGGCEYLCLPAPQISSHSP KYTCACPDTMWLGPDMKRCYRDANEDSKMGSTVTAAVIGIIVPIWIALLCMSGYLIWRNWKRKNTKSMNFD NPVYRKTTEEEDEDELHIGRTAQIGHVYPARVALSLEDDGLP
The disclosed NOV5b amino acid sequence has 345 of 349 amino acid residues (98%) identical to, and 347 of 349 amino acid residues (99%) similar to, the 699 amino acid residue APOLΓPOPROTEIN E RECEPTOR 2 906 protein from Homo sapiens (Human) (SPTREMBL-ACC: Q99876) (E = 0.0). NOV5c
A disclosed NOV5c nucleic acid of 2537 nucleotides (also referred to as sggc_draft_dj784al6_20000723_da3) encoding a novel apolipoprotein e-like protein is shown in Table 5E. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 30-32 and ending with a TGA codon at nucleotides 2505-2507. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5E, and the start and stop codons are in bold letters. Table 5E. NOV5c Nucleotide Sequence (SEQ ID NO:18)
TGTTTTTTTTCTTCAGTACTTTAAATGTCATGCCACTCTCCTGGCCTGTAAGCTTTCCACAGA GAAGCCTGCTGCCAOACCCATTGGAGCTTCTTTGTGTGTTATTTGTTTCTTTTTTCTTGCTGC TTTTACAGCTCCAGCATCTTGCGGCGGCAGCGGCTGATCCGCTGCTCGGCGGCCAAGGGCCGG CCAAGGATTGCGAAAAGGACCAATTCCAGTGCCGGAACGAGCGCTGCATCCCCTCTGTGTGGA GATGCGACGAGGACGATGACTGCTTAGACCACAGCGACGAGGACGACTGCCCTGCAGCCAAGA AGACACCTGTGAGAAGTGACTTCACCTGTGACAACGGCCACTGCATCCACGAACGGTGGAAGT GTGACGGCGAGGAGGAGTGTCCTGATGGCTCCGATGAGTCCGAGGCCACTTGCACCAAGCAGG TGTGTCCTGCAGAGAAGCTGAGCTGTGGACCCACTAGCCACAAGTGTGTACCTGCCTCGTGGC GCTGCGACGGGGAGAAGGACTGCGAGGGTGGAGCGGATGAGGCCGGCTGTGCTACCTCACTGG GCACCTGCCGTGGGGACGAGTTCCAGTGTGGGGATGGGACATGTGTCCTTGCAATCAAGCACT GCAACCAGGAGCAGGACTGTCCAGATGGGAGTGATGAAGCTGGCTGCCTACAGGGGCTGAACG AGTGTCTGCACAACAATGGCGGCTGCTCACACATCTGCACTGACCTCAAGATTGGCTTTGAAT GCACGTGCCCAGCAGGCTTCCAGCTCCTGGACCAGAAGACCTGTGGCGCTGGCAAGAGCCCAT CCCTAATCTTCACCAACCGGCACGAGGTGCGGAGGATCGACCTGGTGAAGCGGAACTATTCAC GCCTCATCCCCATGCTCAAGAATGTCGTGGCACTAGATGTGGAAGTTGCCACCAATCGCATCT ACTGGTGTGACCTCTCCTACCGTAAGATCTATAGCGCCTACATGGACAAGGCCAGTGACCCGA AAGAGCAGGAGGTCCTCATTGACGAGCAGTTGCACTCTCCAGAGGGCCTGGCAGTGGACTGGG TCCACAAGCACATCTACTGGACTGACTCGGGCAATAAGACCATCTCAGTGGCCACAGTTGATG GTGGCCGCCGACGCACTCTCTTCAGCCGTAACCTCAGTGAACCCCGGGCCATCGCTGTTGACC CCCTGCGAGGGTTCATGTATTGGTCTGACTGGGGGGACCAGGCCAAGATTGAGAAATCTGGGC TCAACGGTGTGGACCGGCAAACACTGGTGTCAGACAATATTGAATGGCCCAACGGAATCACCT TGGATCTGCTGAGCCAGCGCTTGTACTGGGTAGACTCCAAGCTACACCAACTGTCCAGCATTG ACTTCAGTGGAGGCAACAGAAAGACGCTGATCTCCTCCACTGACTTCCTGAGCCACCCTTTTG GGATAGCTGTGTTTGAGGACAAGGTGTTCTGGACAGACCTGGAGAACGAGGCCATTTTCAGTG CAAATCGGCTCAATGGCCTGGAAATCTCCATCCTGGCTGAGAACCTGAACAACCCACATGACA TTGTCATCTTCCATGAGCTGAAGCAGCCAAGAGCTCCAGATGCCTGTGAGCTGAGTGTCCAGC CTAATGGAGGCTGTGAATACCTGTGCCTTCCTGCTCCTCAGATCTCCAGCCACTCTCCCAAGT ACACATGTGCCTGTCCTGACACAATGTGGCTGGGTCCAGACATGAAGAGGTGCTACCGAGCAC CTCAATCTACCTCAACTACGACGTTAGCTTCTACCATGACGAGGACAGTACCTGCCACCACAA GAGCCCCCGGGACCACCGTCCACAGATCCACCTACCAGAACCACAGCACAGAGACACCAAGCC TGACAGCTGCAGTCCCAAGCTCAGTTAGTGTCCCCAGGGCTCCCAGCATCAGCCCGTCTACCC TAAGCCCTGCAACCAGCAACCACTCCCAGCACGCAAATGAAGACAGTAAGATGGGCTCAACAG TCACTGCCGCTGTTATCGGGATCATCGTGCCCATAGCAAATGAAGACAGTAAGATGGGCTCAA CAGTCACTGCCGCTGTTATCGGGATCATCGTGCCCATAGGTGATAGCCCTCCTGTGCATGAGT GGATACCTGATCTGGAGAAACTGGAAGCGGAAGAACACCAAAAGCATGAATTTGACAACCCAG TCTACAGGAAAACAACAGAAGAAGAAGACGAAGATGAGCTCCATATAGGGAGAACTGCTCAGA TTGGCCATGTCTATCCTGCAGCAATCAGCAGCTTTGATCGCCCACTGTGGGCAGAGCCCTGTC TTGGGGAGACCAGAGAACCAGAAGACCCAGCCCCTGCCCTGAAGGAGCTTTTTGTCTTGCCGG GGGAACCAAGGTCACAGCTGCACCAACTCCCGAAGAACCCTCTTTCCGAGCTGCCTGTCGTCA AATCCAAGCAGCGAGTGGCATTAAGCCTTGAAGATGATGGACTACCCTGAGGATGGGATCACC CCCTTCGTGCCTCATGG
The NOV5c sequence was identified through an exon linking process. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5c sequence. The NOV5c nucleic acid was identified on chromosome 1 and has 1626 of 1922 bases (84%) identical to a 4468 bp apolipoprotein E receptor 2 mRNA from Homo sapiens (GENBANK-ID:D50678|acc:D50678 ) (E = ie'127)
A disclosed NOV5c polypeptide (SEQ ID NO: 19) encoded by SEQ ID NO: 18 is 825 amino acid residues and is presented using the one-letter code in Table 5F. Signal P, Psort and/or Hydropathy results predict that NOV5c has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000. In other embodiments, NOV5c may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The most likely cleavage site for a NOV5c peptide is between amino acids 44 and 45, at: AAA-DP.
Table 5F. Encoded NOV5c protein sequence (SEQ ID NO:19)
MPLSWPVSFPQRSLLPDPLELLCVLFVSFFLLLLQLQHLAAAAADPLLGGQGPAKDCEKDQFQCR NERCIPSVRCDEDDDCLDHSDEDDCPAAKKTPVRSDFTCDNGHCIHER KCDGEEECPDGSDES EATCTKQVCPAEKLSCGPTSHKCVPASWRCDGEKDCEGGADEAGCATSLGTCRGDEFQCGDGTCV LAIKHCNQEQDCPDGSDEAGCLQGLNΞCLHNNGGCSHICTD KIGFECTCPAGFQ LDQKTCGAG KSPSLIFTNRHEVRRIDLVKRNYSRLIPMLKNVVALDVEVATNRIY CDLSYRKIYSAYMDKASD PKEQEVLIDEQLHSPEGLAVD VHKHIY TDSGNKTISVATVDGGRRRTLFSRNLSEPRAIAVDP LRGFMYWSDWGDQAKIEKSGLNGVDRQTLVSDNIE PNGITLDLLSQRLYWVDSKLHQLSSIDFS GGNRKTLISSTDFLSHPFGIAVFEDKVF TDLENEAIFSANRLNGLEISILAENLNNPHDIVIFH ELKQPRAPDACELSVQPNGGCEYLCLPAPQISSHSPKYTCACPDT WLGPDMKRCYRAPQSTSTT TLAST TRTVPATTRAPGTTVHRSTYQNHSTETPSLTAAVPSSVSVPRAPSISPSTLSPATSNHS QHANEDSKMGSTVTAAVIGIIVPIANEDSKMGSTVTAAVIGIIVPIGDSPPVHEWIPDLEKLEAE EHQKHEFDNPVYRKTTEEEDEDE HIGRTAQIGHVYPAAISSFDRPLAEPC GETREPEDPAPA LKELFVLPGEPRSQLHQLPKNPLSELPWKSKQRVALSLEDDGLP
The disclosed NOV5c amino acid sequence has 586 of 659 amino acid residues (88%) identical to, and 601 of 659 amino acid residues (91%) similar to, the 793 amino acid residue APOER2DELTA4-7 protein from Homo sapiens (SPTREMBL-ACC: 014968) (E = 0.0). NOV5d
A disclosed NOV5d nucleic acid was isolated by subjecting NOV5c to ϋι& exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence ofthe target sequence, or by translated homology ofthe predicted exons to closely related human sequences sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone manow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component ofthe assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for conections if appropriate. The novel NOV5d sequence of 3039 nucleotides (also referred to as CG55256-02) encoding a novel apolipoprotein e-like protein is shown in Table 5G. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TGA codon at nucleotides 3037-3039. The start and stop codons are in bold letters.in Table 5G.
Table 5G. NOV5d Nucleotide Sequence (SEQ ID NO:20)
ATGGGCCTCCCCGAGCCGGGCCCTCTCCGGCTTCTGGCGCTGCTGCTGCTGCTGCTGCTGCTG CTGCTGCTGCGGCTCCAGCATCTTGCGGCGGCAGCGGCTGATCCGCTGCTCGGCGGCCAAGGG CCGGCCAAGGAGTGCGAAAAGGACCAATTCCAGTGCCGGAACGAGCGCTGCATCCCCTCTGTG TGGAGATGCGACGAGGACGATGACTGCTTAGACCACAGCGACGAGGACGACTGCCCCAAGAAG ACCTGTGCAGACAGTGACTTCACCTGTGACAACGGCCACTGCATCCACGAACGGTGGAAGTGT GACGGCGAGGAGGAGTGTCCTGATGGCTCCGATGAGTCCGAGGCCACTTGCACCAAGCAGGTG TGTCCTGCAGAGAAGCTGAGCTGTGGACCCACCAGCCACAAGTGTGTACCTGCCTCGTGGCGC TGCGACGGGGAGAAGGACTGCGAGGGTGGAGCGGATGAGGCCGGCTGTGCTACCTTGTGCGCC CCGCACGAGTTCCAGTGCGGCAACCGCTCGTGCCTGGCCGCCGTGTTCGTGTGCGACGGCGAT GACGACTGTGGTGACGGCAGCGATGAGCGCGGCTGTGCAGACCCGGCCTGCGGGCCCCGCGAG TTCCGCTGCGGCGGCGATGGCGGCGGCGCCTGCATCCCGGAGCGCTGGGTCTGCGACCGCCAG TTTGACTGCGAGGACCGCTCGGACGAGGCAGCCGAGCTCTGCGGCCGCCCGGGCCCCGGGGCC ACGTCCGCGCCCGCCGCCTGCGCCACCGTCTCCCAGTTCGCCTGCCGCAGCGGCGAGTGCGTG CACCTGGGCTGGCGCTGCGACGGCGACCGCGACTGCAAAGACAAATCGGACGAGGCCGACTGC CCACTGGGCACCTGCCGTGGGGACGAGTTCCAGTGTGGGGATGGGACATGTGTCCTTGCAATC AAGCACTGCAACCAGGAGCAGGACTGTCCAGATGGGAGTGATGAAGCTGGCTGCCTACAGGAG TCACCCTGTGAGGGTCCCCGCAGATTTCAGTGTAAGAGTGGCAAGCGCGTGGACGGCGGGAAA GTGTGTGATGTGCAGAGGGACTGCCGGGACTGGTCGGATGAGCTTCTGAAAGTGTGGTGCGTC TCCTACCATCCCCCTCCTGGGCTGAACGAGTGTCTGCACAACAATGGCGGCTGCTCACACATC TGCACTGACCTCAAGATTGGCTTTGAATGCACGTGCCCAGCAGGCTTCCAGCTCCTGGACCAG AAGACCTGTGGCGACATTGATGAGTGCAAGGACCCAGATGCCTGCAGCCAGATCTGTGTCAAT TACAAGGGCTATTTTAAGTGTGAGTGCTACCCTGGCTACGAGATGGACCTACTGACCAAGAAC TGCAAGGCTGCTGCTGGAAAGAGCCCATCCCTAATCTTCACCAACCGGCACGAGGTGCGGAGG ATCGACCTGGTGAAGCGGAACTATTCACGCCTCATCCCCATGCTCAAGAATGTCGTGGCACTA GATGTGGAAGTTGCCACCAATCGCATCTACTGGTGTGACCTCTCCTACCGTAAGATCTATAGC GCCTACATGGACAAGGCCAGTGACCCGAAAGAGCAGGAGGTCCTCATTGACGAGCAGTTGCAC TCTCCAGAGGGCCTGGCAGTGGACTGGGTCCACAAGCACATCTACTGGACTGACTCGGGCAAT AAGACCATCTCAGTGGCCACAGTTGATGGTGGCCGCCGACGCACTCTCTTCAGCCGTAACCTC AGTGAACCCCGGGCCATCGCTGTTGACCCCCTGCGAGGGTTCATGTATTGGTCTGACTGGGGG GACCAGGCCAAGATTGAGAAATCTGGGCTCAACGGTGTGGACCGGCAAACACTGGTGTCAGAC AATATTGAATGGCCCAACGGAATCACCCTGGATCTGCTGAGCCAGCGCTTGTACTGGGTAGAC TCCAAGCTACACCAACTGTCCAGCATTGACTTCAGTGGAGGCAACAGAAAGACGCTGATCTCC TCCACTGACTTCCTGAGCCACCCTTTTGGGATAGCTGTGTTTGAGGACAAGGTGTTCTGGACA GACCTGGAGAACGAGGCCATTTTCAGTGCAAATCGGCTCAATGGCCTGGAAATCTCCATCCTG GCTGAGAACCTCAACAACCCACATGACATTGTCATCTTCCATGAGCTGAAGCAGCCAAGAGCT CCAGATGCCTGTGAGCTGAGTGTCCAGCCTAATGGAGGCTGTGAATACCTGTGCCTTCCTGCT CCTCAGATCTCCAGCCACTCTCCCAAGTACACATGTGCCTGTCCTGACACAATGTGGCTGGGT CCAGACATGAAGAGGTGCTACCGAGCACCTCAATCTACCTCAACTACGACGTTAGCTTCTACC ATGACGAGGACAGTACCTGCCACCACAAGAGCCCCCGGGACCACCGTCCACAGATCCACCTAC CAGAACCACAGCACAGAGACACCAAGCCTGACAGCTGCAGTCCCAAGCTCAGTTAGTGTCCCC AGGGCTCCCAGCATCAGCCCGTCTACCCTAAGCCCTGCAACCAGCAACCACTCCCAGCACTAT GCAAATGAAGACAGTAAGATGGGCTCAACAGTCACTGCCGCTGTTATCGGGATCATCGTGCCC ATAGTGGTGATAGCCCTCCTGTGCATGAGTGGATACCTGATCTGGAGAAACTGGAAGCGGAAG AACACCAAAAGCATGAATTTTGACAACCCAGTCTACAGGAAAACAACAGAAGAAGAAGATGAA GATGAGCTCCATATAGGGAGAACTGCTCAGATTGGCCATGTCTATCCTGCAGCAATCAGCAGC TTTGATCGCCCACTGTGGGCAGAGCCCTGTCTTGGGGAGACCAGAGAACCGGAAGACCCAGCC CCTGCCCTCAAGGAGCTTTTTGTCTTGCCGGGGGAACCAAGGTCACAGCTGCACCAACTCCCG AAGAACCCTCTTTCCGAGCTGCCTGTCGTCAAATCCAAGCGAGTGGCATTAAGCCTTGAAGAT GATGGACTACCCTGA
The NOV5d sequence was identified through an exon linking process. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5d sequence.
The NOV5d nucleic acid was identified to the p34 region of human chromosome 1 and has 2508 of 2995 bases (83%) identical to a gb:GENBANK-ID:D50678|acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E = 0.0) A disclosed NOV5d polypeptide (SEQ ID NO:21) encoded by SEQ ID NO:20 is 1012 amino acid residues and is presented using the one-letter code in Table 5H. Signal P, Psort and/or Hydropathy results predict that NOV5d has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760. In other embodiments, NOV5d may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or the exfracellularly with a certainty of 0.1000. The most likely cleavage site for a NOV5d peptide is between amino acids 32 and 33, at: AAA-AA.
Table 5H. Encoded NOV5d protein sequence (SEQ ID NO:21)
MGLPEPGPLRLLALLLLLLLLLLLRLQHLAAAAADPLLGGQGPAKECEKDQFQCRNERCIPSVWRCDEDDDC LDHSDEDDCPKKTCADSDFTCDNGHCIHERWKCDGEEECPDGSDESEATCTKQVCPAEKLSCGPTSHKCVPA SWRCDGEKDCEGGADEAGCATLCAPHEFQCGNRSCLAAVFVCDGDDDCGDGSDERGCADPACGPREFRCGGD GGGACIPERWVCDRQFDCEDRSDEAAELCGRPGPGATSAPAACATVSQFACRSGECVHLGWRCDGDRDCKDK SDEADCPLGTCRGDEFQCGDGTCVLAIKHCNQEQDCPDGSDEAGCLQESPCEGPRRFQCKSGKRVDGGKVCD VQRDCRDWSDELL-CVWCVSYHPPPGLNECLHNNGGCSHICTDLKIGFECTCPAGFQLLDQKTCGDIDECKDP DACSQICvTSrYKGYFKCECYPGYEMDLLTrasrCKAAAGKSPSLIFTNRHEVRRIDLVKRNYSRLIPMLKNVVAL DVEVATNRIYWCDLSYRKIYSAYMDIC-A.SDPK^QEVLIDEQLHSPEGLAVDWVΗKHIYWTDSGNKTISVATVD GGRRRTLFSRNLSEPRAIAVDPLRGFMYWSDWGDQAKIEKSGLNGVDRQTLVSDNIEWPNGITLDLLSQRLY WVDSKLHQLSSIDFSGGNRKTLISSTDFLSHPFGIAVFEDKVFWTDLENEAIFSANRLNGLEISILAENLNN PHDIVIFHELKQPRAPDACELSVQPNGGCEYLCLPAPQISSHSPKYTCACPDTMWLGPDMKRCYRAPQSTST TTLASTMTRTVPATTRAPGTTVHRSTYQNHSTETPSLTAAVPSSVSVPRAPSISPSTLSPATSNHSQHYANE DSKMGSTVTAAVIGIIVPIWIALLCMSGYLIWRNWKRKNTKSMNFDNPVYRKTTEEEDEDELHIGRTAQIG HVYPAAISSFDRPLWAEPCLGETREPEDPAPALKELFVLPGEPRSQLHQLPKNPLSELPWKSKRVALSLED DGLP
The disclosed NOV5d amino acid sequence has 742 of 931 amino acid residues (79%) identical to, and 777 of 931 amino acid residues (83%) similar to, the 963 amino acid residue ptnr:SPTREMBL-ACC:Q14114 protein from Homo sapiens (Human) (APOLIPOPROTEIN E
RECEPTOR 2 PRECURSOR) (E = 0.0).
NOV5d is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
In addition, the sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:D50678|acc:D50678.1) a closely related Human mRNA for apolipoprotein E receptor 2, complete eds homolog in species Homo sapiens :brain.
NOV5e
A disclosed NOV5e nucleic acid was isolated by subjecting NOV5c to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence ofthe target sequence, or by translated homology ofthe predicted exons to closely related human sequences sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component ofthe assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. The novel NOV5e sequence of 2537 nucleotides (also referced to as CG55256-03) encoding a novel apolipoprotein e-like protein is shown in Table 51. An open reading frame was identified beginning with an ATG imtiation codon at nucleotides 1-3 and ending with a TGA codon at nucleotides 2086-2088. A putative untranslated region downstream from the termination codon is underlined in Table 51, and the start and stop codons are in bold letters.
Table 51. NOV5e Nucleotide Sequence (SEQ ID NO:22)
ATGGGCCTCCCCGAGCCGGGCCCTCTCCGGCTTCTGGCGCTGCTGCTGCTGCTGCTGCTGCTG CTGCTGCTGCGGCTCCAGCATCTTGCGGCGGCAGCGGCTGATCCGCTGCTCGGCGGCCAAGGG CCGGCCAAGGATTGCGAAAAGGACCAATTCCAGTGCCGGAACGAGCGCTGCATCCCCTCTGTG TGGAGATGCGACGAGGACGATGACTGCTTAGACCACAGCGACGAGGACGACTGCCCCAAGAAG ACCTGTGCAGACAGTGACTTCACCTGTGACAACGGCCACTGCATCCACGAACGGTGGAAGTGT GACGGCGAGGAGGAGTGTCCTGATGGCTCCGATGAGTCCGAGGCCACTTGCACCAAGCAGGTG TGTCCTGCAGAGAAGCTGAGCTGTGGACCCACCAGCCACAAGTGTGTACCTGCCTCGTGGCGC TGCGACGGGGAGAAGGACTGCGAGGGTGGAGCGGATGAGGCCGGCTGTGCTACCTCACTGGGC ACCTGCCGTGGGGACGAGTTCCAGTGTGGGGATGGGACATGTGTCCTTGCAATCAAGCACTGC AACCAGGAGCAGGACTGTCCAGATGGGAGTGATGAAGCTGGCTGCCTACAGGGGCTGAACGAG TGTCTGCACAACAATGGCGGCTGCTCACACATCTGCACTGACCTCAAGATTGGCTTTGAATGC ACGTGCCCAGCAGGCTTCCAGCTCCTGGACCAGAAGACCTGTGGCGACATTGATGAGTGCAAG GACCCAGATGCCTGCAGCCAGATCTGTGTCAATTACAAGGGCTATTTTAAGTGTGAGTGCTAC CCTGGCTACGAGATGGACCTACTGACCAAGAACTGCAAGGCTGCTGCTGGAAAGAGCCCATCC CTAATCTTCACCAACCGGCACGAGGTGCGGAGGATCGACCTGGTGAAGCGGAACTATTCACGC CTCATCCCCATGCTCAAGAATGTCGTGGCACTAGATGTGGAAGTTGCCACCAATCGCATCTAC TGGTGTGACCTCTCCTACCGTAAGATCTATAGCGGCTACATGGACAAGGCCAGTGACCCGAAA GAGCAGGAGGTCCTCATTGACGAGCAGTTGCACTCTCCAGAGGGCCTGGCAGTGGACTGGGTC CACAAGCACATCTACTGGACTGACTCGGGCAATAAGAACATCTCTGGTGGCCGCCGACGCACT CTCTTCAGCCGTAACCTCAGTGAACCCCGGGCCATCGCTGTTGACCCCCTGCGAGGGTTCATG TATTGGTCTGACTGGGGGGACCAGGCCAAGATTGAGAAATCTGGGCTCAACGGTGTGGACCGG CAAACACTGGTGTCAGACAATATTGAATGGCCCAACGGAATCACCCTGGATCTGCTGAGCCAG CGCTTGTACTGGGTAGACTCCAAGCTACACCAACTGTCCAGCATTGACTTCAGTGGAGGCAAC AGAAAGACGCTGATCTCCTCCACTGACTTCCTGAGCCACCCTTTTGGGATAGCTGTGTTTGAG GACAAGGTGTTCTGGACAGACCTGGAGAACGAGGCCATTTTCAGTGCAAATCGGCTCAATGGC CTGGAAATCTCCATCCTGGCTGAGAACCTCAACAACCCACATGACATTGTCATCTTCCATGAG CTGAAGCAGCCAAGAGCTCCAGATGCCTGTGAGCTGAGTGTCCAGCCTAATGGAGGCTGTGAA TACCTGTGCCTTCCTGCTCCTCAGATCTCCAGCCACTCTCCCAAGTACACATGTGCCTGTCCT GACACAATGTGGCTGGGTCCAGACATGAAGAGGTGCTACCGAGATGCAAATGAAGACAGTAAG ATGGGCTCAACAGTCACTGCCGCTGTTATCGGGATCATCGTGCCCATAGTGGTGATAGCCCTC CTGTGCATGAGTGGATACCTGATCTGGAGAAACTGGAAGCGGAAGAACACCAAAAGCATGAAT TTTGACAACCCAGTCTACAGGAAAACAACAGAAGAAGAAGATGAAGATGAGCTCCATATAGGG AGAACTGCTCAGATTGGCCATGTCTATCCTGCACGAGTGGCATTAAGCCTTGAAGATGATGGA CTACCCTGAGGATGGGATCACCCCCTTGGTGCCTCATGGAATTCAGTCCCATGCACTACACTC CGGATGGTGTATGACTGGATGAATGGGTTTCTATATATGGGTCTGTGTGAGTGTATGTGTGTG TGTGATTTTTTTTTTTAAATTTATGTTGCGGAAAGGTAACCACAAAGTTATGATGAACTGCAA. ACATCCAAAGGATGTGAGAGTTTTTCTATGTATAATGTTTTATACACTTTTTAACTGGTTGCA CTACCCATGAGGAATTCGTGGAATGGCTACTGCTGACTAACATGATGCACATAACCAAATGGG GGCCAATGGCACAGTACCTTACTCATCATTTAAAAACTATATTTACAGAAGATGTTTGGTTGC TGGGGGGCTTTTTTAGGTTTTGGGCATTTGTTTTTTGTAflATAAGATGATTATGCTTTGTGGC TATCCATCAACATAAGT
The NOV5e sequence was identified through an exon linking process. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5e sequence. The NOV5e nucleic acid was identified to the p34 region of human chromosome 1 and has 1359 of 1359 bases (100%) identical to a gb:GENBANK-ID:HSZ75190|acc:Z75190.1 mRNA from Homo sapiens (H.sapiens mRNA for apolipoprotein E receptor 2) (E = 0.0)
A disclosed NOV5e polypeptide (SEQ ID NO:23) encoded by SEQ ID NO:22 is 695 amino acid residues and is presented using the one-letter code in Table 5 J. Signal P, Psort and/or Hydropathy results predict that NOV5e has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760. In other embodiments, NOV5e may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or the extracellularly with a certainty of 0.1000. The most likely cleavage site for a NOV5e peptide is between amino acids 32 and 33, at: AAA-AA.
Table 5J. Encoded NOV5e protein sequence (SEQ ID NO:23)
MGLPEPGPLRLLALLLLLLLLLLLRLQHLAAAAADPLLGGQGPAKDCEKDQFQCRNERCIPSVWRCDEDDDC LDHSDEDDCPKKTCADSDFTCDNGHCIHERWKCDGEEECPDGSDESEATCTKQVCPAEKLSCGPTSHKCVPA SWRCDGEKDCEGGADEAGCATSLGTCRGDEFQCGDGTCVLAIKHCNQEQDCPDGSDEAGCLQGLNECLHNNG GCSHICTDLKIGFECTCPAGFQLLDQKTCGDIDECKDPDACSQICVNYKGYFKCECYPGYEMDLLTKNCKAA AGKSPSLIFTNRHEVRRIDLVKRNYSRLIPMLKNVVALDVEVATNRIYWCDLSYRKIYSAYMDIASDPKEQE VLIDEQLHSPEGLAVDWVHKHIYWTDSGNKNISGGRRRTLFSRNLSEPRAIAVDPLRGFMYWSDWGDQAKIE KSGLNGVDRQTLVSDNIEWPNGITLDLLSQRLYWVDSKLHQLSSIDFSGGNRKTLISSTDFLSHPFGIAVFE DKVFWTDLENEAIFSANRLNGLEISILAENLNNPHDIVIFHELKQPRAPDACELSVQPNGGCEYLCLPAPQI SSHSPKYTCACPDTMWLGPDMKRCYRDANEDSKMGSTVTAAVIGIIVPIWIALLCMSGYLIWRNWKRKNTK SMNFDNPVYRKTTEEEDEDELHIGRTAQIGHVYPARVALSLEDDGLP
The disclosed NOV5e amino acid sequence has 683 of 699 amino acid residues (97%) identical to, and 685 of 699 amino acid residues (97%) similar to, the 699 amino acid residue ρtnr:SPTREMBL-ACC:Q99876 protein from Homo sapiens (Human) (APOLIPOPROTEIN E RECEPTOR 2 906) (E = 0.0).
NOV5e is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, and/or RACE sources. In addition, the sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-JD:HSZ75190|acc:Z75190.1) a closely related H.sapiens mRNA for apolipoprotein E receptor 2 homolog in species Homo sapiens : umbilical vein endothelial cells. The presence of identifiable domains in NOV5e was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). The results indicate that this protein contains the following protein domains (as defined by Interpro) at the indicated positions: low density lipoprotein receptor domains at amino acid positions 45 to 83, 84 to 165, 168 to 206; low density receptor repeat at amino acid ρositions333 to 378, 380 to 416, 418 to 460, 462 to 546; EGF-like domains at amino acid positions 86 tol22, 170 to 204, 211 to 245, 251 to 285, 556 to 600, 739 to 783; and trypsin inhibitor like cysteine rich domain at amino acid positions 195 to 251. This indicates that the sequence ofthe invention has properties similar to those of other proteins known to contain this/these domain(s) and similar to the properties of these domains. NOV5f
A disclosed NOV5f nucleic acid was isolated by subjecting NOV5c to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence ofthe target sequence, or by translated homology ofthe predicted exons to closely related human sequences sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component ofthe assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. The novel NO V5f sequence of 2474 nucleotides (also referred to as CG55256-04) encoding a novel apolipoprotein e-like protein is shown in Table 5K. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 30-32 and ending with a TGA codon at nucleotides 2442-2444. A putative untranslated region upstream from the initiation codon and downsfream from the termination codon is underlined in Table 5K, and the start and stop codons are in bold letters.
Table 5K. NO 5f Nucleotide Sequence (SEQ ID NO:24)
TGTTTTTTTTCTTCAGTACTTTAAATGTCATGCCACTCTCCTGGCCTGTAAGCTTTCCACAGA GAAGCCTGCTGCCAGACCCATTGGAGCTTCTTTGTGTGTTATTTGTTTCTTTTTTCTTGCTGC TTTTACAGCTCCAGCATCTTGCGGCGGCAGCGGCTGATCCGCTGCTCGGCGGCCAAGGGCCGG CCAAGGATTGCGAAAAGGACCAATTCCAGTGCCGGAACGAGCGCTGCATCCCCTCTGTGTGGA GATGCGACGAGGACGATGACTGCTTAGACCACAGCGACGAGGACGACTGCCCTGCAGCCAAGA AGACACCTGTGAGAAGTGACTTCACCTGTGACAACGGCCACTGCATCCACGAACGGTGGAAGT GTGACGGCGAGGAGGAGTGTCCTGATGGCTCCGATGAGTCCGAGGCCACTTGCACCAAGCAGG TGTGTCCTGCAGAGAAGCTGAGCTGTGGACCCACTAGCCACAAGTGTGTACCTGCCTCGTGGC GCTGCGACGGGGAGAAGGACTGCGAGGGTGGAGCGGATGAGGCCGGCTGTGCTACCTCACTGG GCACCTGCCGTGGGGACGAGTTCCAGTGTGGGGATGGGACATGTGTCCTTGCAATCAAGCACT GCAACCAGGAGCAGGACTGTCCAGATGGGAGTGATGAAGCTGGCTGCCTACAGGGGCTGAACG AGTGTCTGCACAACAATGGCGGCTGCTCACACATCTGCACTGACCTCAAGATTGGCTTTGAAT GCACGTGCCCAGCAGGCTTCCAGCTCCTGGACCAGAAGACCTGTGGCGCTGGCAAGAGCCCAT CCCTAATCTTCACCAACCGGCACGAGGTGCGGAGGATCGACCTGGTGAAGCGGAACTATTCAC GCCTCATCCCCATGCTCAAGAATGTCGTGGCACTAGATGTGGAAGTTGCCACCAATCGCATCT ACTGGTGTGACCTCTCCTACCGTAAGATCTATAGCGCCTACATGGACAAGGCCAGTGACCCGA AAGAGCAGGAGGTCCTCATTGACGAGCAGTTGCACTCTCCAGAGGGCCTGGCAGTGGACTGGG TCCACAAGCACATCTACTGGACTGACTCGGGCAATAAGACCATCTCAGTGGCCACAGTTGATG GTGGCCGCCGACGCACTCTCTTCAGCCGTAACCTCAGTGAACCCCGGGCCATCGCTGTTGACC CCCTGCGAGGGTTCATGTATTGGTCTGACTGGGGGGACCAGGCCAAGATTGAGAAATCTGGGC TCAACGGTGTGGACCGGCAAACACTGGTGTCAGACAATATTGAATGGCCCAACGGAATCACCT TGGATCTGCTGAGCCAGCGCTTGTACTGGGTAGACTCCAAGCTACACCAACTGTCCAGCATTG ACTTCAGTGGAGGCAACAGAAAGACGCTGATCTCCTCCACTGACTTCCTGAGCCACCCTTTTG GGATAGCTGTGTTTGAGGACAAGGTGTTCTGGACAGACCTGGAGAACGAGGCCATTTTCAGTG CAAATCGGCTCAATGGCCTGGAAATCTCCATCCTGGCTGAGAACCTGAACAACCCACATGACA TTGTCATCTTCCATGAGCTGAAGCAGCCAAGAGCTCCAGATGCCTGTGAGCTGAGTGTCCAGC CTAATGGAGGCTGTGAATACCTGTGCCTTCCTGCTCCTCAGATCTCCAGCCACTCTCCCAAGT ACACATGTGCCTGTCCTGACACAATGTGGCTGGGTCCAGACATGAAGAGGTGCTACCGAGCAC CTCAATCTACCTCAACTACGACGTTAGCTTCTACCATGACGAGGACAGTACCTGCCACCACAA GAGCCCCCGGGACCACCGTCCACAGATCCACCTACCAGAACCACAGCACAGAGACACCAAGCC TGACAGCTGCAGTCCCAAGCTCAGTTAGTGTCCCCAGGGCTCCCAGCATCAGCCCGTCTACCC TAAGCCCTGCAACCAGCAACCACTCCCAGCACTATGCAAATGAAGACAGTAAGATGGGCTCAA CAGTCACTGCCGCTGTTATCGGGATCATCGTGCCCATAGTGGTGATAGCCCTCCTGTGCATGA GTGGATACCTGATCTGGAGAAACTGGAAGCGGAAGAACACCAAAAGCATGAATTTTGACAACC CAGTCTACAGGAAAACAACAGAAGAAGAAGACGAAGATGAGCTCCATATAGGGAGAACTGCTC AGATTGGCCATGTCTATCCTGCAGCAATCAGCAGCTTTGATCGCCCACTGTGGGCAGAGCCCT GTCTTGGGGAGACCAGAGAACCGGAAGACCCAGCCCCTGCCCTCAAGGAGCTTTTTGTCTTGC CGGGGGAACCAAGGTCACAGCTGCACCAACTCCCGAAGAACCCTCTTTCCGAGCTGCCTGTCG TCAAATCCAAGCGAGTGGCATTAAGCCTTGAAGATGATGGACTACCCTGAGGATGGGATCACC CCCTTCGTGCCTCATGG The NOV5f sequence was identified through an exon linking process. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was. encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5f sequence.
The NOV5f nucleic acid was identified to the p34 region of human chromosome 1 and has 2047 of 2341 bases (87%) identical to a gb:GENBANK-JX>:D50678|acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E = 0.0) A disclosed NOV5f polypeptide (SEQ ID NO:25) encoded by SEQ ID NO:24 is 804 amino acid residues and is presented using the one-letter code in Table 5L. Signal P, Psort and/or Hydropathy results predict that NOV5f has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000. In other embodiments, NOV5f may also be localized to the Golgi body with acertainty of 0.4000, the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the microbody (peroxisome) with a certainty of 0.3000. The most likely cleavage site for a NOV5f peptide is between amino acids 44 and 45, at: AAA-DP.
Table 5L. Encoded NO V5f protein sequence (SEQ ID NO:25)
MPLSWPVSFPQRSLLPDPLELLCVLFVSFFLLLLQLQHLAAAAADPLLGGQGPAKDCEKDQFQCRNERCIPS VWRCDEDDDCLDHSDEDDCPAAKKTPVRSDFTCDNGHCIHERWKCDGEEECPDGSDESEATCTKQVCPAEKL SCGPTSHKCVPASWRCDGEKDCEGGADEAGCATSLGTCRGDEFQCGDGTCVLAIKHCNQEQDCPDGSDEAGC LQGLNECLHNNGGCSHICTDLKIGFECTCPAGFQLLDQKTCGAGKSPSLIFTNRHEVRRIDLVKRNYSRLIP MLKNVVALDVEVATNRIY CDLSYRKIYSAYMDKASDPKEQEVLIDEQLHSPEGLAVDWVHKHIYWTDSGNK TISVATVDGGRRRTLFSRNLSEPRAIAVDPLRGFMYWSDWGDQAKIEKSGLNGVDRQTLVSDNIEWPNGITL DLLSQRLYWVDSKLHQLSSIDFSGGNRKTLISSTDFLSHPFGIAVFEDKVFWTDLENEAIFSANRLNGLEIS ILAENLNNPHDIVIFHELKQPRAPDACELSVQPNGGCEYLCLPAPQISSHSPKYTCACPDTMWLGPDMKRCY RAPQSTSTTTLASTMTRTVPATTRAPGTTVHRSTYQNHSTETPSLTAAVPSSVSVPRAPSISPSTLSPATSN HSQHYANEDSKMGSTVTAAVIGIIVPIWIALLCMSGYLIWRNWKRKNTKSMNFDNPVYRKTTEEEDEDELH IGRTAQIGHVYPAAISSFDRPLWAEPCLGETREPEDPAPALKELFVLPGEPRSQLHQLPKNPLSELPWKSK RVALSLEDDGLP
The disclosed NOV5f amino acid sequence has 716 of 789 amino acid residues (90%) identical to, and 731 of 789 amino acid residues (92%) similar to, the 793 amino acid residue ptnr:SPTREMBL-ACC:O14968 protein from Homo sapiens (Human) (APOER2DELTA4-7) (E = 0.0).
NOV5f is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. This information was derived by determining the tissue sources ofthe sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Literature sources, and/or RACE sources.
In addition, the sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:D50678|acc:D50678.1) a closely related Human mRNA for apolipoprotein E receptor 2, complete eds homolog in species Homo sapiens: brain. The presence of identifiable domains in NOV5f was determined by searches using algorithms such as PROSITE, Blocks, Pfam, ProDomain, Prints and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/interpro/). The results indicate that this protein contains the following protein domains (as defined by Interpro) at the indicated positions: domain name low density lipoprotein receptor domains at amino acid positions 55 to 93, 96 to 177, 180 to 218; low density receptor repeat domains at amino acid positions 303 to 348, 350 to 391, 393 to 435, 437 to 521; EGF-like domains at amino acid positions 182 to 216, 223 to 257, 531 to 575; and fusion glycoprotein FO domain at amino acid positions 650 to 683. This indicates that the sequence ofthe invention has properties similar to those of other proteins known to contain this/these domain(s) and similar to the properties of these domains. NOV5g
A disclosed NOV5g nucleic acid was isolated by The sequence of NOV5g was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence. cDNA fragments covering either the full length ofthe DNA sequence, or part ofthe sequence, or both, were cloned. In silico prediction was based on sequences available in Curagen's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof. The novel NOV5g sequence of 4150 nucleotides (also referred to as CG55256-05) encoding a novel apolipoprotein e-like protein is shown in Table 5M. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 103-105 and ending with a TGA codon at nucleotides 2674-2676. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5M, and the start and stop codons are in bold letters.
Table 5M. NOV5g Nucleotide Sequence (SEQ ID NO:26)
GCAGCGGCAACCCCGGCGCCGCGGCAAGGACTCGGAGGGCTGAGACGCGGCGGCGGCGGCGCG GGGAGCGCGGGGCGCGGCGGCCGGAGCCCCGGGCCCGCCATGGGCCTCCCCGAGGCGGGCCCT CTCCGGCTTCTGGCGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCGGCTCCAGCATCTT GCGGCGGCAGCGGCTGATCCGCTGCTCGGCGGCCAAGGGCCGGCCAAGGAGTGCGAAAAGGAC CAATTCCAGTGCCGGAACGAGCGCTGCATCCCCTCTGTGTGGAGATGCGACGAGGACGATGAC TGCTTAGACCACAGCGACGAGGACGACTGCCCCAAGAAGACCTGTGCAGACAGTGACTTCACC TGTGACAACGGCCACTGCATCCACGAACGGTGGAAGTGTGACGGCGAGGAGGAGTGTCCTGAT GGCTCCGATGAGTCCGAGGCCACTTGCACACTGGGCACCTGCCGTGGGGACGAGTTCCAGTGT GGGGATGGGACATGTGTCCTTGCAATCAAGCACTGCAACCAGGAGCAGGACTGTCCAGATGGG AGTGATGAAGCTGGCTGCCTACAGGAGTCACCCTGTGAGGGTCCCCGCAGATTTCAGTGTAAG AGTGGCAAGCGCGTGGACGGCGGGAAAGTGTGTGATGTGCAGAGGGACTGCCGGGACTGGTCG GATGAGCTTCTGAAAGTGTGGTGCGGTGCCTGTCTACGCCCACTGGCTGGACTCAGTCTCCTA CCATCCCCCTCCTGGAGCCCATGTCCCTCCCCAGGGCTGAACGAGTGTCTGCACAACAATGGC GGCTGCTCACACATCTGCACTGACCTCAAGATTGGCTTTGAATGCACGTGCCCAGCAGGCTTC CAGCTCCTGGACCAGAAGACCTGTGGCGACATTGATGAGTGCAAGGACCCAGATGCCTGCAGC CAGATCTGTGTCAATTACAAGGGCTATTTTAAGTGTGAGTGCTACCCTGGCTACGAGATGGAC CTACTGACCAAGAACTGCAAGGCTGCTGCTGGCAAGAGCCCATCCCTAATCTTCACCAACCGG CACGAGGTGCGGAGGATCGACCTGGTGAAGCGGAACTATTCACGCCTCATCCCCATGCTCAAG AATGTCGTGGCACTAGATGTGGAAGTTGCCACCAATCGCATCTACTGGTGTGACCTCTCCTAC CGTAAGATCTATAGCGCCTACATGGACAAGGCCAGTGACCCGAAAGAGCAGGAGGTCCTCATT GACGAGCAGTTGCACTCTCCAGAGGGCCTGGCAGTGGACTGGGTCCACAAGCACATCTACTGG ACTGACTCGGGCAATAAGACCATCTCAGTGGCCACAGTTGATGGTGGCCGCCGACGCACTCTC TTCAGCCGTAACCTCAGTGAACCCCGGGCCATCGCTGTTGACCCCCTGCGAGGGTTCATGTAT TGGTCTGACTGGGGGGACCAGGCCAAGATTGAGAAATCTGGGCTCAACGGTGTGGACCGGCAA ACACTGGTGTCAGACAATATTGAATGGCCCAACGGAATCACCCTGGATCTGCTGAGCCAGCGC TTGTACTGGGTAGACTCCAAGCTACACCAACTGTCCAGCATTGACTTCAGTGGAGGCAACAGA AAGACGCTGATCTCCTCCACTGACTTCCTGAGCCACCCTTTTGGGATAGCTGTGTTTGAGGAC AAGGTGTTCTGGACAGACCTGGAGAACGAGGCCATTTTCAGTGCAAATCGGCTCAATGGCCTG GAAATCTCCATCCTGGCTGAGAACCTCAACAACCCACATGACATTGTCATCTTCCATGAGCTG AAGCAGCCAAGAGCTCCAGATGCCTGTGAGCTGAGTGTCCAGCCTAATGGAGGCTGTGAATAC CTGTGCCTTCCTGCTCCTCAGATCTCCAGCCACTCTCCCAAGTACACATGTGCCTGTCCTGAC ACAATGTGGCTGGGTCCAGACATGAAGAGGTGCTACCGAGCACCTCAATCTACCTCAACTACG ACGTTAGCTTCTACCATGACGAGGACAGTACCTGCCACCACAAGAGCCCCCGGGACCACCGTC CACAGATCCACCTACCAGAACCACAGCACAGAGACACCAAGCCTGACAGCTGCAGTCCCAAGC TCAGTTAGTGTCCCCAGGGCTCCCAGCATCAGCCCGTCTACCCTAAGCCCTGCAACCAGCAAC CACTCCCAGCACTATGCAAATGAAGACAGTAAGATGGGCTCAACAGTCACTGCCGCTGTTATC GGGATCATCGTGCCCATAGTGGTGATAGCCCTCCTGTGCATGAGTGGATACCTGATCTGGAGA AACTGGAAGCGGAAGAACACCAAAAGCATGAATTTTGACAACCCAGTCTACAGGAAAACAACA GAAGAAGAAGATGAAGATGAGCTCCATATAGGGAGAACTGCTCAGATTGGCCATGTCTATCCT GCAGCAATCAGCAGCTTTGATCGCCCACTGTGGGCAGAGCCCTGTCTTGGGGAGACCAGAGAA CCGGAAGACCCAGCCCCTGCCCTCAAGGAGCTTTTTGTCTTGCCGGGGGAACCAAGGTCACAG CTGCACCAACTCCCGAAGAACCCTCTTTCCGAGCTGCCTGTCGTCAAATCCAAGCGAGTGGCA TTAAGCCTTGAAGATGATGGACTACCCTGAGGATGGGATCACCCCCTTCGTGCCTCATGGAAT TCAGTCCCATGCACTACACTCTGGATGGTGTATGACTGGATGAATGGGTTTCTATATATGGGT CTGTGTGAGTGTATGTGTGTGTGTGATTTTTTTTTTAAATTTATGTTGCGGAAAGGTAACCAC AAAGTTATGATGAACTGCAAACATCCAAAGGATGTGAGAGTTTTTCTATGTATAATGTTTTAT ACACTTTTTAACTGGTTGCAGTACCCATGAGGAATTCGTGGAATGGCTACTGCTGACTAACAT GATGCACATAACCAAATGGGGGCCAATGGCACAGTACCTTACTCATCATTTAAAAACTATATT TACAGAAGATGTTTGGTTGCTGGGGGGGCTTTTTTGGGTTTTGGGGCATTTGTTTTTTGTAAA TAAGATGATTATGCTTTGTGGCTATCCATCAACATAAGTAAAAAAAAAAAAAAAACACTTCAA CTCCCTCCCCCATTTAGATTATTTATTAACATATTTTAAAAATCAGATGAGTTCTATAAATAA TTTAGAGAAGTGAGAGTATTTATTTTTGGCATGTTTGGCCCACCACACAGACTCTGTGTGTGT ATGTGTGTGTTTATATGTGTATGTGTGTGACAGGAAAATCTGTAGAGAAGAGGCACATCTATG GCTACTGTTCAAATACATAAAGATAAATTTATTTTCACACAGTCCACAAGGGGTATATCTTGT AGTTTTCAGAAAAGCCTTTGGAAATCTGGATCAGGAAATAGATACCATGGTTTGTGCAATTAT GTAGTAAAAAAGGCAAATCTTTTCACCTCTGGCTATTCCTGAGACCCCAGGAAGTCAGGAAAA GCCTTTCAGCTCACCCATGGCTGCTGTGACTCCTACCAGGGCTTTCTTGGCTTTGGCGAAGGT CAGTGTACAGACATTCCATGGTACCAGAGTGCTCAGAAAGTCAAGATAGGATATGCCTCACCC TCAGCTACTCCTTGTTTTAAAGTTCAGCTCTTTGAGTAACTTCTTCAATTTCTTTCAGGACAC TTGGGTTGAATTCAGTAAGTTTCCTCTGAAGCACCCTGAAGGGTGCCATCCTTACAGAGCTAA GTGGAGACGTTTCCAGATCAGCCCAAGTTTACTATAGAGACTGGCCCAGGCACTGAATGTCTA GGACATGCTGTGGATGAAGATAAAGATGGTGGAATAGGTTTTATCACATCTCTTATTTCTCTT TTCCCCTTACTCTCTACCATTTCCTTTATGTGGGGAAACATTTTAAGGTAATAAATAGGTTAC TTACCATCATATGTTCATATAGATGAΑACTAATTTTTGGCTTAAGTCAGAACAACTGGCCCCC AATTGAAGTCATATTTGTGGGGGGAAATGGCATACGCAATATTATATTATATTGGATATTTAT GTTCACACAGGAATTTGGTTTACTGCTTTGTAAATAAAAGGGAAAACTCCGGGTA
The NOV5g sequence was identified through an exon linking process. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library contaimng a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5g sequence.
The NOV5g nucleic acid was identified to the p34 region of human chromosome 1 and has 3358 of 3361 bases (99%) identical to a gb:GENBANK-ID:D50678|acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E = 0.0) A disclosed NOV5g polypeptide (SEQ ID NO:27) encoded by SEQ ID NO:26 is 857 amino acid residues and is presented using the one-letter code in Table 5N. Signal P, Psort and/or Hydropathy results predict that NOV5g has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760. i other embodiments, NOV5g may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or exfracellularly with a certainty of 0.1000. The most likely cleavage site for a NOV5g peptide is between amino acids 32 and 33, at: AAA-DP.
Table 5N. Encoded NOV5g protein sequence (SEQ ID NO:27)
MGLPEPGPLRLLALLLLLLLLLLLRLQHLAAAAADPLLGGQGPAKECEKDQFQCRNERCIPSVWRCDEDDDC LDHSDEDDCPKKTCADSDFTCDNGHCIHERWKCDGEEECPDGSDESEATCTLGTCRGDEFQCGDGTCVLAIK HCNQEQDCPDGSDEAGCLQESPCEGPRRFQCKSGKRVDGGKVCDVQRDCRDWSDELLKVWCGACLRPLAGLS LLPSPSWSPCPSPGLNECLHNNGGCSHICTDLKIGFECTCPAGFQLLDQKTCGDIDECKDPDACSQICVNYK GYFKCECYPGYEMDLLTK-NrCKAAAGKSPSLIFTNRHEVRRIDLVKRNYSRLIPMLKNVVALDVEVATNRIYW CDLSYRKIYSAYMDKASDPKEQEVLIDΞQLHSPEGLAVDWVHKHIYWTDSGNKTISVATVDGGRRRTLFSRN LSEPRAIAVDPLRGFMYWSDWGDQAKIEKSGLNGVDRQTLVSDNIEWPNGITLDLLSQRLYWVDSKLHQLSS IDFSGGNRKTLISSTDFLSHPFGIAVFEDKVFWTDLENEAIFSANRLNGLEISILAENLNNPHDIVIFHELK QPRAPDACELSVQPNGGCEYLCLPAPQISSHSPKYTCACPDTMWLGPDMKRCYRAPQSTSTTTLASTMTRTV PATTRAPGTTVHRSTYQNHSTETPSLTAAVPSSVSVPRAPSISPSTLSPATSNHSQHYANEDSKMGSTVTAA VIGIIVPIWIALLCMSGYLIWRNWKRKNTKSMNFDNPVYRKTTEEEDEDELHIGRTAQIGHVYPAAISSFD RPLWAEPCLGETREPEDPAPALKELFVLPGEPRSQLHQLPKNPLSELPWKSKRVALSLEDDGLP
The disclosed NOV5g amino acid sequence has 684 of 816 amino acid residues (83%) identical to, and 703 of 816 amino acid residues (86%) similar to, the 963 amino acid residue ρtnr:SPTREMBL-ACC:Q14114 protein from Homo sapiens (Human) (APOLΓPOPROTEIN E RECEPTOR 2 PRECURSOR) (E = 0.0).
NOV5g is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus, Cerebral Medulla/Cerebral white matter, Colon, Lung, Lymphoid tissue, Parietal Lobe, Tonsils, Whole Organism. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOV5g.
The presence of identifiable domains in NOV5g was determined by searches versus domain databases such as Pfam, PROSITE, ProDom, Blocks or Prints and then identified by the Interpro domain accession number. Significant domains are summarized below.
IPR000033, YWTD repeat, from amino acid 356 to amino acid 401, from amino acid 403 to amino acid 444, from amino acid 446 to amino acid 488, from amino acid 490 to amino acid 533, from amino acid 534 to amino acid 574. This domain is also known as the YWTD motif after the most conserved region ofthe repeat. The YWTD repeat is found in multiple tandem repeats and has been predicted to form a beta-propeller structure. It is found in a variety of proteins that include, vitellogenin receptor from Drosophila, low-density lipoprotein (LDL) receptor, preproepidermal growth factor, nidogen (entactin) and others.
IPR002172, Low density lipoprotein (LDL)-receptor class A (LDLRA) domain, from amino acid 45 to amino acid 83, from amino acid 84 to amino acid 124, from amino acid 125 to amino acid 163, from amino acid 165 to amino acid 207. Low density lipoprotein (LDL) is the major cholesterol-carrying lipoprotein of plasma. The receptor protein binds LDL and transports it into cells by endocytosis. In order to be internalised, the receptor-ligand complex must first cluster into clathrin-coated pits. Seven successive cysteine-rich repeats of about 40 amino acids are present in the N-terminal of this multidomain membrane protein. The LDL- receptor class A domain contains 6 disulfide-bound cysteines and a highly conserved cluster of negatively charged amino acids, of which many are clustered on one face ofthe module. In LDL-receptors the class A domains form the binding site for LDL and calcium. The acidic residues between the fourth and sixth cysteines are important for high-affinity binding of positively charged sequences in LDLR's ligands. The repeat has been shown to consist of a beta-hairpin structure followed by a series of beta turns. The binding of calcium seems to induce no significant conformational change. Following these repeats is a 350 residue domain that resembles part ofthe epidermal growth factor (EGF) precursor. Similar domains have been found in several extracellular and membrane proteins.
IPR000561, EGF-like domain, from amino acid 234 to amino acid 268, from amino acid 274 to amino acid 308 A sequence of about thirty to forty amino-acid residues long found in the sequence of epidermal growth factor (EGF) has been shown to be present, in a more or less conserved form, in a large number of other, mostly animal proteins. The list of proteins currently known to contain one or more copies of an EGF-like pattern is large and varied. The functional significance of EGF domains in what appear to be unrelated proteins is not yet clear. However, a common feature is that these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G H synthase). The EGF domain includes six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds. The main structure is a two-stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet. Subdomains between the conserved cysteines vary in length. NOV5h A disclosed NOV5h nucleic acid was isolated by The sequence of NOV5h was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence. cDNA fragments covering either the full length ofthe DNA sequence, or part ofthe sequence, or both, were cloned. In silico prediction was based on sequences available in Curagen's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof. The novel NOV5h sequence of 4180 nucleotides (also refereed to as CG55256-06) encoding a novel apolipoprotein e-like protein is shown in Table 5O. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 163-165 and ending with a TGA codon at nucleotides 2704-2706. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 5O, and the start and stop codons are in bold letters.
Table 5O. NOV5h Nucleotide Sequence (SEQ ID NO:28)
ATGGCCTCCCCGAACGTATGGGCCTCCCCGAGCGTATGGGCCTCCCCGAGCGTATGGGCCTCC CCGAGCGTATGGGCCTCACCCGAGCGTATGGGCCTCCCCGAGCGTATGGGCCTCCCCGAGCGT ATGGGCCTCCCCGAGCGTATGGGCCTCCCCGAGCGTATGGGCCTCCCCGAGCCGGGCCCTCTC CGGCTTCTGGCGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCAGCTCCAGCATCTTGCG GCGGCAGCGGCTGATCCGCTGCTCGGCGGCCAAGGGCCGGCCAAGGATTGCGAAAAGGACCAA TTCCAGTGCCGGAACGAGCGCTGCATCCCCTCTGTGTGGAGATGCGACGAGGACGATGACTGC TTAGACCACAGCGACGAGGACGACTGCCCCAAGAAGACCTGTGCAGACAGTGACTTCACCTGT GACAACGGCCACTGCATCCACGAACGGTGGAAGTGTGACGGCGAGGAGGAGTGTCCTGATGGC TCCGATGAGTCCGAGGCCACTTGCACCAAGCAGGTGTGTCCTGCAGAGAAGCTGAGCTGTGGA CCCACCAGCCACAAGTGTGTACCTGCCTCGTGGCGCTGCGACGGGGAGAAGGACTGCGAGGGT GGAGCGGATGAGGCCGGCTGTGCTACCTCACTGGGCACCTGCCGTGGGGACGAGTTCCAGTGT GGGGATGGGACATGTGTCCTTGCAATCAAGCACTGCAACCAGGAGCAGGACTGTCCAGATGGG AGTGATGAAGCTGGCTGCCTACAGGTTCCGCCAACATTCCTGGGAAACAGGAGGAGGCCCAGG GGGCTGAACGAGTGTCTGCACAACAATGGCGGCTGCTCACACATCTGCACTGACCTCAAGATT GGCTTTGAATGCACGTGCCCAGCAGGCTTCCAGCTCCTGGACCAGAAGACCTGTGGCGACATT GATGAGTGCAAGGACCCAGATGCCTGCAGCCAGATCTGTGTCAATTACAAGGGCTATTTTAAG TGTGAGTGCTACCCTGGCTACGAGATGGACCTACTGACCAAGAACTGCAAGGCTGCTGCTGGA AAGAGCCCATCCCTAATCTTCACCAACCGGCACGAGGTGCGGAGGATCGACCTGGTGAAGCGG AACTATTCACGCCTCATCCCCATGCTCAAGAATGTCGTGGCACTAGATGTGGAAGTTGCCACC AATCGCATCTACTGGTGTGACCTCTCCTACCGTAAGATCTATAGCGCCTACATGGACAAGGCC AGTGACCCGAAAGAGCAGGAGGTCCTCATTGACGAGCAGTTGCACTCTCCAGAGGGCCTGGCA GTGGACTGGGTCCACAAGCACATCTACTGGACTGACTCGGGCAATAAGACCATCTCAGTGGCC ACAGTTGATGGTGGCCGCCGACGCACTCTCTTCAGCCGTAACCTCAGTGAACCCCGGGCCATC GCTGTTGACCCCCTGCGAGGGTTCATGTATTGGTCTGACTGGGGGGACCAGGCCAAGATTGAG AAATCTGGGCTCAACGGTGTGGACCGGCAAACACTGGTGTCAGACAATATTGAATGGCCCAAC GGAATCACCCTGGATCTGCTGAGCCAGCGCTTGTACTGGGTAGACTCCAAGCTACACCAACTG TCCAGCATTGACTTCAGTGGAGGCAACAGAAAGACGCTGATCTCCTCCACTGACTTCCTGAGC CACCCTTTTGGGATAGCTGTGTTTGAGGACAAGGTGTTCTGGACAGACCTGGAGAACGAGGCC ATTTTCAGTGCAAATCGGCTCAATGGCCTGGAAATCTCCATCCTGGCTGAGAACCTCAACAAC CCACATGACATTGTCATCTTCCATGAGCTGAAGCAGCCAAGAGCTCCAGATGCCTGTGAGCTG AGTGTCCAGCCTAATGGAGGCTGTGAATACCTGTGCCTTCCTGCTCCTCAGATCTCCAGCCAC TCTCCCAAGTACACATGTGCCTGTCCTGACACAATGTGGCTGGGTCCAGACATGAAGAGGTGC TACCGAGCACCTCAATCTACCTCAACTACGACGTTAGCTTCTACCATGACGAGGACAGTACCT GCCACCACAAGAGCCCCCGGGACCACCGTCCACAGATCCACCTACCAGAACCACAGCACAGAG ACACCAAGCCTGACAGCTGCAGTCCCAAGCTCAGTTAGTGTCCCCAGGGCTCCCAGCATCAGC CCGTCTACCCTAAGCCCTGCAACCAGCAACCACTCCCAGCACTATGCAAATGAAGACAGTAAG ATGGGCTCAACAGTCACTGCCGCTGTTATCGGGATCATCGTGCCCATAGTGGTGATAGCCCTC CTGTGCATGAGTGGATACCTGATCTGGAGAAACTGGAAGCGGAAGAACACCAAAAGCATGAAT TTTGACAACCCAGTCTACAGGAAAACAACAGAAGAAGAAGATGAAGATGAGCTCCATATAGGG AGAACTGCTCAGATTGGCCATGTCTATCCTGCAGCAATCAGCAGCTTTGATCGCCCACTGTGG GCAGAGCCCTGTCTTGGGGAGACCAGAGAACCGGAAGACCCAGCCCCTGCCCTCAAGGAGCTT TTTGTCTTGCCGGGGGAACCAAGGTCACAGCTGCACCAACTCCCGAAGAACCCTCTTTCCGAG CTGCCTGTCGTCAAATCCAAGCGAGTGGCATTAAGCCTTGAAGATGATGGACTACCCTGAGGA TGGGATCACCCCCTTCGTGCCTCATGGAATTCAGTCCCATGCACTACACTCTGGATGGTGTAT GACTGGATGAATGGGTTTCTATATATGGGTCTGTGTGAGTGTATGTGTGTGTGTGATTTTTTT TTTAAATTTATGTTGCGGAAAGGTAACCACAAAGTTATGATGAACTGCAAACATCCAAAGGAT GTGAGAGTTTTTCTATGTATAATGTTTTATACACTTTTTAACTGGTTGCACTACCCATGAGGA ATTCGTGGAATGGCTACTGCTGACTAACATGATGCACATAACCAAATGGGGGCCAATGGCACA GTACCTTACTCATCATTTAAAAACTATATTTACAGAAGATGTTTGGTTGCTGGGGGGGCTTTT TTGGGTTTTGGGGCATTTGTTTTTTGTAAATAAGATGATTATGCTTTGTGGCTATCCATCAAC ATAAGTAAAAAAAAAAAAAAAACACTTCAACTCCCTCCCCCATTTAGATTATTTATTAACATA TTTTAAAAATCAGATGAGTTCTATAAATAATTTAGAGAAGTGAGAGTATTTATTTTTGGCATG TTTGGCCCACCACACAGACTCTGTGTGTGTATGTGTGTGTTTATATGTGTATGTGTGTGACAG GAAAATCTGTAGAGAAGAGGCACATCTATGGCTACTGTTCAAATACATAAAGATAAATTTATT TTCACACAGTCCACAAGGGGTATATCTTGTAGTTTTCAGAAAAGCCTTTGGAAATCTGGATCA GGAAATAGATACCATGGTTTGTGCAATTATGTAGTAAAAAAGGCAAATCTTTTCACCTCTGGC TATTCCTGAGACCCCAGGAAGTCAGGAAAAGCCTTTCAGCTCACCCATGGCTGCTGTGACTCC TACCAGGGCTTTCTTGGCTTTGGCGAAGGTCAGTGTACAGACATTCCATGGTACCAGAGTGCT CAGAAAGTCAAGATAGGATATGCCTCACCCTCAGCTACTCCTTGTTTTAAAGTTCAGCTCTTT GAGTAACTTCTTCAATTTCTTTCAGGACACTTGGGTTGAATTCAGTAAGTTTCCTCTGAAGCA CCCTGAAGGGTGCCATCCTTACAGAGCTAAGTGGAGACGTTTCCAGATCAGCCCAAGTTTACT ATAGAGACTGGCCCAGGCACTGAATGTCTAGGACATGCTGTGGATGAAGATAAAGATGGTGGA ATAGGTTTTATCACATCTCTTATTTCTCTTTTCCCCTTACTCTCTACCATTTCCTTTATGTGG GGAAACATTTTAAGGTAATAAATAGGTTACTTACCATCATATGTTCATATAGATGAAACTAAT TTTTGGCTTAAGTCAGAACAACTGGCCCCCAATTGAAGTCATATTTGTGGGGGGAAATGGCAT ACGCAATATTATATTATATTGGATATTTATGTTCACACAGGAATTTGGTTTACTGCTTTGTAA ATAAAAGGGAAAACTCCGGGTA
The NOV5h'sequence was identified through. an exon linking process. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5h sequence.
The NOV5h nucleic acid was identified to the p34 region of human chromosome 1 and has 3634 of 3851 bases (94%) identical to a gb:GENBANK-ID:D50678|acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E = 0.0) A disclosed NOV5h polypeptide (SEQ ID NO:29) encoded by SEQ ID NO:28 is 847 amino acid residues and is presented using the one-letter code in Table 5P. Signal P, Psort and/or Hydropathy results predict that NOV5h has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760. In other embodiments, NOV5h may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or exfracellularly with a certainty of 0.1000. The most likely cleavage site for a NOV5h peptide is between amino acids 32 and 33, at: AAA-AA.
Table 5P. Encoded NOV5h protein sequence (SEQ ID NO:29)
MGLPEPGPLRLLALLLLLLLLLLLQLQHLAAAAADPLLGGQGPAKDCEKDQFQCRNERCIPSVWRCDEDDDC LDHSDEDDCPKKTCADSDFTCDNGHCIHERWKCDGEEECPDGSDESEATCTKQVCPAEKLSCGPTSHKCVPA SWRCDGEKDCEGGADEAGCATSLGTCRGDEFQCGDGTCVLAIKHCNQEQDCPDGSDEAGCLQVPPTFLGNRR RPRGLNECLHNNGGCSHICTDLKIGFECTCPAGFQLLDQKTCGDIDECKDPDACSQICVNYKGYFKCECYPG YEMDLLTKNC-AAAGKSPSLIFTNRHEVRRIDLVKRNYSRLIPMLKNVVALDVEVATNRIYWCDLSYRKIYS AYMDKASDPKEQEVLIDEQLHSPEGLAVDWVH-<SIYWTDSGNKTISVATVDGGRRRTLFSRNLSEPRAIAVD PLRGFMYWSDWGDQAKIEKSGLNGVDRQTLVSDNIEWPNGITLDLLSQRLYWVDSKLHQLSSIDFSGGNRKT LISSTDFLSHPFGIAVFEDKVFWTDLENEAIFSANRLNGLEISILAENLNNPHDIVIFHELKQPRAPDACEL SVQPNGGCEYLCLPAPQISSHSPKYTCACPDTMWLGPDMKRCYRAPQSTSTTTLASTMTRTVPATTRAPGTT VHRSTYQNHSTETPSLTAAVPSSVSVPRAPSISPSTLSPATSNHSQHYANEDSKMGSTVTAAVIGIIVPIW IALLCMSGYLI RNWKRKNTKSMNFDNPVYRKTTEEEDEDELHIGRTAQIGHVYPAAISSFDRPLWAEPCLG ETREPEDPAPALKELFVLPGEPRSQLHQLPKNPLSELPWKSKRVALSLEDDGLP
The disclosed NOV5h amino acid sequence has 719 of 806 amino acid residues (89%) identical to, and 735 of 806 amino acid residues (91%) similar to, the 963 amino acid residue ρtnr:SPTREMBL-ACC:Q14114 protein from Homo sapiens (Human) (APOLIPOPROTEIN E RECEPTOR 2 PRECURSOR) (E = 0.0).
NOV5h is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus, Cerebral Medulla/Cerebral white matter, Colon, Lung, Lymphoid tissue, Parietal Lobe, Tonsils, Whole Organism. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOV5h
The sequence is predicted to be expressed in placenta because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:D50678|acc:D50678.1) a closely related Human mRNA for apolipoprotein E receptor 2, complete eds homolog in species Homo sapiens.
The presence of identifiable domains in NOV5h was determined by searches versus domain databases such as Pfam, PROSITE, ProDom, Blocks or Prints and then identified by the Interpro domain accession number. Significant domains are summarized below. IPR002172, Low density lipoprotein (LDL)-receptor class A (LDLRA) domain, from amino acid 45 to amino acid 83, from amino acid 84 to amino acid 124, from amino acid 125 to amino acid 165, from amino acid 168 to amino acid 206. Low density lipoprotein (LDL) is the major cholesterol-carrying lipoprotein of plasma. The receptor protein binds LDL and transports it into cells by endocytosis. In order to be internalised, the receptor-ligand complex must first cluster into clathrin-coated pits. Seven successive cysteine-rich repeats of about 40 amino acids are present in the N-terminal of this multidomain membrane protein. The LDL- receptor class A domain contains 6 disulfide-bound cysteines and a highly conserved cluster of negatively charged amino acids, of which many are clustered on one face ofthe module. In LDL-receptors the class A domains form the binding site for LDL and calcium. The acidic residues between the fourth and sixth cysteines are important for high-affinity binding of positively charged sequences in LDLR's ligands. The repeat has been shown to consist of a beta-hairpin structure followed by a series of beta turns. The binding of calcium seems to induce no significant confonnational change. Following these repeats is a 350 residue domain that resembles part ofthe epidermal growth factor (EGF) precursor. Similar domains have been found in several extracellular and membrane proteins.
TPR000033, YWTD repeat, from amino acid 346 to amino acid 391, from amino acid 393 to amino acid 434, from amino acid 436 to amino acid 478, from amino acid 480 to amino acid 523, from amino acid 524 to amino acid 564. This domain is also known as the YWTD motif after the most conserved region ofthe repeat. The YWTD repeat is found in multiple tandem repeats and has been predicted to form a beta-propeller structure. It is found in a variety of proteins that include, vitellogenin receptor from Drosophila, low-density lipoprotein (LDL) receptor, preproepidermal growth factor, nidogen (entactin) and others.
IPR000561, EGF-like domain, from amino acid 224 to amino acid 258, from amino acid 264 to amino acid 298, from amino acid 574 to amino acid 618. A sequence of about thirty to forty amino-acid residues long found in the sequence of epidermal growth factor (EGF) has been shown to be present, in a more or less conserved form, in a large number of other, mostly animal proteins. The list of proteins currently known to contain one or more copies of an EGF-like pattern is large and varied. The functional significance of EGF domains in what appear to be unrelated proteins is not yet clear. However, a common feature is that these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G/H synthase). The EGF domain includes six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds. The main structure is a two-stranded beta-sheet followed by a loop to a C-terminal short two- stranded sheet. Subdomains between the conserved cysteines vary in length.
This indicates that the sequence ofthe invention has properties similar to those of other proteins known to contain this/these domain(s) and similar to the properties of these domains. NOV5i A disclosed NOV5i nucleic acid was isolated by The sequence of NOV5i was derived by laboratory cloning of cDNA fragments, by in silico prediction ofthe sequence. cDNA fragments covering either the full length ofthe DNA sequence, or part ofthe sequence, or both, were cloned. In silico prediction was based on sequences available in Curagen's proprietary sequence databases or in the public human sequence databases, and provided either the full length DNA sequence, or some portion thereof. The novel NOV5i sequence of 4294 nucleotides (also referred to as CG55256-07) encoding a novel apolipoprotein e-like protein is shown in Table 5Q. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 103-105 and ending with a TGA codon at nucleotides 2818-2820. A putative untranslated region upsfream from the initiation codon and downstream from the termination codon is underlined in Table 5Q, and the start and stop codons are in bold letters.
Table 5Q. NOV5i Nucleotide Sequence (SEQ ID NO:30)
GCAGCGGCAACCCCGGCGCCGCGGCAAGGACTCGGAGGGCTGAGACGCGGCGGCGGCGGCGCG GGGAGCGCGGGGCGCGGCGGCCGGAGCCCCGGGCCCGCCATGGGCCTCCCCGAGCCGGGCCCT CTCCGGCTTCTGGCGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCGGCTCCAGCATCTT GCGGCGGCAGCGGCTGATCCGCTGCTCGGCGGCCAAGGGCCGGCCAAGGAGTGCGAAAAGGAC CAATTCCAGTGCCGGAACGAGCGCTGCATCCCCTCTGTGTGGAGATGCGACGAGGACGATGAC TGCTTAGACCACAGCGACGAGGACGACTGCCCCAAGAAGACCTGTGCAGACAGTGACTTCACC TGTGACAACGGCCACTGCATCCACGAACGGTGGAAGTGTGACGGCGAGGAGGAGTGTCCTGAT GGCTCCGATGAGTCCGAGGCCACTTGCACACTGGGCACCTGCCGTGGGGACGAGTTCCAGTGT GGGGATGGGACATGTGTCCTTGCAATCAAGCACTGCAACCAGGAGCAGGACTGTCCAGATGGG AGTGATGAAGCTGGCTGCCTACAGGAGTCACCCTGTGAGGGTCCCCGCAGATTTCAGTGTAAG AGTGGCAAGCGCGTGGACGGCGGGAAAGTGTGTGATGTGCAGAGGGACTGCCGGGACTGGTCG GATGAGCTTCTGAAAGTGTGGTGCGGTGCCTGTCTACGCCCACTGGCTGGACTCAGTCTCCTA CCATCCCCCTCCTGGTATCTAGGCTCAAGGCCCTCCAGTGCCCCCTGCCCTGACACTTTCTGC TCTGACCCTCTCTTTGGATTCATGTGCCGTCCTATGGCTTCACATGGGGCTTTTCGCCCCCAG GCCTCTGGTCTACATCTCTACAAAGTGCTAAGAGCTTGTCCATCCCAGAGGAGGCTGAACGAG TGTCTGCACAACAATGGCGGCTGCTCACACATCTGCACTGACCTCAAGATTGGCTTTGAATGC ACGTGCCCAGCAGGCTTCCAGCTCCTGGACCAGAAGACCTGTGGCGACATTGATGAGTGCAAG GACCCAGATGCCTGCAGCCAGATCTGTGTCAATTACAAGGGCTATTTTAAGTGTGAGTGCTAC CCTGGCTACGAGATGGACCTACTGACCAAGAACTGCCAAGCTGCTGCTGGAAAGAGCCCATCC CTAATCTTCACCAACCGGTACGAGGTGCGGAGGATCGACCTGGTGAAGCGGAACTATTCACGC CTCATCCCCATGCTCAAGAATGTCGTGGCACTAGATGTGGAAGTTGCCACCAATCGCATCTAC TGGTGTGACCTCTCCTACCGTAAGATCTATAGCGCCTACATGGACAAGGCCAGTGACCCGAAA GAGCAGGAGGTCCTCATTGACGAGCAGTTGCACTCTCCAGAGGGCCTGGCAGTGGACTGGGTC CACAAGCACATCTACTGGACTGACTCGGGCAATAAGACCATCTCAGTGGCCACAGTTGATGGT GGCCGCCGACGCACTCTCTTCAGCCGTAACCTCAGTGAACCCCGGGCCATCGCTGTTGACCCC CTGCGAGGGTTCATGTATTGGTCTGACTGGGGGGACCAGGCCAAGATTGAGAAATCTGGGCTC AACGGTGTGGACCGGCAAACACTGGTGTCAGACAATATTGAATGGCCCAACGGAATCACCCTG GATCTGCTGAGCCAGCGCTTGTACTGGGTAGACTCCAAGCTACACCAACTGTCCAGCATTGAC TTCAGTGGAGGCAACAGAAAGACGCTGATCTCCTCCACTGACTTCCTGAGCCACCCTTTTGGG ATAGCTGTGTTTGAGGACAAGGTGTTCTGGACAGACCTGGAGAACGAGGCCATTTTCAGTGCA AATCGGCTCAATGGCCTGGAAATCTCCATCCTGGCTGAGAACCTCAACAACCCACATGACATT GTCATCTTCCATGAGCTGAAGCAGCCAAGAGCTCCAGATGCCTGTGAGCTGAGTGTCCAGCCT AATGGAGGCTGTGAATACCTGTGCCTTCCTGCTCCTCAGATCTCCAGCCACTCTCCCAAGTAC ACATGTGCCTGTCCTGACACAATGTGGCTGGGTCCAGACATGAAGAGGTGCTACCGAGCACCT CAATCTACCTCAACTACGACGTTAGCTTCTACCATGACGAGGACAGTACCTGCCACCACAAGA GCCCCCGGGACCACCGTCCACAGATCCACCTACCAGAACCACAGCACAGAGACACCAAGCCTG ACAGCTGCAGTCCCAAGCTCAGTTAGTGTCCCCAGGGCTCCCAGCATCAGCCCGTCTACCCTA AGCCCTGCAACCAGCAACCACTCCCAGCACTATGCAAATGAAGACAGTAAGATGGGCTCAACA GTCACTGCCGCTGTTATCGGGATCATCGTGCCCATAGTGGTGATAGCCCTCCTGTGCATGAGT GGATACCTGATCTGGAGAAACTGGAAGCGGAAGAACACCAAAAGCATGAATTTTGACAACCCA GTCTACAGGAAAACAACAGAAGAAGAAGATGAAGATGAGCTCCATATAGGGAGAACTGCTCAG ATTGGCCATGTCTATCCTGCAGCAATCAGCAGCTTTGATCGCCCACTGTGGGCAGAGCCCTGT CTTGGGGAGACCAGAGAACCGGAAGACCCAGCCCCTGCCCTCAAGGAGCTTTTTGTCTTGCCG GGGGAACCAAGGTCACAGCTGCACCAACTCCCGAAGAACCCTCTTTCCGAGCTGCCTGTCGTC AAATCCAAGCGAGTGGCATTAAGCCTTGAAGATGATGGACTACCCTGAGGATGGGATCACCCC CTTCGTGCCTCATGGAATTCAGTCCCATGCACTACACTCTGGATGGTGTATGACTGGATGAAT GGGTTTCTATATATGGGTCTGTGTGAGTGTATGTGTGTGTGTGATTTTTTTTTTAAATTTATG TTGCGGAAAGGTAACCACAAAGTTATGATGAACTGCAAACATCCAAAGGATGTGAGAGTTTTT CTATGTATAATGTTTTATACACTTTTTAACTGGTTGCACTACCCATGAGGAATTCGTGGAATG GCTACTGCTGACTAACATGATGCACATAACCAAATGGGGGCCAATGGCACAGTACCTTACTCA TCATTTAAAAACTATATTTACAGAAGATGTTTGGTTGCTGGGGGGGCTTTTTTGGGTTTTGGG GCATTTGTTTTTTGTAAATAAGATGATTATGCTTTGTGGCTATCCATCAACATAAGTAAAAAA AAAAAAAAAACACTTCAACTCCCTCCCCCATTTAGATTATTTATTAACATATTTTAAAAATCA GATGAGTTCTATAAATAATTTAGAGAAGTGAGAGTATTTATTTTTGGCATGTTTGGCCCACCA CACAGACTCTGTGTGTGTATGTGTGTGTTTATATGTGTATGTGTGTGACAGGAAAATCTGTAG AGAAGAGGCACATCTATGGCTACTGTTCAAATACATAAAGATAAATTTATTTTCACACAGTCC ACAAGGGGTATATCTTGTAGTTTTCAGAAAAGCCTTTGGAAATCTGGATCAGGAAATAGATAC CATGGTTTGTGCAATTATGTAGTAAAAAAGGCAAATCTTTTCACCTCTGGCTATTCCTGAGAC CCCAGGAAGTCAGGAAAAGCCTTTCAGCTCACCCATGGCTGCTGTGACTCCTACCAGGGCTTT CTTGGCTTTGGCGAAGGTCAGTGTACAGACATTCCATGGTACCAGAGTGCTCAGAAAGTCAAG ATAGGATATGCCTCACCCTCAGCTACTCCTTGTTTTAAAGTTCAGCTCTTTGAGTAACTTCTT CAATTTCTTTCAGGACACTTGGGTTGAATTCAGTAAGTTTCCTCTGAAGCACCCTGAAGGGTG CCATCCTTACAGAGCTAAGTGGAGACGTTTCCAGATCAGCCCAAGTTTACTATAGAGACTGGC CCAGGCACTGAATGTCTAGGACATGCTGTGGATGAAGATAAAGATGGTGGAATAGGTTTTATC ACATCTCTTATTTCTCTTTTCCCCTTACTCTCTACCATTTCCTTTATGTGGGGAAACATTTTA AGGTAATAAATAGGTTACTTACCATCATATGTTCATATAGATGAAACTAATTTTTGGCTTAAG TCAGAACAACTGGCCCCCAATTGAAGTCATATTTGTGGGGGGAAATGGCATACGCAATATTAT ATTATATTGGATATTTATGTTCACACAGGAATTTGGTTTACTGCTTTGTAAATAAAAGGGAAA ACTCCGGGTA
The NOV5i sequence was identified through an exon linking process. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such suitable sequences were then employed as the forward and reverse primers in a PCR amplification based on a library containing a wide range of cDNA species. The resulting amplicon was gel purified, cloned and sequenced to high redundancy to provide the NOV5i sequence.
The NOV5i nucleic acid was identified to the p34 region of human chromosome 1 and has 3375 of 3389 bases (99%) identical to a gb:GENBANK-ID:D50678|acc:D50678.1 mRNA from Homo sapiens (Human mRNA for apolipoprotein E receptor 2, complete eds) (E = 0.0) A disclosed NOV5i polypeptide (SEQ ID NO:31) encoded by SEQ ID NO:30 is 905 amino acid residues and is presented using the one-letter code in Table 5R. Signal P, Psort and/or Hydropathy results predict that NOV5i has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6760. In other embodiments, NOV5i may also be localized to the endoplasmic reticulum (membrane) with acertainty of 0.1000, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or extracellularly with a certainty of 0.1000. The most likely cleavage site for a NOV5i peptide is between amino acids 32 and 33, at: AAA-AA. Table 5R. Encoded NOV5i protein sequence (SEQ ID NO:31)
MGLPEPGPLRLLALLLLLLLLLLLRLQHLAAAAADPLLGGQGPAKECEKDQFQCRNERCIPSVWRCDEDDDC LDHSDEDDCPKKTCADSDFTCDNGHCIHERWKCDGEEECPDGSDESEATCTLGTCRGDEFQCGDGTCVLAIK HCNQEQDCPDGSDEAGCLQESPCEGPRRFQCKSGKRVDGGKVCDVQRDCRDWSDELLKVWCGACLRPLAGLS LLPSPSWYLGSRPSSAPCPDTFCSDPLFGFMCRPMASHGAFRPQASGLHLYKVLRACPSQRRLNECLHNNGG CSHICTDLKIGFECTCPAGFQLLDQKTCGDIDECKDPDACSQICVNYKGYFKCECYPGYEMDLLTKNCQAAA GKSPSLIFTNRYEVRRIDLVKKNYSRLIPMLKNVVALDVEVATNRIYWCDLSYRKIYSAYMDKASDPKEQEV LIDEQLHSPEGLAVDWVHKHIYWTDSGNKTISVATVDGGRRRTLFSRNLSEPRAIAVDPLRGFMYWSDWGDQ AKIEKSGLNGVDRQTLVSDNIEWPNGITLDLLSQRLYWVDSKLHQLSSIDFSGGNRKTLISSTDFLSHPFGI AVFEDKVFWTDLENEAIFSANRLNGLEISI AENLNNPHDIVIFHELKQPRAPDACELSVQPNGGCEYLCLP APQISSHSPKYTCACPDTMWLGPDMKRCYRAPQSTSTTTLASTMTRTVPATTRAPGTTVHRSTYQNHSTETP SLTAAVPSSVSVPRAPSISPSTLSPATSNHSQHYANEDSKMGSTVTAAVIGIIVPIWIALLCMSGYLIWRN WKRKNTKSMNFDNPVYRKTTEEEDEDELHIGRTAQIGHVYPAAISSFDRPLWAEPCLGETREPEDPAPALKE LFVLPGEPRSQLHQLPKNPLSELPWKSKRVALSLEDDGLP
The disclosed NOV5i amino acid sequence has 625 of 627 amino acid residues (99%) identical to, and 626 of 627 amino acid residues (99%) similar to, the 963 amino acid residue ptnr:SPTREMBL-ACC:Q14114 protein from Homo sapiens (Human) (APOLIPOPROTEIN E RECEPTOR 2 PRECURSOR) (E = 0.0).
NOV5i is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus, Cerebral Medulla/Cerebral white matter, Colon, Lung, Lymphoid tissue, Parietal Lobe, Tonsils, Whole Organism. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOV5L The sequence is predicted to be expressed in placenta because ofthe expression pattern of (GENBANK-ID: gb:GENBANK-ID:D50678|acc:D50678.1) a closely related Human mRNA for apolipoprotein E receptor 2, complete eds homolog in species Homo sapiens.
The presence of identifiable domains in NOV5i was determined by searches versus domain databases such as Pfam, PROSITE, ProDom, Blocks or Prints and then identified by the Interpro domain accession number. Significant domains are summarized below.
IPR002172, Low density lipoprotein (LDL)-receptor class A (LDLRA) domain, from amino acid 45 to amino acid 83, from amino acid 84 to amino acid 124, from amino acid 125 to amino acid 163, from amino acid 165 to amino acid 207. Low density lipoprotein (LDL) is the major cholesterol-carrying lipoprotein of plasma. The receptor protein binds LDL and transports it into cells by endocytosis. In order to be internalised, the receptor-ligand complex must first cluster into clathrin-coated pits. Seven successive cysteine-rich repeats of about 40 amino acids are present in the N-terminal of this multidomain membrane protein. The LDL- receptor class A domain contains 6 disulfide-bound cysteines and a highly conserved cluster of negatively charged amino acids, of which many are clustered on one face ofthe module. In LDL-receptors the class A domains form the binding site for LDL and calcium. The acidic residues between the fourth and sixth cysteines are important for high-affinity binding of positively charged sequences in LDLR's ligands. The repeat has been shown to consist of a beta-hairpin structure followed by a series of beta turns. The binding of calcium seems to induce no significant conformational change. Following these repeats is a 350 residue domain that resembles part ofthe epidermal growth factor (EGF) precursor. Similar domains have been found in several extracellular and membrane proteins.
IPR000033, YWTD repeat, from amino acid 404 to amino acid 449, from amino acid 451 to amino acid 492, from amino acid 494 to amino acid 536, from amino acid 538 to amino acid 581, from amino acid 582 to amino acid 622. This domain is also known as the YWTD motif after the most conserved region of the repeat. The YWTD repeat is found in multiple tandem repeats and has been predicted to form a beta-propeller structure. It is found in a variety of proteins that include, vitellogenin receptor from Drosophila, low-density lipoprotein (LDL) receptor, preproepidermal growth factor, nidogen (entactin) and others.
IPR000561, EGF-like domain, from amino acid 322 to amino acid 356, from amino acid 632 to amino acid 676. A sequence of about thirty to forty amino-acid residues long found in the sequence of epidermal growth factor (EGF) has been shown to be present, in a more or less conserved form, in a large number of other, mostly animal proteins. The list of proteins currently known to contain one or more copies of an EGF-like pattern is large and varied. The functional significance of EGF domains in what appear to be unrelated proteins is not yet clear. However, a common feature is that these repeats are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G/H synthase). The EGF domain includes six cysteine residues which have been shown (in EGF) to be involved in disulfide bonds. The main structure is a two-stranded beta- sheet followed by a loop to a C-terminal short two-stranded sheet. Subdomains between the conserved cysteines vary in length.
This indicates that the sequence ofthe invention has properties similar to those of other proteins known to contain this/these domain(s) and similar to the properties of these domains. NOV5a also has homology to the amino acid sequences shown in the BLASTP data listed in Table 5S.
Figure imgf000085_0002
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 5T.
Table 5T Clustal W Sequence Alignment i) NOV5a (SEQ ID NO: 15)
2) NOV5b (SEQ ID NO: 17) 3) NOV5C (SEQ ID NO: 19) N0V5d (SEQ ID NO: 21) NOV5e (SEQ ID NO: 23) N0V5f (SEQ ID NO: 5)
7) N0V5g (SEQ ID NO: 7) NOV5h (SEQ ID NO: 29)
9) N0V5i (SEQ ID Nθ:31)
10) Q99876 homo sapiens (human) apolipoprotein e receptor 2 906 (SEQ ID NO: 54)
11) 014968 homo sapiens (human) apoer2delta4-7 (SEQ ID NO: 55)
12) Q14114 homo sapiens (human) apolipoprotein e receptor 2 precursor (SEQ ID
NO: 56)
13) Q98931 gallus gallus (chicken) . Idl receptor homolog lr8b protein (SEQ ID
NO: 57)
Figure imgf000085_0001
Q98931 UJCRΓJJA- -HARHW-HnHHHfflκHlϊfll- 21
Figure imgf000086_0001
130 140 150 160 170 180
N0V5a 152
N0V5b JFTCDNGHCIHERWKCDGEEECPDGSDESEATCl 152
N0V5C JFTCDNGHCIHERWKCDGEEECPDGSDESEATCT. 135
N0V5d IFTCDNGHCIHERWKCDGEEECPDGSDESEATCTiKQVCPAEKIiSCGPTSHKCVPASWRCD 149
N0V5e 1FTCDNGHCIHERWKCDGEEECPDGSDESEATCT 123
N0V5f IFTCDNGHCIHERWKCDGEEECPDGSDESEATCT 135
N0V5g 1FTCDNGHCIHERWKCDGEEECPDGSDESEATCT 123
N0V5h JFTCDNGHCIHERWKCDGEEECPDGSDESEATCT 123
N0V51 1FTCDNGHCIHERWKCDGEEECPDGSDESEATCT 23
Q99876 1FTCDNGHCIHERWKCDGEEECPDGSDESEATCT 123
014968 IFTCDNGHCIHERWKCDGEEECPDGSDESEATCT 123
Q14114 1FTCDNGHCIHERWKCDGEEECPDGSDESEATCT 123
Q98931 irtwt>tw.pnig 107
190 200 210 220 230 240
N0V5a 152
N0V5b 152
N0V5c 35
N0V5d GE DCEGGADEAGCAT CAPHEFQCGNRSCLAAVFVCDGDDDCGDGSDERGCADPACGPR 209
N0V5e 123
N0V5f 135
N0V5g 123
N0V5h 123
N0V5i 123
Q99876 123
014968 123
Q14114 23
Q98931 07
250 260 270 280 290 300
N0V5a 152
N0V5b 152
N0V5C 135
N0V5d EFRCGGDGGGACIPER VCDRQFDCEDRSDEAAE CGRPGPGATSAPAACATVSQFACRS 269
N0V5e 123
N0V5f 135
N0V5g . 123
N0V5h 123
N0V5i 123
Q99876 123
014968 123
Q14114 123
Q98931 107
Figure imgf000086_0002
UJ t t o O O O O o
Figure imgf000087_0001
-J Q\ CJ\ 4^ 4i* t to o O o o o
Figure imgf000088_0002
Figure imgf000088_0001
Figure imgf000089_0001
o a a o a q q a o COoHoμioCOaOaO aOaOaOaO aOaOaO oo oOoHoHoCOaOaOaOaO OaaOaOaOaO
< < < co rf* rf* co < < < < < ! <J co rf* rf» co <i <i < <l < <J <J | cπ < cπ cπ cπ co H co co cπ cπ cn cπ cπ cπ cti cπ cη co H co co cπ cπ cπ cπ cπ cπ cπ cπ Cπ p. n tr u u μ m «ι μ triq Hi m p, tr p ω μ owl μ p1 m cD n o tr f» μ» rf* co en
Figure imgf000089_0002
μ* rf* co en
Figure imgf000089_0003
Figure imgf000090_0001
Tables 5U-Y list the domain description from DOMAIN analysis results against NOV5a. This indicates that the NOV5a sequence has properties similar to those of other proteins known to contain this domain.
Table 5U. Domain Analysis of NOV5a qnl 1 Smart | smart00135 , LY, Low-density lipoprotein-receptor YWTD domain; Type "B" repeats in low-density lipoprotein (LDL) receptor that plays a central role in mammalian cholesterol metabolism. Also present in a variety of molecules similar to gp300/megalin (SEQ ID NO: 83)
CD-Length = 43 residues, 100.0% aligned Score = 62.8 bits (151), Expect = 6e-ll
Query: 389 EVLIDEQLHSPEGLAVDWVHKHIYWTDSGNKTISVATVDGGRR 431
11+ I I I +1111 I I II +11 I
Sbjct: 1 RVLLSEGLGHPNGLAVDWIEGRLYWTDWGLDVIEVANLDGTNR 43
CD-Length = 43 residues, 100.0% aligned Score = 56.6 bits (135), Expect = 5e-09 Query: 432 RTLFSR LSEPRAIAVDPLRGFMYWSDWGDQAKIEKSGLNGVDR 475
I I I I I +111 + I +11+111 II + l+l +1
Sbjct: 1 RVLLSEGLGHPNGLAVDWIEGRLYWTDWG-LDVIEVANLDGTNR 43
CD-Length = 43 residues, 100.0% aligned Score = 55.5 bits (132), Expect = le-08
Query: 476 QTLVSDNIEWPNGITLDLLSQRLYWVDSKLHQLSSIDFSGGNR 518
+ 1 + 1+ 111+ +1 + MM 1 1 + + 1 11
Sbjct: 1 RVLLSEGLGHPNGLAVDWIEGRLYWTDWGLDVIEVANLDGTWR 43
CD-Length = 43 residues, 81.4% aligned Score = 38.1 bits (87), Expect = 0.002
Query: 345 LIPMLKNWALDVEVATNRIYWCDLSYRKIYSAYI 379
I I + 1 1+ l+ll I 1 1 +
Sbjct: 4 LSEGLGHPNGLAVDWIEGRLYWTDWGLDVIEVANL 38 CD-Length = 43 residues, 93.0% aligned Score = 37.4 bits (85), Expect = 0.003
Query: 519 KTLISSTDFLSHPFGIAV--FEDKVFWTDLENEAIFSANRLNG 559
+ l+l I II l+ll I +++III + I II l+l Sbjct: 1 RVLLSEG--LGHPNGLAVDWIEGRLYWTDWGLDVIEVAN-LDG 40
Table 5V. Domain Analysis of NOV5 gnl] Pfam|pfam00058, ldl_recept_b, Low-density lipoprotein receptor repeat class B. (SEQ ID NO: 84)
CD-Length = 43 residues, 95.3% aligned Score = 62.4 bits (150), Expect = 8e-ll
Query: 452 GFMY SDWGDQAKIEKSGLNGVDRQTLVSDNIEWPNGITLD 492
I +11 + 1 +1 I + l + l l + ll I++ MM +1 Sbjct: 1 GRLYWTDSSLRASISVADLDGSSRRTLFSEDLQHPNGIAVD 41
CD-Length = 43 residues, 93.0% aligned Score = 55.5 bits (132), Expect = le-08 Query: 411 IYWTDSGN-KTISVATVDGGRRRTLFSRNLSEPRAIAVDP 449
+I11M +IIM +ιι linn +ι i nm
Sbjct: 3 LYWTDSSLRASISVADLDGSSRRTLFSEDLQHPNGIAVDP 42
CD-Length = 43 residues, 100.0% aligned Score = 46.6 bits (109), Expect = 5e-06
Query: 539 DKVFWTDLENEAIFSANRLNGL-EISILAENLNNPHDIVIFHE 580
+++111 I I l + l ++ +1 + 1 +1+ I +
Sbjct: 1 GRLYWTDSSLRASISVADLDGSSRRTLFSEDLQHPNGIAVDPI 43
CD-Length = 43 residues, 97.7% aligned Score = 46.2 bits (108), Expect = 6e-06
Query: 496 QRLYWVDSKLHQ-LSSIDFSGGNRKTLISSTDFLSHPFGIAVFE 538
MM 11 1 +1 1 1 +1 + 11 I + I II MM
Sbjct: 1 GRLYWTDSSLRASISVADLDGSSRRTLFS--EDLQHPNGIAVDP 42
CD-Length = 43 residues, 100.0% aligned Score = 40.8 bits (94), Expect = 3e-04 Query : 362 NRIYWCDLSYRKIYSAYIGQGQDPKEQEVLIDEQLHSPEGLAVDWV 407 l+ll I I I 1 I + 1 I I l+MI +
Sbjct: 1 GRLY TDSSLRASISVADLDGSS---RRTLFSEDLQHPNGIAVDPI 43
Table 5W. Domain Analysis of NOV5 gnl I Smart | smart00192 , LDLa, Low-density lipoprotein receptor domain class A; Cysteine-rich repeat in the low-density lipoprotein (LDL) receptor that plays a central role in mammalian cholesterol metabolism. The N-terminal type A repeats in LDL receptor bind the lipoproteins. Other homologous domains occur in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement . Mutations in the LDL receptor gene cause familial hypercholesterolemia. (SEQ ID NO:85)
CD-Length = 38 residues, 86.8% aligned Score = 50.8 bits (120), Expect = 2e-07
Query: 114 TCADSDFTCDNGHCIHERWKCDGEEECPDGSDE 146
II +1 I II II I Ml ++I Mill
Sbjct: 1 TCPPGEFQCKNGRCIPLSWVCDGVDDCGDGSDE 33
CD-Length = 38 residues, 94.7% aligned Score = 50.4 bits (119), Expect = 3e-07
Query: 198 TCRGDEFQCGDGTCVLAIKHCNQEQDCPDGSDEAGC 233 | | M M + 1 1 + 1 + I I M i l l I
Sbjct: 1 TCPPGEFQCKNGRCIPLSWVCDGVDDCGDGSDEENC 36
CD-Length = 38 residues, 97.4% aligned Score = 44.7 bits (104), Expect = 2e-05
Query: 156 CPAEKLSCGPTSHKCVPASWRCDGEKDCEGGADEAGCAT 194
II + 1 + +1+1 II III II l+ll I +
Sbjct: 2 CPPGEFQCK--NGRCIPLSWVCDGVDDCGDGSDEENCPS 38
Table 5X. Domain Analysis of NOV5 gnl 1 Pfam| pfam00057, ldl_recept_a, Low-density lipoprotein receptor domain class A (SEQ ID NO: 86)
CD-Length = 39 residues, 87.2% aligned Score = 47.8 bits (112), Expect = 2e-06
Query: 113 KTCADSDFTCDNGHCIHERWKCDGEEECPDGSDE 14£
II ++I I +1 II I 111+ +1 Mill
Sbjct: 1 STCEPNEFQCGSGECIPMSWVCDGDPDCRDGSDE 34
CD-Length = 39 residues, 94.9% aligned Score = 45.1 bits (105), Expect = le-05 Query: 197 GTCRGDEFQCGDGTCVLAIKHCNQEQDCPDGSDEAGC 233
II +IIIII 1 1+ 1+ + II Mill I
Sbjct: 1 STCEPNEFQCGSGECIPMSWVCDGDPDCRDGSDEKNC 37
CD-Length = 39 residues, 92.3% aligned Score = 39.7 bits (91), Expect = 6e-04
Query: 156 CPAEKLSCGPTSHKCVPASWRCDGEKDCEGGADEAGCA 193
I + II I +1+1 II 111+ II +11 I
Sbjct: 3 CEPNEFQCG--SGECIPMSWVCDGDPDCRDGSDEKNCP 38
Table 5Y. Domain Analysis of NOV5 gnl I Smart | smart00179 , EGF_CA, Calcium-binding EGF-like domain (SEQ ID Nθ:87)
CD-Length = 41 residues, 87.8% aligned Score = 37.0 bits (84), Expect = 0.004 Query: 276 DIDECKDPDACSQ--ICVNYKGYFKC-ECYPGYEMD 30£
Mill + 1 111 I +1 II III +1
Sbjct: 1 DIDECASGNPCQNGGTCVNTVGSYRCEECPPGYTLD 36
NOV5a also has high homology to members ofthe patp database shown in Table 5Z.
Figure imgf000093_0001
Figure imgf000094_0001
NOV5c also has high homology to members ofthe patp database shown in Table 5AA.
Figure imgf000094_0002
Apolipoprotein E (apoE) is a 34-kDa lipophilic protein that circulates in the plasma primarily as a major component of various lipoproteins including chylomicron remnants, intermediate density lipoprotein, very low density lipoprotein (VLDL), β-migrating VLDL (β- VLDL), and high density lipoprotein (with apoE). It is a key molecule responsible for the cellular recognition and internalization of these lipoproteins. Biochemical and genetic studies have demonstrated that apoE is involved in the hepatic clearance of chylomicron remnants and VLDL remnants from the plasma. Apolipoprotein E receptor 2 binds apoE-rich β-VLDL with high affinity and internalizes it into the cells.
Apolipoprotein E receptor 2 is a receptor belonging to a family of low and very low density lipoprotein receptors (LDL-R). This family consists of cell-surface receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. The family members contain four major structural modules: the cysteine-rich complement-type repeats, epidermal growth factor precursor-like repeats, a transmembrane domain, and a cytoplasmic domain. Each structural module serves distinct and important functions. These receptors bind several structurally dissimilar ligands. It is proposed that instead of a primary sequence, positive electrostatic potential in different ligands constitutes a receptor bmding domain. This family of receptors plays crucial roles in various physiologic functions. LDL-R plays an important role in cholesterol homeostasis. Mutations cause familial hypercholesterolemia and premature coronary artery disease. LDL-R-related protein plays an important role in the clearance of plasma-activated alpha 2-macroglobulin and apolipoprotein E-enriched lipoproteins. It is essential for fetal development and has been associated with Alzheimer's disease. The regulation of expression of these receptors occurs at the transcriptional level.
Expression ofthe LDL-R is regulated by intracellular sterol levels involving novel membrane- bound transcription factors. All the members are, however, regulated by hormones and growth factors, but the mechanisms of regulation by hormones have not been elucidated.
The NOV5 nucleic acids and proteins ofthe invention are useful in potential therapeutic applications implicated in atherosclerosis, dysbetalipoproteinemia, hyperlipoproteinemia type III, xanthomatosis, coronary and/or peripheral vascular disease, hypothyroidism, systemic lupus erythematosus, diabetic acidosis, hypercholesterolemia, planar and tendon xanthomas, dysbetalipoproteinemia, hypercholesterolemia, accelerated vascular disease, Alzheimer disease, familial amyloidotic polyneuropathy, as well as other diseases, disorders and conditions. The LDL receptor related protein, the apolipoprotein E receptor 2 and the very low density lipoprotein (VLDL) receptor are preferentially expressed in the brain and they have been suspected to be involved in Alzheimer disease at various levels (Microsc Res Tech 2000 Aug 15;50(4):273-7)., and/or other pathologies and disorders. For example, a cDNA encoding the apolipoprotein e-like protein may be useful in gene therapy, and the apolipoprotein e-like protein may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from atherosclerosis, dysbetalipoproteinemia, hyperlipoproteinemia type III, xanthomatosis, coronary and or peripheral vascular disease, hypothyroidism, systemic lupus erythematosus, diabetic acidosis, hypercholesterolemia, planar and tendon xanthomas, dysbetalipoproteinemia, hypercholesterolemia, accelerated vascular disease, Alzheimer disease, familial amyloidotic polyneuropathy, as well as other diseases, disorders and conditions. The NOV5 nucleic acid, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. NOV5 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. For example the disclosed NOV5a, e, and f proteins have multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, contemplated NOV5a, e, and f epitopes are from about amino acids 80 to 220. In other embodiments, NOV5a, e, and f epitopes are from about amino acids 250 to 340, from about amino acids 370 to 540, from about amino acids 590 to 650, or from about amino acids 680 to 720. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders. The disclosed NOV5b protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOV5b epitope is from about amino acids 80 to 280. In other embodiments, a NOV5b epitope is from about amino acids 290 to 350, from about amino acids 390 to 560, from about amino acids 600 to 670, or from about amino acids 690 to 750. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
The disclosed NOV5c protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOV5c epitope is from about amino acids 50 to 220. In other embodiments, a NOV5c epitope is from about amino acids
250 to 280, from about amino acids 300 to 480, from about amino acids 550 to 630,from about amino acids 650 to 670, from about amino acids 690 to 760, or from about amino acids 770 to 800. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
The disclosed NOV5d protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOV5d epitope is from about amino acids 50 to 180. In other embodiments, a NOV5d epitope is from about amino acids 190 to 230, from about amino acids 250 to 390, from about amino acids 420 to 550,from about amino acids 470 to 650, from about amino acids 780 to 850, from amino acids 870 to 900, from about amino acids 920 to 980, or from about amino acids 990 to 1050. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
The disclosed NOV5g and h proteins have multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, contemplated NOV5g and h epitopes are from about amino acids 30 to 220. In other embodiments, NOV5g and h epitopes are from about amino acids 250 to 330, from about amino acids 350 to 540, from about amino acids 550 to 680, from about amino acids 690 to 720, from about amino acids 750 to 780, or from about amino acids 790 to 850. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
The disclosed NOV5i protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOV5i epitope is from about amino acids 5 to 150. In other embodiments, a NOV5i epitope is from about amino acids 190 to 230, from about amino acids 170 to 320, or from about amino acids 360 to 450. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
NOV6
A disclosed NOV6 nucleic acid of 843 nucleotides (also referred to as SC_129296119- A) encoding a novel mastocytoma protease precursor -like protein is shown in Table 6A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 26- 28 and ending with a TAG codon at nucleotides 836-838. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 6A, and the start and stop codons are in bold letters.
Table 6A. NOV6 Nucleotide Sequence (SEQ ID NO.32) GAGGTGGAGGTTGCAGTAAGCCAAGATGGCGCCACTGCACTCTAGCCTGTTTCTGCTGAGCGGGACCATG AGCCCAAAAGTGGGCATCACTGGGGGCTGCGACGTCTCGGCCAGGAGGCACCCCTGGCAGGTCAGCCTGA GGTTCTACAGCATGAAGAAGGGTCTGTGGGAGCCCATCTGTGGGGGCTCCCTCATCCACCCAGAGTGGGT GCTGACCGCCGCCCACTGCGTCGAGCTTGAGGAGTTGGAGGCTTGCGCGTTTAGAGTGCAGGTGGGGCAG CTGAGGCTCTATGAGGACGACCAGCGGACGAAGGTGGTTGAGATCGTCCGTCACCCCCAGTACAACGAGA GCCTGTCTGCCCAGGGCGGTGCGGACATCGCCCTGCTGAAGCTGGAGGCCCCGGTGCCGCTGTCTGAGCT CATCCACCCGGTCTCGCTCCCGTCTGCCTCCCTGGACGTGCCCTCGGGGAAGACCTGCTGGGTGACCGGC TGGGGTGTCATTTGGGGACACGTTTTCCTGCTCCCGCCACCCCACCTCAGGGCAGCGGAAGGTCCAATCA TGAGGACCCGAGCTTGCGAGAGGATGTATCACAAAGGCCCCACTGCCCACGTCACCATCATCAAGGCTGC CATGCCGTGTGCAGGGGCTGAGCGCCATCTCTCCCCACAGGGCGACAACGGGGGCCCCCTCCTGTGCAGG CGGAATTGCACCTGGGTCCAGGTGGAGGTGGTGAGCTGGGGCAAACTCTGCGGCCTTCGCTATCCCGGCA TGTACACCCGCGTGACGAGCTACGTGTCCTGGATCCGCCAGTACGTCCCGCCGTTCCCCAGACGCTAGCT GGG
The disclosed NOV6 nucleic acid sequence has 570 of 763 bases (74 %) identical to a Canis familiaris (Dog) MASTOCYTOMA PROTEASE PRECURSOR mRNA (GENBANK- ID: DOGMCTRPB) (E = Lie-80).
A disclosed NOV6 polypeptide (SEQ ID NO:33) encoded by SEQ ID NO:32 is 270 amino acid residues and is presented using the one-letter amino acid code in Table 6B. Signal P, Psort and/or Hydropathy results predict that NOV6 contains a signal peptide and is likely to be localized in thecytosol with a certainty of 0.4500. The most likely cleavage site for a NOV6 peptide is between amino acids 20 and 21, at: KVG-IT.
Table 6B. Encoded NOV6 protein sequence (SEQ ID NO.33).
MAPLHSSLFLLSGTMSPKVGITGGCDVSARRHP QVSLRFYSMKKGLWEPICGGSLIHPE VLTAAHCVELEELEA CAFRVQVGQLRLYEDDQRTKWEIVRHPQYNESLSAQGGADIALLKLEAPVPLSELIHPVSLPSASLDVPSGKTCW VTG GVIWGHVFLLPPPHLRAAEGPIMRTRACERMYHKGPTAHVTIIKAAMPCAGAERHLSPQGDNGGPLLCRRNC TWVQVEWSWGKLCGLRYPGMYTRVTSYVSWIRQYVPPFPRR
The disclosed NOV6 amino acid sequence has 165 of 268 amino acid residues (61 %) identical to, and 186 of 268 residues (69 %) positive with, the 269 amino acid residue MASTOCYTOMA PROTEASE PRECURSOR protein from Canis familiaris (Dog) (ptnr:SPTREMBL-ACC: P19236) (E = 3.0e"sl). The global sequence homology (as defined by FASTA alignment with the full length sequence of NOV6) is 66.171 % amino acid homology and 61.338 % amino acid identity. In addition, this protein contains the following protem domains (as defined by Interpro) at the indicated nucleotide positions: Trypsin domain (IPR001254) at amino acid positions 21 to 260.
NOV6 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 6F.
Figure imgf000099_0002
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 6G.
Table 6G Information for the ClustalW proteins
1) N0V6 (SEQ ID NO: 33)
2) TRYM_CANFA canis familiaris (dog), mastocytoma protease precursor (ec 3.4.21.-) (SEQ ID NO: 58)
3) TRYM_RAT rattus norvegicus (rat), mast cell tryptase precursor (ec 3.4.21.59) (SEQ ID NO: 59)
4) MCT7_RAT rattus norvegicus (rat), mast cell protease 7 precursor (ec 3.4.21.-) (rmcp-7) (tryptase, skin) (SEQ ID NO: 60)
5) MCT6_MOUSE mus musculus (mouse), mast cell protease 6 precursor (ec 3.4.21.-) (mmcp-6) (tryptase) (SEQ ID NO: 61)
6) Q9N2D1 sus scrofa (pig) . miniature swine mast cell tryptase precursor (SEQ ID NO: 62)
Figure imgf000099_0001
Figure imgf000100_0001
Table 6H lists the domain description from DOMAIN analysis results against NOV6.
This indicates that the NOV6 sequence has properties similar to those of other proteins known to contain this domain.
Table 6H. Domain Analysis of NOV6 gnl I Smart | smart00020, Tryp_SPc, Trypsin-like serine protease; Many of these are synthesised as inactive precursor zymogens that are cleaved during limited proteolysis to generate their active forms. A few, however, are active as single chain molecules, and others are inactive due to substitutions of the catalytic triad residues.. (SEQ ID Nθ:88) CD-Length = 230 residues, 99.6% aligned Score = 198 bits (503) , Expect = 4e-52
Query: 21 ITGGCDVSARRHP QVSLRFYSM KGLWEPICGGSLIHPEWVLTAAHCVELEELEACAFR 80
I I I ++ + 1 I I I M ++ + M U M I I I M U M I + 1
Sbjct: 2 IVGGSEANIGSFP QVSLQYRGGR HFCGGSLISPRWVLTAAHCVYGSAPS--SIR 54 Query: 81 VQVGQLRL--YEDDQRTKVVEIVRHPQYNESLSAQGGADIALLKLEAPVPLSELIHPVSL 138 ι++ι i ι+ i M++++ II II i mini i n+ ι+ i
Sbjct: 55 VRLGSHDLSSGEETQTVKVSKVIVHPNYNPSTY DNDIALLKLSEPVTLSDTVRPICL 111
Query: 139 PSASLDVPSGKTCWVTG GVIWGHVFLLPPPHLRAAEGPIMRTRACERMYHKGPTAHVTI 198
11+ +11+1 II i+lll 1 1+ 11+ I I I II
Sbjct: 112 PSSGYNVPAGTTCTVSGWGRTSE-SSGSLPDTLQEVNVPIVSNATCRRAYSGGP A 165
Query: 199 IKAAMPCAGAER--HLSPQGDNGGPLLCRRNCTWVQVEVVS GKL-CGL-RYPGMYTRVT 254 i i III + in+ini+i n i +nn i 11+1111+
Sbjct: 166 ITDNMLCAGGLEGGKDACQGDSGGPLVCNDP-RWVLVGIVSWGSYGCARPNKPGVYTRVS 224
Query: 255 SYVSWI 260 Sbjct: 225 SlYlL+D+WlI1 230
Table 61. Domain Analysis of NOV6 gnl I famI pfamO0089, trypsin, Trypsin. (SEQ ID NO: 89)
CD-Length = 217 residues, 100.0% aligned
Score = 154 bits (390) , Expect = 5e-39
Query: 21 ITGGCDVSARRHPWQVSLRFYSMKKGLWEPICGGSLIHPEWVLTAAHCVELEELEACAFR 80 i ii + i II i in+ i II nn iiniini ι++ i Sbjct: 1 IVGGREAQAGSFP QVSLQVSS GHFCGGSLISENVLTAAHCVS GASSVR 50
Query: 81 VQVGQLRLYEDD QRTKWEIVRHPQYNESLSAQGGADIALLKLEAPVPLSELIHPVS 137
I +1+ I + l+ l +1 II II I + 1+
Sbjct: 51 WLGEHNLGTREGTEQKFDVKKIIVHPNYNPD TNDIALL L SPVTLGDTVRPIC 105
Query: 138 LPSASLDVPSGKTCWVTG GVI GHVFLLPPPHLRAAEGPIMRTRACERMYHKGPTAHVT 197 lllll+1+l I II l+lll I 1+ 11+ I I I
Sbjct: 106 LPSASSDLPVGTTCSVSGWGRTK NLGTSDTLQEVWPIVSRETCRSAYGGTVTDT-- 160 Query: 198 IIKAAMPCAGAERHLSP-QGDNGGPLLCRRNCT VQVEVVS GKLCGL-RYPGMYTRVTS 255 i nn ni+iin+i 1++1111 1 111+1111+
Sbjct: 161 MICAGALGGKDACQGDSGGPLVCSDG ELVGIVSWGYGCAVGNYPGVYTRVSR 212
Query: 256 YVS I 260
1+ II Sbjct: 213 YLD I 217
Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation. The tryptase sequence includes the essential residues ofthe catalytic triad and an aspartic acid at the base ofthe putative substrate binding pocket that confers PI Arg and Lys specificity on tryptic serine proteases. The apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Mast cell activation in vivo is often associated with areas of oedema and connective- tissue degradation. Tryptase and chymase are the major serine proteinases released by mast cells, but they appear to have little activity on most components ofthe extracellular matrix. The matrix metalloproteinases (MMP) are purported to degrade almost all connective tissue elements and are secreted by cells in the form of inactive precursors. Since the mechanisms of MMP activation in vivo are poorly understood we have examined the potential of mast cell proteinases to activate the precursor forms of human collagenase (MMP-1), stromelysin (MMP-3), gelatinase A (MMP-2) and gelatinase B (MMP-9). Mast cell proteinases prepared from purified dog mastocytoma cells were shown to process and activate purified precursor forms of both MMP-1 and MMP-3. Using antipain and chymostatin, inhibitors for tryptase and chymase, respectively, it was demonstrated that both pMMP-1 and pMMP-3 were effectively processed and activated by the chymase component. By contrast, tryptase activated only pMMP-3. The mast cell proteinases were unable to process or activate purified precursor forms of MMP-2 and MMP-9. However, MMP-3 previously activated by mast cell proteinases was shown to activate pMMP-9, but not pMMP-2. Since we have no evidence that mast cells express these four metalloenzymes, the release of mast cell serine proteinases following activation/degranulation could contribute to local metalloproteinase activation and subsequent matrix degradation.
The mast cell proteases tryptase and chymase have long been known to constitute one- fifth ofthe total protein in mast cells. However, their biological functions have not been easy to study because ofthe difficulty in obtaining sufficient amounts ofthe enzymes to study their biological functions. Recently, we have been fortunate to have available a permanent line of dog mastocytoma cells to purify both enzymes to homogeneity, and we have used the purified enzymes in two ways. First, in a series of biological studies, we have discovered unique and potent actions ofthe enzymes that may provide important insights into the pathogenesis of diseases such as asthma and cystic fibrosis. Important biological activities are also likely to exist in other tissues. Because of their structures, mast cell proteases are likely to act in proximity to their sites of release. Thus, the presence and amounts of tryptase and chymase in specific loci may play important roles in tissue responses. In diseases such as asthma and cystic fibrosis, there is evidence that the expression of these mast cell enzymes changes, and these changes have important pathogenetic implications. Second, we have begun to perform structural studies ofthe enzymes. The recent cloning of tryptase by our group should assist in the better understanding of its functions. Crystallography ofthe pure proteins should provide further insights and could be the basis of rational development of potent and selective drugs that will inhibit their actions. The above defined information for this invention suggests that these Voltage- Dependent Anion Channel-like proteins (NOV6) may function as a member of a "Voltage- Dependent Anion Channel family". Therefore, the NOV6 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below. The potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
The nucleic acids and proteins of NOV6 are useful in potential therapeutic applications implicated in immune disorders and airway pathologies such as allergies, asthma and cystic fibrosis, cancers such as mastocytomas, vascular diseases, and/or other pathologies and disorders For example, a cDNA encodingNOV6 may be useful in gene therapy, and NOV6 may be useful when administered to a subject in need thereof. By way of nonlimiting example, NOV6 will have efficacy for treatment of patients suffering from immune disorders and airway pathologies such as allergies, asthma and cystic fibrosis, cancers such as mastocytomas, and vascular diseases. The novel NOV6 nucleic acid encoding NOV6 protein,, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
NOV6 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOV6 substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders. NOV7
A disclosed NOV7 nucleic acid of 239 nucleotides (also referred to GM10d7_A) encoding a novel thymosin beta-4-like receptor protein is shown in Table 7A. An open reading frame was identified beginning with a TCA initiation codon at nucleotides 40-42 and ending with a TGA codon at nucleotides 190-092. In Table 7A, and the start and stop codons are in bold letters.
Table 7A. NOV7 Nucleotide Sequence (SEQ ID NO.34)
CACTAAGCAGACCAGATTCCACTTGCTAGTGGGCATTGCTCAGCTTCCTCTGTGACTATGTCTGACAAGTC CAATATGGATGAGATCGAGAAATTCAGTAAGTCGAAACTGAAGAAGACAGAAATGCAAGAGAAAAATCCAC AGCCTTCCAAGGAATGGATCGAACAGGAGAAGCAAGCAGGCTTCGTATGAGGCGTGCATCGCCAATATGC ACTGTTCATTCCACAAAGCATTGCTTT
The disclosed NO V7 nucleic acid sequence has 205 of 245 bases (83 %) identical to a Homo sapiens THYMOSIN BETA-4 mRNA (GENBANK-ID: M17733) (E = 1.4e"30).
A disclosed NOV7 polypeptide (SEQ ID NO:35) encoded by SEQ ID NO:34 is 50 amino acid residues and is presented using the one-letter amino acid code in Table 7B. Signal P, Psort and/or Hydropathy results predict that NOV7 has no signal peptide and is likely to be localized in the cytoplasm with a certainty of 0.4500.
Table 7B. Encoded NOV7 protem sequence (SEQ ID NO:35).
SASSVTMSDKSNMDEIEKFSKSKLKKTEMQEKNPQPSKEWIEQE QAGFV
NOV7 maps to chromosome 5p35 and was found to be expressed in at least the following tissues: mammary gland, small intestine, colon, testis and leukocytes.
The disclosed NOV7 amino acid sequence has 26 of 43 amino acid residues (83 %) identical to, and 37 of 43 residues (86 %) positive with, the 50 amino acid residue
THYMOSIN BETA-4 protein ftom Mus musculus (ptnr:SPTREMBL-ACC: P20065) ( 2.2e" 12). The global sequence homology (as defined by FASTA alignment with the full length sequence of this protein) is 72.000% amino acid homology and 72.000% amino acid identity.
In addition, NOV7 contains the following protein domains (as defined by Interpro) at the indicated nucleotide positions: Thymosin domain(JPR001152) at amino acid positions 8 to 48 NOV7 also has homology to the amino acid sequence shown in the BLASTP data listed in Table 7C.
Figure imgf000105_0002
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 7D.
Table 7D. Information for the ClustalW proteins
1 ) NOV7 (SEQ ID Nθ : 35 )
2) TYB4_M0USE mus musculus (mouse) . thymosin beta-4] (SEQ ID Nθ:63) 3) Q9NQQ5 homo sapiens (human). djl071110.1 (thymosin/interferon-inducible multigene family) (SEQ ID NO: 64)
4) Q95274 sus scrofa (pig) . thymosin beta-4 (fragment) (SEQ ID NO: 65)
5) TYB4_HUMAN homo sapiens (human) , bos taurus (bovine) , rattus norvegicus (rat) , and equus caballus (horse) . thymosin beta-4 (fx) [contains: hematopoietic system regulatory peptide] (SEQ ID NO: 66)
6) TYB4_RABIT oryctolagus cuniculus (rabbit) . thymosin beta-4 (SEQ ID NO: 67)
Figure imgf000105_0001
TYB4 RABIT - MAEIEKFDKΞKLKKTETOEKNPLPSKETIEQE 43
NOV7 also has high homology to members ofthe patp database shown in Table 7E.
Figure imgf000106_0001
Thymosin beta-4 is a small polypeptide whose exact physiological role is not yet known . It was first isolated as a thymic hormone that induces terminal deoxynucleotidyltransferase. It is found in high quantity in thymus and spleen but is widely distributed in many tissues. It has also been shown to bind to actin monomers and thus to inhibit actin polymerization .It has inhibitory activity on the proliferation of hematopoeitic pluripotent stem cells, and is localized in the cytosol of cells. It is induced by alpha interferon, nerve and fibroblast growth factors. It belongs to the thymosin beta family.
Atkinson MJ et al. reported that thymosin beta 4 is expressed in ROS 17/2.8 osteosarcoma cells in a regulated manner. The differential expression of rnRNAs between the closely related rat osteosarcoma cell lines ROS 17/2.8 and ROS 25/1 was used to identify genes whose expression is associated with the osteoblast phenotype. Thymosin beta 4 cDNA was cloned from an ROS 17/2.8 complimentary DAN library on the basis of its differential hybridization with radiolabeled cDNA prepared from ROS 17/2.8 and ROS 25/1 cells. Northern blot analysis confirmed that thymosin beta 4, hitherto a putative immunodulatory hormone, was indeed differentially expressed. Steady state mRNA levels were severalfold higher in ROS 17/2.8 cells exhibiting an osteoblast-like phenotype, compared with the less osteoblast-like ROS 25/1. Thymosin beta 4 transcripts were also detected in rat UMR 106 osteosarcoma cells and in intact neonatal and fetal rat calvaria. Sequence analysis ofthe cDNA indicated that thymosin beta 4 transcripts may arise by processing at a more distal polyadenylation signal. Treatment of ROS 17/2.8 cells with dexamethasone increased, while addition of 1,25-dihydroxyvitamin D3 decreased thymosin beta 4 mRNA. The phenotype- dependent expression in the ROS cells and the response to steroid hormone suggest that thymosin beta 4 expression contributes to the osteoblast phenotype.
Thymosin-beta(4) (Tbeta(4)) binds actin monomers stoichiometrically and maintains the bulk ofthe actin monomer pool in metazoan cells. Tbeta(4) binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylenediamine (AEDANS) conjugated to Cys(374) of actin monomers. The K(d) ofthe actin-Tbeta(4) complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation. At 25 degrees C, the free energy of Tbeta(4) binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on subdomain 2 is >12 times faster when Tbeta(4) is bound. The C terminus of Tbeta(4) contacts actin near this cleavage site, at His(40). By tritium exchange, Tbeta(4) slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that Tbeta(4) changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of Tbeta(4) to sequester actin monomers and inhibit nucleotide exchange.
The fact that glucocorticoids upregulate the expression of anti-inflammatory mediators is an exciting prospect for therapy in inflammatory diseases, because these molecules could give the therapeutic benefits of steroids without toxic side effects. Supernatants from monocytes and macrophages cultured in the presence of glucocorticoids increase the dispersion of neutrophils from a cell pellet in the capillary tube migration assay. This supernatant factor, unlike other neutrophil agonists, promotes dispersive locomotion of neutrophils at uniform concentration, lowers their adhesion to endothelial cells, inhibits their chemotactic response to fMLP and induces distinctive morphological changes. Here we show that thymosin beta4 sulfoxide is generated by monocytes in the presence of glucocorticoids and acts as a signal to inhibit an inflammatory response. In vitro, thymosin beta4 sulfoxide inhibited neutrophil chemotaxis, and in vivo, the oxidized peptide, but not the native form, was a potent inhibitor of carrageenin-induced edema in the mouse paw. Thymosin beta4 is unique, because oxidation attenuates its intracellular G-actin sequestering activity, but greatly enhances its extracellular signaling properties. This description of methionine oxidation conferring extracellular function on a cytosolic protein may have far-reaching implications for future strategies of ai ti-inflammatory therapy.
Angiogenesis is an essential step in the repair process that occurs after injury. In this study, we investigated whether the angiogenic thymic peptide thymosin beta4 (Tbeta4) enhanced wound healing in a rat full thickness wound model. Addition of Tbeta4 topically or intraperitoneally increased reepithelialization by 42% over saline controls at 4 d and by as much as 61 % at 7 d post- wounding. Treated wounds also contracted at least 11 % more than controls by day 7. Increased collagen deposition and angiogenesis were observed in the treated wounds. We also found that Tbeta4 stimulated keratinocyte migration in the Boyden chamber assay. After 4-5 h, migration was stimulated 2-3-fold over migration with medium alone when as little as 10 pg of Tbeta4 was added to the assay. These results suggest that Tbeta4 is a potent wound healing factor with multiple activities.
The protein similarity information, expression pattern, and map location for the thymosin beta-4-like protein and nucleic acid (NOV7) disclosed herein suggest that NOV7 may have important structural and/or physiological functions characteristic ofthe thymosin beta-4-like family. Therefore, the NOV7 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo. The NOV7 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from prostate cancer, immunological and autoimmune disorders (ie hyperthyroidism), angiogenesis and wound healing, modulation of apoptosis, neurodegenerative and neuropsychiatric disorders, age-related disorders, pathological disorders involving spleen, thymus, lung, and peritoneal macrophages and/or other pathologies and disorders. The NOV7 nucleic acid, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
NOV7 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances ofthe invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. For example the disclosed NOV7 protein has multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
NOV8
A disclosed NOV8 nucleic acid of 1348 nucleotides (also referred to as GMba550p23_A) encoding a novel Protein-tyrosine-phosphatase -like protein is shown in Table 8A. An open reading frame was identified beginning with a ATG initiation codon at nucleotides 57-59 and ending with a TAA codon at nucleotides 1302-1304. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 8A, and the start and stop codons are in bold letters.
Table 8A. NOV8 Nucleotide Sequence (SEQ ID NO:36)
CTTAGGAGCCAACCTCACTCTTCCATTTCTCTTAAGATTCACTATTTGTCCCAGCCATGCCCAC CATCATTGAGCGGGAGTTCAAGGAGTTGGATGCTCAGCATCGCTGGCAGCTGCCGTACTTGGAA ATTCGAAATGAGTCCCATGACTATCTTCATAGAGTGGCCAAGTTTCCAGAAAACAGAAATCGAA ATAGATACAGAGATGTAAGCCCATATGATCACAGTCGTGTTAAACTGCAGAATACTGAGAATGA TTATATTAATGCCAGCTTAGTTGACATAGAAGAGGCACGAAGGAGTTACATCTTAACACCGGGT CCACTTCCTAACATGTGCTGCCATTTCTGGCTTACGGTTTGGCAGCAGAAGACCAAAGCAGTTG TTGTGCTGAACCGCATTGTGGAGAAAGAATCGGTTAAATGTGCGCAGTACTGGCCAACAGATGA CCAAAAGATGCTGTTTAAAGAAACGGGATTCAATGTGAAGCTCCTTTCAGAAGATGTGAAGTCA TATTACACAGTACATCTACTACGATTAGAAAATATCAATAGTGGTGAAACCAGAACAATATCTC ACTTGCATTATACGACCTGGCCAGATTTTGGAGTCCCTGAATCACCAGCTTCATTTCTCAATTT CTTGTTGAAAGTGAGAGAATCTGGCTCCTTGAACCCTGACCATGGGCCTGCAGTGATCCATTGT AGCGAAGGCACTGGGTGCACGGGCACCTTCTCTCTGGTAGACGCTTGTCTTGTTTTGATGGGAA AAGGAGATTATATTAACATTAAACAAGTGTTATTGAACATGAGAAAATACCGAATGGGTCTTAT TCAGACCCCAGATCAACTGAGATTCTCATACATGGCTATAATAGAAGGAGCAAAATGTATAAAG GTAGATTCTAGTATACAGAAACGGTGGAAAGAATTTTCTAAGGAAAACCTTAGCGCTTGCTTTG ATCATTCACCGAACAAAATAATGACTGAAAAATACAATGGGAACAGAATTGGTCTAGAAGAAGA AAAACTGACAGGTGACCGATGTACAGGACTTTCCTCTAAAATGCAAGATACAATGGAAGAGAAC AGTGAGAATGCTCTACGGAAACATATTCTAGAGGACAGAAAGGCCACCACAGCTCAGAAGGTGC AGCAGATGAAACAGAGGCTAAATGAGAATGAACAAAAAAGAAAAAGGTGGTTATATTGGCAACC TATTCTCACTAAGATGGGGTTTATGTCAGTCATTTTGGTTGGCGCTTTTGTTGGCTGGACACTG TTTTTTCAGCAAAATGCCCTATAAACAATTAATTTTGCCCAGCAAGCTTCTGCACTAGTAAGTG ACAG
The disclosed NOV 8 nucleic acid sequence maps to chromosome 13 and has 1237 of 1299 bases (95%) identical to a Homo sapiens Protein-tyrosine-phosphatase mRNA (GENBANK-ID: HUMPTPASE) (E = 6.8e"262).
A disclosed NOV8 protein (SEQ ID NO:37) encoded by SEQ ID NO:36 has 415 amino acid residues, and is presented using the one-letter code in Table 8B. Signal P, Psort and/or Hydropathy results predict that NOV8 is likely to be localized to the endoplasmic reticulum (membrane) with a certainty of 0.8500.
Table 8B. Encoded NOV8 protein sequence (SEQ ID NO:37).
MPTIIEREFKELDAQHRWQLPYLEIRNESHDYLHRVAKFPENRNRNRYRDVSPYDHSRVKLQNT ENDYINAS VDIEEARRSYILTPGPLP MCCHFWLTVWQQKTKAVVVLNRIVE ESVKCAQY P TDDQKMLFKETGFNVKLLSEDVKSYYTVHL RLENINSGETRTISHLHYTT PDFGVPESPASP LNFLLKVRESGSLNPDHGPAVIHCSEGTGCTGTFSLVDACLVLMGKGDYINIKQV LNMRKYRM GLIQTPDQLRFSYMAIIEGAKCIKVDSSIQ R KEFSKENLSACFDHSPNKIMTEKYNGNRIGL EEEKLTGDRCTGLSSl^QDTMEENSENALRKHILEDRKATTAQK QQMKQRLNENEQKR R LY QPILTKMGFMSVILVGAFVG TLFFQQNAL
The disclosed NOV8 amino acid has 381 of 415 amino acid residues (91 %) identical to, and 391 of 415 residues (94 %) positive with, the 415 amino acid residue protein-tyrosine- phosphatase, nonreceptor type 2 protein from Homo sapiens (ptnr:SPTREMBL-ACC: A33899) (E= 4.9e-207).
NOV8 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 8C.
Figure imgf000111_0002
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 8D.
Table 8D. Information for the ClustalW proteins 1) NOV8 (SEQ ID NO: 37)
2) PTN2_HUMAN homo sapiens (human) . t-cell protein-tyrosine phosphatase (ec 3.1.3.48) (tcptp) (SEQ ID NO: 68)
3) PTN2_M0USE mus musculus (mouse) . protein-tyrosine phosphatase ptp-2 (ec 3.1.3.48) (mptp) (SEQ ID Nθ:69) 4) PTN2_RAT rattus norvegicus (rat) . protein-tyrosine phosphatase ptp-s (ec 3.1.3.48) (SEQ ID Nθ:70)
5) PTN1_CHICK gallus gallus (chicken) . protein-tyrosine phosphatase lb (ec 3.1.3.48) (ptp-lb) (cptpl). (Pa (SEQ ID N0:71)
6) PTN1_M0USE mus musculus (mouse), protein-tyrosine phosphatase lb (ec 3.1.3.48) (ptp-lb) (protein- protein-tyrosine phosphatase ha2) (ptp-ha2) (SEQ ID NO: 72)
Figure imgf000111_0001
Figure imgf000112_0001
190 200 210 220 230 240
■ ■■■| ....| ....| ....|....|....| ....|.... I .... | .. - | ..
NOV8 239
PTN2_HUMAN FPDFGVPESPASFLNFLFKVRESGSLNPDHGPAVIHCSAGIGRSGTFSLVDTCLVLI 239
PTN2_MOUSE WPDFGVPESPASFLNFLF VRESGGLØPDHGPAVIHCSAGIGRSGTFSLVDTCLVLMIKG 239
PTN2_RAT WPDFGVPESPASFLNFLFKVRESGSLNPDHGPAVIHCSAGIGRSGTFSLVDTCLVLMIKG 239
PTN1_CHICK iSR 238
PTN1 MOUSE IPDFGVPESPASFLNFLFKVRESGS 8R 238
Figure imgf000112_0002
PTNl_MOUSE
Figure imgf000113_0001
432
Tables 8E-8G list the domain description from DOMAIN analysis results against NOV8. This indicates that the NOV8 sequence has properties similar to those of other proteins known to contain this domain.
Table 8E. Domain Analysis of NOV8 gnl I Smart | smart00194 , PTPc, Protein tyrosine phosphatase, catalytic domain (SEQ ID NO: 90)
CD-Length = 264 residues, 98.5% aligned
Score = 263 bits (672) , Expect = le-71
Query: 5 IEREFKELDAQHRWQLPYLEIRNESHDYLHRVAKFPENRNRNRYRDVSPYDHSRVKLQNT 64
+1 II++I I II I II l I II IIII+IIII+ Sbjct: 2 LEEEFEKLQRLTPDDLSC TVAILPENRDKNRYKDVLPYDHTRVKLKPP 49 Query : 65 E NDYINASLVDIEEARRSYILTPGPLPNMCCHFWLTV QQKTKAVVVLNRIVEKESV 1 1
+IIIII1 +1 ++II I 1111+ II II++I +1+1 +111 Sbjct: 50 PGEGSDYINASYIDGPNRPKAYIATQGPLPSTVEDF RMV EEKVPVI MLTELVEKGRE 109 Query : 122 KCAQY PTD-DQKMLFKETGFNVKLLSEDVKSYYTVHLLRLENINSGETRTISHLHYTT 180 + + 1 1 + 11+ I + I IIII++I III I Sbjct: 110 KCAQYWPE EGGSLTYG--DITVTLKSVEKVDDYTIRTLEVTNTGGSETRTVTHYHYTNW 167 Query: 181 PDFGVPESPASFLNFLLKVRESGSLNPDHGPAVIHCSEGTGCTGTFSLVDACLVLMGKGD 240 ii null i ι+ + n+i i + n i+ni i i nn +ι i + i Sbjct: 168 PDHGVPESPKSLLDLVRAVRKSQSTLRNSGPIWHCSAGVGRTGTFIAIDILLQQLEAGK 227 Query: 241 YINIKQVLLNMRKYRMGLIQTPDQLRFSYMAIIE 274
++l +++ +| I i+H-ii +| i I M + i Sbjct: 228 EVDIFEIVKELRSQRPGMVQTEEQYIFLYRAILE 261
Table 8F. Domain Analysis of NOV8 gnl |Pfam|pfam00102, Yjphosphatase, Protein-tyrosine phosphatase. (SEQ
ID NO: 91)
CD-Length = 235 residues, 100.0% aligned
Score = 235 bits (599) , Expect = 4e-63
Query: 42 NRNRNRYRDVSPYDHSRVKLQNTEN---DYINASLVDIEEARRSYILTPGPLPNMCCHFW 98 i +IM+I+ MII+MM+ + mm n + ++ιι 1 mil 11
Sbjct: 1 NLE NRYKDILPYDHTRV- oKPLGDEDSDYINASYVDGYKKPKAYIATQGPLPNTIEDF 60
Query: 99 LTvWQQKTKAVvVLNRIVE ESVKCAQY P-TDDQKMLF-^TGFNV LLSED-VKSYYTV 156
M++I + +1 + 1 +111 + + + * I I +1 I III
Sbjct: 61 RMV EE VRVIVMLTELVEKGREKCAQYWPEKEGGPLTYGD--FTVTCVSVEKKKDDYTV 118 Query: 157 HLLRLENINSGETRTISHLHYTTWPDFGVPESPASFLNFLLKVRESGSLNPDHGPAVIHC 216 i i IIH+ i III III mm i ι+ i n n n mi
Sbjct: 119 RTFELTNSGDEETRTVKHYHYTG PDHGVPESPKSILDLLRKVRKS-KGTPDDGPIWHC 177 Query: 217 SEGTGCTGTFSLVDACLVLMGKGDYINIKQVLLNMRKYRMGLIQTPDQLRFSYMAIIE 274
I I I llll +1 I +1 ++ + l I MM I I n + i
Sbjct: 178 SAGIGRTGTFIAIDILLQQLEKEGWDVFDTVKELRSQRPGMVQTLEQYLFIYDAILE 235 Table 8G. Domain Analysis of NOV8 gnl I Smart | smart00404, PTPc_motif, Protein tyrosine phosphatase, catalytic domain motif SEQ ID NO: 92)
CD-Length = 105 residues, 100.0% aligned
Score = 108 bits (269) , Expect = 8e-25
Query: 171 TISHLHYTTWPDFGVPESPASFLNFLLKVRESGSLNPDHGPAVIHCSEGTGCTGTFSLVD 230 ι+ i I I I I I I m i l l i i n ι++ι + + ++ι 1+1 1 1 1 1 1 1 1 1 +1 Sbjct: 1 TVi YHYTG PDHGVPESPDSILEFLRAVK SLNKSANNGPVVVHCSAGVGRTGTFVAID 60 Query: 231 ACLVLMGKGDY-INIKQVLLNMRKYRMGLIQTPDQLRFSYMAIIE 274 l + l ++I ++ +1 I I +11 +1 I I I++I Sbjct: 61 ILLQQLEAGTGEVDIFDIVKELRSQRPGAVQTLEQYLFLYRALLE 105
NOV8 also has high homology to members ofthe patp database shown in Table 8H.
Figure imgf000114_0001
Many transmembrane receptors for polypeptide growth factors and oncogenes catalyze the phosphorylation of proteins on tyrosine residues, suggesting a regulatory role in cell growth and possibly differentiation. Whereas many tyrosine kinases had been characterized, relatively little was known about enzymes that remove phosphate specifically from tyrosine residues. Both kinases and phosphatases are involved in the phosphorylation of proteins on tyrosine. The importance of tyrosine dephosphorylation in regulating enzymatic activity is demonstrated, for example, for the SRC oncogene (190090), where dephosphorylation at tyrosine 527 in the carboxy terminus is associated with increased activity. The protein-tyrosine phosphatases for which genes have been cloned and mapped include placental PTPase IB
(PTP1B; 176885). CD45 (151460), and LAR (179590). T-cell PTPase was cloned by Cool et al. (1989) who showed that it shares 72% amino acid sequence identity with PTP1B in a 236- amino acid core region present in all PTPases. Sakaguchi et al. (1991, 1992) used a human PTPT cDNA to assign the gene to human chromosome 18 and mouse chromosome 18 by Southern analysis of human/mouse and mouse/Chinese hamster somatic cell hybrids, respectively. Johnson et al. (1993) mapped the PTPN2 gene to 18pll.3-pl 1.2 by fluorescence in situ hybridization. They identified pseudogenes on Iq22-q24 and 13ql2-ql3. Direct comparison ofthe specificity of genomic and cDNA probes demonstrated that under identical conditions the genomic probes (containing both exon and intron sequences) readily identified a single specific site of hybridization, whereas the cDNA identified sites of both the gene and its pseudogenes. They suggested that this is a strategy for addressing the recurrent problem in gene mapping studies where highly related sequences exist within the genome.
Protein tyrosine phosphatases (PTPs) are a family of enzymes that modulate the cellular level of tyrosine phosphorylation. Based on cellular location, they are classified as receptor like or intracellular PTPs. Structure and function studies have led to the understanding of the enzymatic mechanism of this class of enzymes . Proper targeting of PTPs is essential for many cellular signalling events including antigen induced proliferative responses of B and T cells. The physiological significance of PTPs is further unveiled through mice gene knockout studies and human genome sequencing and mapping projects. Several PTPs are shown to be critical in the pathogenesis of human diseases. PMED: 10723800, UI: 20188875
During neuronal development, cells respond to a variety of environmental cues through cell surface receptors that are coupled to a signaling transduction machinery based on protein tyrosine phosphorylation and dephosphorylation. Receptor and non-receptor tyrosine kinases have received a great deal of attention; however, in the last few years, receptor (plasma membrane associated) and non-receptor protein-tyrosine phosphatases (PTPs) have also been shown to play important roles in development ofthe nervous system. In many cases PTPs have provocative distribution patterns or have been shown to be associated with specific cell adhesion and growth factor receptors. Additionally, altering PTP expression levels or activity impairs neuronal behavior. In this review we outline what is currently known about the role of PTPs in development, differentiation and neuronal physiology.
The protein similarity information, expression pattern, and map location for the protein-tyrosine-phosphatase-like protein and nucleic acid (NOV8) disclosed herein suggest that NOV8 protein may have important structural and/or physiological functions characteristic ofthe protein-tyrosine-phosphatase F family. Therefore, the NOV8 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo. The NOV8 nucleic acids and proteins ofthe invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions ofthe present invention will have efficacy for treatment of patients suffering from hematopoiesic disorders; leukemogenesis; polycystic kidney disease; Oncogenesis such as lung, liver and ovarian cancers; apoptosis; type 2 diabetes and obesity; cell growth and possibly differentiation; non-Hodgkin lymphomas; musculoskeletal disorders; and CNS development disorders. NOV8 nucleic acid, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
NOV8 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. For example the disclosed NOV8 protein has multiple hydrophilic regions, each of which can be used as an immunogen. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
NOVX Nucleic Acids and Polypeptides
One aspect ofthe invention pertains to isolated nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g. , mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double- stranded DNA.
An NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage ofthe N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal ofthe N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+1 to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
The term "probes", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies. The term "isolated" nucleic acid molecule, as utilized herein, is one, which is separated from other nucleic acid molecules which are present in the natural source ofthe nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3 '-termini ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule ofthe invention, e.g., a nucleic acid molecule having the nucleotide sequence SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion ofthe nucleic acid sequence of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et αl., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et αl., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
A nucleic acid ofthe invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g. , using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment ofthe invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or a complement thereof. Oligonucleotides maybe chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule that is a complement ofthe nucleotide sequence shown in SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an NOVX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence shown SEQ JD NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19 is one that is sufficiently complementary to the nucleotide sequence shown SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species. Derivatives and analogs maybe full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs ofthe nucleic acids or proteins ofthe invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins ofthe invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues ofthe same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for an NOVX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations ofthe nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, as well as a polypeptide possessing NOVX biological activity. Various biological activities ofthe NOVX proteins are described below.
An NOVX polypeptide is encoded by the open reading frame ("ORF") of an NOVX nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG "start" codon and terminates with one ofthe three "stop" codons, namely, TAA, TAG, or TGA. For the purposes of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bonafide cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning ofthe human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or cloning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19; or an anti-sense strand nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19; or of a naturally occurring mutant of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36.
Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis- express an NOVX protein, such as by measuring a level of an NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX niRNA levels or determining whether a genomic NOVX gene has been mutated or deleted. "A polypeptide having a biologically-active portion of an NOVX polypeptide" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide ofthe invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically- active portion of NOVX" can be prepared by isolating a portion SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17 or 19, that encodes a polypeptide having an NOVX biological activity (the biological activities ofthe NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g. , by recombinant expression in vitro) and assessing the activity ofthe encoded portion of NOVX.
NOVX Nucleic Acid and Polypeptide Variants
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 due to degeneracy ofthe genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36. In another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
In addition to the human NOVX nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g. , the human population). Such genetic polymorphism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an NOVX protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence ofthe NOVX genes. Any and all such nucleotide variations and resulting amino acid polymorphisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity ofthe NOVX polypeptides, are intended to be within the scope ofthe invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from the human SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 are intended to be within the scope ofthe invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNAs ofthe invention can be isolated based on their homology to the human NOVX nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule ofthe mvention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Homologs (i.e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion ofthe particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% ofthe probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% ofthe probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al, (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequences SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5%) SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C. Other conditions of moderate stringency that maybe used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, thereby leading to changes in the amino acid sequences ofthe encoded NOVX proteins, without altering the functional ability of said NOVX proteins. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence SEQ ID NOS :2, 4, 6, 8, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31, 33, 35, or 37. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences ofthe NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the NOVX proteins ofthe invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art.
Another aspect ofthe invention pertains to nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37. Preferably, the protein encoded by the nucleic acid molecule is at least about 60% homologous to SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37; more preferably at least about 70% homologous SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37; still more preferably at least about 80% homologous to SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37; even more preferably at least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37; and most preferably at least about 95% homologous to SEQ ID NOS.2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37. An isolated nucleic acid molecule encoding an NOVX protein homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
Mutations can be introduced into SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, the encoded protein can be expressed by any recombinant technology known in the art and the activity ofthe protein can be determined. The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues may be any one ofthe following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the "weak" group of conserved residues may be any one ofthe following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLJJVI, HFY, wherein the letters within each group represent the single letter amino acid code.
In one embodiment, a mutant NOVX protein can be assayed for (i) the ability to form proteimprotein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (ii) complex formation between a mutant NOVX protein and an NOVX ligand; or (iii) the ability of a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
In yet another embodiment, a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
Antisense Nucleic Acids
Another aspect ofthe invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of an NOVX protein of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37, or antisense nucleic acids complementary to an NOVX nucleic acid sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding an NOVX protein. The term "coding region" refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding the NOVX protein. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding the NOVX protein disclosed herein, antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of NOVX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid ofthe invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
.Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5 -methylaminomethyluracil, 5 -methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules ofthe invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an NOVX protein to thereby inhibit expression of the protein (e.g. , by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisense nucleic acid molecules ofthe invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β -units, the strands run parallel to each other. See, e.g., Gaultier, et al, 1987. Nucl. Acids Res. 15: 6625-6641. The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (See, e.g., ϊnoue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue (See, e.g., Inoue, et al, 1987. FEBS Lett. 215: 327-330.
Ribozymes and PNA Moieties
Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid ofthe invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in
Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for an NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of an NOVX cDNA disclosed herein (i.e., SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in an NOVX-encoding mRNA. See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al NOVX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Barrel et al, (1993) Science 261:1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe NOVX nucleic acid (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe NOVX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6: 569-84; Helene, et al 1992. Ann. NY. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
In various embodiments, the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule. For example, the deoxyribose phosphate backbone ofthe nucleic acids can be modified to generate peptide nucleic acids. See, e.g., Hyrup, et al, 1996. Bioorg Med Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al, 1996. supra; Perry-O'Keefe, et al, 1996. Proc. Natl Acad. Sci. USA 93: 14670-14675. PNAs of NOVX can be used in therapeutic and diagnostic applications. For example,
PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., Si nucleases (See, Hyrup, et al, 1996.supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al, 1996, supra; Perry-O'Keefe, et al, 1996. supra).
In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996. supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al, 1996. supra and Finn, et al, 1996. Nucl Acids Res 24: 3357-3363. For example, aDNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5,-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al, 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al, 1996. supra. Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al, 1975. Bioorg. Med. Chem. Lett. 5: 1119-11124. In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al, 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al, 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549). To this end, the oligonucleotide maybe conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
NOVX Polypeptides
A polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37 while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof.
In general, an NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues ofthe parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect ofthe invention pertains to isolated NOVX proteins, and biologically- active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies. In one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques, hi another embodiment, NOVX proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, an NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of NOVX proteins in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly-produced. h one embodiment, the language "substantially free of cellular material" includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX proteins. When the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% ofthe volume of the NOVX protein preparation.
The language "substantially free of chemical precursors or other chemicals" includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis ofthe protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals.
Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences ofthe NOVX proteins (e.g., the amino acid sequence shown in SEQ JD NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of an NOVX protein. Typically, biologically- active portions comprise a domain or motif with at least one activity ofthe NOVX protein. A biologically-active portion of an NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native NOVX protein.
In an embodiment, the NOVX protein has an amino acid sequence shown SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37, and retains the functional activity ofthe protein of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37, and retains the functional activity of the NOVX proteins of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37.
Determining Homology Between Two or More Sequences
To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. JMol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region ofthe analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part ofthe DNA sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, ' 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36. The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
Chimeric and Fusion Proteins The invention also provides NOVX chimeric or fusion proteins. As used herein, an
NOVX "chimeric protein" or "fusion protein" comprises an NOVX polypeptide operatively- linked to a non-NOVX polypeptide. An "NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an NOVX protein SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37), whereas a "non-NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within an NOVX fusion protein the NOVX polypeptide can correspond to all or a portion of an NOVX protein, hi one embodiment, an NOVX fusion protein comprises at least one biologically-active portion of an NOVX protein. In another embodiment, an NOVX fusion protein comprises at least two biologically- active portions of an NOVX protein. In yet another embodiment, an NOVX fusion protein comprises at least three biologically-active portions of an NOVX protein. Within the fusion protein, the term "operatively-linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another. The non-NOVX polypeptide can be fused to the N-terminus or C-terminus ofthe NOVX polypeptide. In one embodiment, the fusion protein is a GST-NO VX fusion protein in which the
NOVX sequences are fused to the C-terminus ofthe GST (glutathione S -transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX polypeptides.
In another embodiment, the fusion protein is an NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g. , mammalian host cells), expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence.
In yet another embodiment, the fusion protein is an NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member ofthe immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an NOVX ligand and an NOVX protein on the surface of a cell, to thereby suppress NOVX-mediated signal transduction in vivo. The NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of an NOVX cognate ligand. Inhibition of the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with an NOVX ligand.
An NOVX chimeric or fusion protein ofthe invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
NOVX Agonists and Antagonists
The invention also pertains to variants ofthe NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists. Variants ofthe NOVX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation ofthe NOVX protein). An agonist ofthe NOVX protein can retain substantially the same, or a subset of, the biological activities ofthe naturally occurring form ofthe NOVX protein. An antagonist of the NOVX protein can inhibit one or more ofthe activities ofthe naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe NOVX proteins.
Variants ofthe NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g. , truncation mutants) of the NOVX proteins for NOVX protein agonist or antagonist activity. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all ofthe sequences encoding the desired set of potential NOVX sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al, 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al, 1984. Science 198: 1056; Ike, et al, 1983. Nucl. Acids Res. 11: 477.
Polypeptide Libraries
In addition, libraries of fragments ofthe NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of an NOVX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Si nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes ofthe NOVX proteins.
Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of NOVX proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation ofthe vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave, et al, 1993. Protein Engineering 6:327-331.
Anti-NOVX Antibodies
Also included in the invention are antibodies to NOVX proteins, or fragments of NOVX proteins. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fa , Fab* and F(a ')2 fragments, and an Fab expression library. In general, an antibody molecule obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature ofthe heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGi, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species. An isolated NOVX-related protein ofthe invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments ofthe antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues ofthe amino acid sequence ofthe full length protein and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions ofthe protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments ofthe invention, at least one epitope encompassed by the antigenic peptide is a region of NOVX-related protein that is located on the surface ofthe protein, e.g., a hydrophilic region. A hydrophobicity analysis ofthe human NOVX-related protein sequence will indicate which regions of a NOVX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78:
3824-3828; Kyte and Doolittle 1982, J. Mol. Biol. 157: 105-142, each of which is incorporated herein by reference in its entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein. A protein ofthe invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein ofthe invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow and Lane, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Some of these antibodies are discussed below.
Polyclonal Antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative ofthe foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by inimunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
Monoclonal Antibodies The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope ofthe antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al, MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES AND APPLICATIONS, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells ofthe invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nαtwre 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody ofthe invention to create a chimeric bivalent antibody.
Humanized Antibodies
The antibodies directed against the protein antigens ofthe invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen- binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues ofthe human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin and all or substantially all ofthe framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al, 1988; and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)).
Human Antibodies
Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 hnmunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, hie, pp. 77-96). Human monoclonal antibodies may be utilized in the practice ofthe present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication
WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encodmg human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement ofthe modifications. The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker. A method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049.
Fab Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein ofthe invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fa fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(a ')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment; (iii) an Fab fragment generated by the treatment ofthe antibody molecule with papain and a reducing agent and (iv) Fv fragments.
Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one ofthe binding specificities is for an antigenic protein ofthe invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because ofthe random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, 1991 EMBO J, 10:3655-3659.
Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).
According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part ofthe CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield ofthe heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence ofthe dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One ofthe Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount ofthe other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J Immunol. 148(5): 1547- 1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary V and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J Immunol 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen ofthe invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
Heteroconjugate Antibodies
Heteroconjugate antibodies are also within the scope ofthe present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells
(U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO
92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
For example, immunotoxins can be constructed using a disulfide exchange reaction or by foπning a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.
4,676,980.
Effector Function Engineering It can be desirable to modify the antibody ofthe invention with respect to effector function, so as to enhance, e.g., the effectiveness ofthe antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989). Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPL PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 1311, 13Ifrι, 90Y, and 186Re.
Conjugates ofthe antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent. In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of an NOVX protein is facilitated by generation of hybridomas that bind to the fragment of an NOVX protein possessing such a domain. Thus, antibodies that are specific for a desired domain within an NOVX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
Anti-NOVX antibodies may be used in methods known within the art relating to the localization and/or quantitation of an NOVX protein (e.g., for use in measuring levels of the NOVX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like), h a given embodiment, antibodies for NOVX proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds (hereinafter "Therapeutics").
An anti-NOVX antibody (e.g., monoclonal antibody) can be used to isolate an NOVX polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-NOVX antibody can facilitate the purification of natural NOVX polypeptide from cells and of recombinantly-produced NOVX polypeptide expressed in host cells. Moreover, an anti-NOVX antibody can be used to detect NOVX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe NOVX protein. Anti-NOVX antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, 131I, 35S or 3H.
NOVX Recombinant Expression Vectors and Host Cells Another aspect ofthe invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an NOVX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are refeπed to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. The recombinant expression vectors ofthe invention comprise a nucleic acid ofthe invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression ofthe nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.).
The recombinant expression vectors ofthe invention can be designed for expression of NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein. Such fusion vectors typically serve three purposes: (i) to increase expression of recombinant protein; (ii) to increase the solubility ofthe recombinant protein; and (iii) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Ine; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N. J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET lld (Studier et α ., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al, 1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques. hi another embodiment, the NOVX expression vector is a yeast expression vector.
Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al, 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif). Alternatively, NOVX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid ofthe invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDMδ (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. In another embodiment, the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al, 1987. Genes Dev. 1 : 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al, 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al, 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Grass, 1990. Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule ofthe invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription ofthe DNA molecule) of an RNA molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression ofthe antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion ofthe regulation of gene expression using antisense genes see, e.g., Weintraub, et al, "Antisense RNA as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986. Another aspect ofthe invention pertains to host cells into which a recombinant expression vector ofthe invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope ofthe term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, NOVX protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NOVX or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell ofthe invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NOVX protein. Accordingly, the invention further provides methods for producing NOVX protein using the host cells ofthe invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals
The host cells ofthe invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more ofthe cells ofthe animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome ofthe mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell ofthe animal, prior to development ofthe animal.
A transgenic animal ofthe invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NOVX cDNA sequences SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue ofthe human NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operably-linked to the NOVX transgene to direct expression of NOVX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan, 1986. hi: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence ofthe NOVX transgene in its genome and/or expression of NOVX mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals caπying the transgene. Moreover, transgenic animals carrying a transgene- encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of an NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX gene can be a human gene (e.g., the cDNA of SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36), but more preferably, is a non-human homologue of a human NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ JO NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome, hi one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NOVX protein). In the homologous recombination vector, the altered portion ofthe NOVX gene is flanked at its 5'- and 3 '-termini by additional nucleic acid ofthe NOVX gene to allow for homologous recombination to occur between the exogenous NOVX gene carried by the vector and an endogenous NOVX gene in an embryonic stem cell. The additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5'- and 3'-termini) are included in the vector. See, e.g., Thomas, et al, 1987. Cell 51: 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX gene has homologously-recombined with the endogenous NOVX gene are selected. See, e.g., Li, et al, 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells ofthe animal contain the homologously-recombined DNA by gennline transmission ofthe transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI. For a description ofthe cre/loxP recombinase system, See, e.g., Lakso, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of
Saccharomyces cerevisiae. See, O'Gorman, et al, 1991. Science 251:1351-1355. Ifa cre/loxP recombinase system is used to regulate expression ofthe transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones ofthe non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, et al, 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter G0 phase. The quiescent cell can then be fused, e.g. , through the use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone ofthe animal from which the cell (e.g., the somatic cell) is isolated.
Pharmaceutical Compositions The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also referred to herein as "active compounds") ofthe invention, and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical admimstration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Prefeπed examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS), hi all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an NOVX protein or anti-NOVX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition. The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For admimstration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellent, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic admimstration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S.
Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Natl Acad. Sci. USA 91: 3054-3057).
The pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g. , retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Screening and Detection Methods The isolated nucleic acid molecules ofthe invention can be used to express NOVX protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in an NOVX gene, and to modulate NOVX activity, as described further, below. In addition, the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or abeπant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies ofthe invention can be used to detect and isolate NOVX proteins and modulate NOVX activity. In yet a further aspect, the invention can be used in methods to influence appetite, absorption of nutrients and the disposition of metabolic substrates in both a positive and negative fashion.
The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra.
Screening Assays
The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX protein activity.
The invention also includes compounds identified in the screening assays described herein. In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity ofthe membrane-bound form of an NOVX protein or polypeptide or biologically-active portion thereof. The test compounds ofthe invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the
"one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997 '. Anticancer Drug Design 12: 145. A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any ofthe assays ofthe invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al, 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb, et al, 1994. Proc. Natl Acad. Sci. U.S.A. 91: 11422; Zuckermann, et al, 1994. J Med. Chem. 37: 2678; Cho, et al, 1993. Science 261: 1303; Carrell, et al, 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et al, 1994. Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop, et al, 1994. J. Med. Chem. 37: 1233.
Libraries of compounds maybe presented in solution (e.g., Houghten, 1992. Biotechniques 13: 412-421), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al, 1990. Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to an NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding ofthe test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with I, S, C, or H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an NOVX protein, wherein determining the ability ofthe test compound to interact with an NOVX protein comprises determining the ability ofthe test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity ofthe NOVX protein or biologically-active portion thereof. Determining the ability ofthe test compound to modulate the activity of NOVX or a biologically-active portion thereof can be accomplished, for example, by determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule. As used herein, a "target molecule" is a molecule with which an NOVX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an NOVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. An NOVX target molecule can be a non-NOVX molecule or an NOVX protein or polypeptide ofthe invention. In one embodiment, an NOVX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding of a compound to a membrane-bound NOVX molecule) through the cell membrane and into the cell. The. target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NOVX.
Determimng the ability ofthe NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by one ofthe methods described above for determining direct binding. In one embodiment, determining the ability ofthe NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by determimng the activity ofthe target molecule. For example, the activity ofthe target molecule can be determined by detecting induction of a cellular second messenger ofthe target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity ofthe target an appropriate substrate, detecting the induction of a reporter gene (comprising an NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g. , luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation. In yet another embodiment, an assay ofthe invention is a cell-free assay comprising contacting an NOVX protein or biologically-active portion thereof with a test compound and determining the ability ofthe test compound to bind to the NOVX protein or biologically- active portion thereof. Binding ofthe test compound to the NOVX protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an NOVX protein, wherein determining the ability ofthe test compound to interact with an NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX or biologically-active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting NOVX protein or biologically-active portion thereof with a test compound and determimng the ability ofthe test compound to modulate (e.g. stimulate or inhibit) the activity ofthe NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX can be accomplished, for example, by determining the ability ofthe NOVX protein to bind to an NOVX target molecule by one ofthe methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX protein can be accomplished by determining the ability of the NOVX protein further modulate an NOVX target molecule. For example, the catalytic/enzymatic activity ofthe target molecule on an appropriate substrate can be determined as described, supra.
In yet another embodiment, the cell-free assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an NOVX protein, wherein determining the ability ofthe test compound to interact with an NOVX protein comprises determining the ability ofthe NOVX protein to preferentially bind to or modulate the activity of an NOVX target molecule. The cell-free assays ofthe invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, N-dodecyl--N,N-dimethyl-3-ammonio-l -propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-l-propane sulfonate (CHAPSO).
In more than one embodiment ofthe above assay methods ofthe invention, it may be desirable to immobilize either NOVX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both ofthe proteins, as well as to accommodate automation ofthe assay. Binding of a test compound to NOVX protein, or interaction of NOVX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both ofthe proteins to be bound to a matrix. For example, GST-NO VX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays ofthe invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g. , biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding ofthe NOVX protein to its target molecule, can be derivatized to the wells ofthe plate, and unbound target or NOVX protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.
In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX mRNA or protein in the cell is determined. The level of expression of NOVX mRNA or protein in the presence ofthe candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence ofthe candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i.e., statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of NOVX mRNA or protein expression. Alternatively, when expression of NOVX mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression. The level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein.
In yet another aspect ofthe invention, the NOVX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos, et al, 1993. Cell 72: 223-232; Madura, et al, 1993. J. Biol. Chem. 268: 12046-12054; Bartel, et al, 1993. Biotechniques 14: 920-924; Iwabuchi, et al, 1993. Oncogene 8:
1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with NOVX ("NOVX-binding proteins" or "NOVX-bp") and modulate NOVX activity. Such NOVX-binding proteins are also likely to be involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements ofthe NOVX pathway. The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain ofthe known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming an NOVX-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. This proximity allows franscription of a reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX.
The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
Detection Assays
Portions or fragments ofthe cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below.
Chromosome Mapping
Once the sequence (or a portion ofthe sequence) of a gene has been isolated, this sequence can be used to map the location ofthe gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments ofthe NOVX sequences, SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or fragments or derivatives thereof, can be used to map the location ofthe NOVX genes, respectively, on a chromosome. The mapping ofthe NOVX sequences to chromosomes is an important first step in coπelating these sequences with genes associated with disease. Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 bp in length) from the NOVX sequences. Computer analysis ofthe NOVX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene coπesponding to the NOVX sequences will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al, 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub- localization can be achieved with panels of fragments from specific chromosomes.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al, HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions ofthe genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data. Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al, 1987. Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
Tissue Typing
The NOVX sequences ofthe invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences ofthe invention are useful as additional DNA markers for RFLP ("restriction fragment length polymorphisms," described in U.S. Patent No. 5,272,057). Furthermore, the sequences ofthe invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini ofthe sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences ofthe invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences ofthe invention uniquely represent portions ofthe human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much ofthe allelic variation is due to single nucleotide polymorphisms (SNPs), which include restriction fragment length polymorphisms (RFLPs).
Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect ofthe invention relates to diagnostic assays for determining NOVX protein and/or nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g. , blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant NOVX expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in an NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.
Another aspect ofthe invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (refeπed to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g. , drugs) for therapeutic or prophylactic treatment of an individual based on the genotype ofthe individual (e.g., the genotype ofthe individual examined to determine the ability ofthe individual to respond to a particular agent.)
Yet another aspect ofthe invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials.
These and other agents are described in further detail in the following sections.
Diagnostic Assays
An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid of SEQ ID NOS.l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.
An agent for detecting NOVX protein is an antibody capable of binding to NOVX protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, pr a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently- labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method ofthe invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of NOVX mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immuno fluorescence. In vitro techniques for detection of NOVX genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging teclrniques. In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting NOVX protein, mRNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect NOVX protein or nucleic acid.
Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant NOVX expression or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue. Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant NOVX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g., wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant NOVX expression or activity).
The methods ofthe invention can also be used to detect genetic lesions in an NOVX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by aberrant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an NOVX-protein, or the misexpression ofthe NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: (i) a deletion of one or more nucleotides from an NOVX gene; (ii) an addition of one or more nucleotides to an NOVX gene; (iii) a substitution of one or more nucleotides of an NOVX gene, (z'v) a chromosomal rearrangement of an NOVX gene; (v) an alteration in the level of a messenger RNA transcript of an NOVX gene, (vi) aberrant modification of an NOVX gene, such as ofthe methylation pattern ofthe genomic DNA, (vii) the presence of a non- wild-type splicing pattern of a messenger RNA transcript of an NOVX gene, (viii) a non-wild-type level of an NOVX protein, (ix) allelic loss of an NOVX gene, and (x) inappropriate post-translational modification of an NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in an NOVX gene. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
In certain embodiments, detection ofthe lesion involves the use of a probe/primer in a poiymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et αl., 1988. Science 241: 1077-1080; andNakazawa, et αl, 1994. Proc. Nαtl Acαd. Sci. USA 91 : 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et αl., 1995. Nucl. Acids Res. 23: 675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an NOVX gene under conditions such that hybridization and amplification ofthe NOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size ofthe amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe techniques used for detecting mutations described herein. Alternative amplification methods include: self sustained sequence replication (see,
Guatelli, et al, 1990. Proc. Natl. Acad. Sci. USA 87: 1874-1878), franscriptional amplification system (see, Kwoh, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177); Qβ Replicase (see, Lizardi, et al, 1988. BioTechnology 6: 1197), or any other nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in an NOVX gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent No. 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. In other embodiments, genetic mutations in NOVX can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes. See, e.g., Cronin, et al, 1996. Human Mutation 7: 244-255; Kozal, et al, 1996. Nat. Med. 2: 753-759. For example, genetic mutations in NOVX can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, et al, supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene. In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the NOVX gene and detect mutations by comparing the sequence ofthe sample NOVX with the corresponding wild-type (confrol) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977. Proc. Natl. Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e.g., Naeve, et al, 1995. Biotechniques 19: 448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen, et al, 1996. Adv. Chromatography 36: 127-162; and Griffin, et al, 1993. Appl Biochem. Biotechnol. 38: 147-159).
Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA RNA or RNA/DNA heteroduplexes. See, e.g., Myers, et al, 1985. Science 230: 1242. In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions ofthe duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with Si nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion ofthe mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton, et al, 1988. Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al, 1992. Methods Enzymol. 217: 286-295. hi an embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches. See, e.g., Hsu, et al, 1994. Carcinogenesis 15: 1657-1662. According to an exemplary embodiment, a probe based on an NOVX sequence, e.g., a wild-type NOVX sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039. In other embodiments, alterations in elecfrophoretic mobility will be used to identify mutations in NOVX genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in elecfrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et αl, 1989. Proc. Nαtl Acαd. Sci. USA: 86: 2766; Cotton, 1993. Mutαt. Res. 285: 125-144; Hayashi, 1992. Genet. Anal Tech. Appl 9: 73-79. Single-stranded DNA fragments of sample and confrol NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in elecfrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity ofthe assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in elecfrophoretic mobility. See, e.g., Keen, et al, 1991. Trends Genet. 1: 5. hi yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al, 1985. Nature 313: 495. When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. hi a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of confrol and sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al, 1986. Nature 324: 163; Saiki, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center ofthe molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al, 1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11: 238). hi addition it may be desirable to introduce a novel restriction site in the region ofthe mutation to create cleavage-based detection. See, e.g., Gasparini, et al, 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189. In such cases, ligation will occur only if there is a perfect match at the 3'-terminus ofthe 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an NOVX gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which NOVX is expressed may be utilized in the prognostic assays described herein. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
Pharmacogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on NOVX activity (e.g., NOVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.) In conjunction with such treatment, the pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drag) ofthe individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration ofthe pharmacologically active drag. Thus, the pharmacogenomics ofthe individual permits the selection of effective agents (e.g., drags) for prophylactic or therapeutic treatments based on a consideration ofthe individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. Pharmacogenomics deals with clinically significant hereditary variations in the response to drags due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol, 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drag action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drag metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drags (anti-malarials, sulfonamides, analgesics, nifrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drag action. The discovery of genetic polymorphisms of drag metabolizing enzymes (e.g., N-acetylfransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drag effects or show exaggerated drag response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among .different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drag response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification. Thus, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drag selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an NOVX modulator, such as a modulator identified by one ofthe exemplary screening assays described herein.
Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drags, compounds) on the expression or activity of NOVX (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase NOVX gene expression, protein levels, or upregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting decreased NOVX gene expression, protein levels, or downregulated NOVX activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NOVX gene expression, protein levels, or downregulate NOVX activity, can be monitored in clinical frails of subjects exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity. In such clinical trials, the expression or activity of NOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers ofthe immune responsiveness of a particular cell.
By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NOVX activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one ofthe methods as described herein, or by measuring the levels of activity of NOVX or other genes. In this mam er, the gene expression pattern can serve as a marker, indicative ofthe physiological response ofthe cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment ofthe individual with the agent. In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drag candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of an NOVX protein, mRNA, or genomic DNA in the preadminisfration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity ofthe NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity ofthe NOVX protein, mRNA, or genomic DNA in the pre-administration sample with the NOVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (yi) altering the administration ofthe agent to the subject accordingly. For example, increased administration ofthe agent may be desirable to increase the expression or activity of NOVX to higher levels than detected, i.e., to increase the effectiveness ofthe agent. Alternatively, decreased administration ofthe agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i.e., to decrease the effectiveness ofthe agent. Methods of Treatment
The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with abeπant NOVX expression or activity. The disorders include cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodysfrophy, congenital adrenal hyperplasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sclerosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions ofthe like. These methods of treatment will be discussed more fully, below.
Disease and Disorders
Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysftinctionaP' (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and antagonists, including additional peptide mimetic ofthe invention or antibodies specific to a peptide ofthe invention) that alter the interaction between an aforementioned peptide and its binding partner. Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity ofthe expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemisfry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).
Prophylactic Methods
In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an abeπant NOVX expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVX activity. Subjects at risk for a disease that is caused or contributed to by aberrant NOVX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe NOVX aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of NOVX aberrancy, for example, an NOVX agonist or NOVX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods ofthe invention are further discussed in the following subsections.
Therapeutic Methods
Another aspect ofthe invention pertains to methods of modulating NOVX expression or activity for therapeutic purposes. The modulatory method ofthe invention involves contacting a cell with an agent that modulates one or more ofthe activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of an NOVX protein, a peptide, an NOVX peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NOVX protein activity. Examples of such stimulatory agents include active NOVX protein and a nucleic acid molecule encoding NOVX that has been introduced into the cell. In another embodiment, the agent inhibits one or more NOVX protein activity. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an NOVX protein or nucleic acid molecule, h one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) NOVX expression or activity. In another embodiment, the method involves administering an NOVX protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NOVX expression or activity.
Stimulation of NOVX activity is desirable in situations in which NOVX is abnormally downregulated and/or in which increased NOVX activity is likely to have a beneficial effect. One example of such a situation is where a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders). Another example of such a situation is where the subject has a gestational disease (e.g., preclampsia).
Determination of the Biological Effect of the Therapeutic
In various embodiments ofthe invention, suitable in vitro or in vivo assays are performed to determine the effect of a specific Therapeutic and whether its administration is indicated for treatment ofthe affected tissue. In various specific embodiments, in vitro assays may be performed with representative cells ofthe type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s). Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any ofthe animal model system known in the art may be used prior to administration to human subjects. Prophylactic and Therapeutic Uses of the Compositions of the Invention
The NOVX nucleic acids and proteins ofthe invention are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.
As an example, a cDNA encoding the NOVX protein ofthe invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions ofthe invention will have efficacy for treatment of patients suffering from: metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyslipidemias.
Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein ofthe invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed. A further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of antibodies, which immunospecifically-bind to the novel substances ofthe invention for use in therapeutic or diagnostic methods.
The invention will be further described in the following examples, which do not limit the scope ofthe invention described in the claims.
Examples
Example 1. Identification of NOVX clones
The novel NOVX target sequences identified in the present invention were subjected to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. Table 17A shows the sequences ofthe PCR primers ~wd tor obtaimng different clones. In each case, the sequence was examined, walk ng inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence ofthe target sequence, or by translated homology ofthe predicted exons to closely related human sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The PCR product derived from exon linking was cloned into the pCR2.1 vector from Invitrogen. The resulting bacterial clone has an insert covering the entire open reading frame cloned into the pCR2.1 vector. Table 17B shows a list of these bacterial clones. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component ofthe assembly was at least 95%) over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. These procedures provide the sequence reported herein.
Table 10A. PCR Primers for Exon Linking
Figure imgf000189_0001
Physical clone: Exons were predicted by homology and the infron/exon boundaries were determined using standard genetic rules. Exons were further selected and refined by means of similarity determination using multiple BLAST (for example, tBlastN, BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein.
Table 10B. Physical Clones for PCR products
NOVX Clone Bacterial Clone
NOVl Physical clone hRPK.474_N 24, 211,945 bp
NOV2 Physical clone RP11-60L3, 172,244 bp
NOV3 Physical clone AP000501
NOV4 Physical clone ba56pl0.698056.K10 FLC EL
NOV5a-c Physical clone RP4-784A16, 139,557 bp
NOV5d-i Physical clone sggc_draft_dj784al6 20000723 da3
NOV6 Physical clone AL031711.25
NOV7 Genomic file: 10d7
NOV8 Genomic file: ba550p23
Example 2. Quantitative expression analysis of clones in various cells and tissues
The quantitative expression of various clones was assessed using microtiter plates containing RNA samples from a variety of normal and pathology-derived cells, cell lines and tissues using real time quantitative PCR (RTQ PCR). RTQ PCR was performed on a Perkin- Elmer Biosystems ABI PRISM® 7700 Sequence Detection System. Various collections of samples are assembled on the plates, and referred to as Panel 1 (containing cells and cell lines from normal and cancer sources), Panel 2 (containing samples derived from tissues, in particular from surgical samples, from normal and cancer sources), Panel 3 (containing samples derived from a wide variety of cancer sources), Panel 4 (containing cells and cell lines from normal cells and cells related to inflammatory conditions) and Panel CNSD.01 (containing samples from normal and diseased brains).
First, the RNA samples were normalized to reference nucleic, acids such as constitutively expressed genes (for example, β-actin and GAPDH). Normalized RNA (5 ul) was converted to cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix Reagents (PE Biosystems; Catalog No. 4309169) and gene-specific primers according to the manufacturer's instructions. Probes and primers were designed for each assay according to
Perkin Elmer Biosystem's Primer Express Software package (version I for Apple Computer's Macintosh Power PC) or a similar algorithm using the target sequence as input. Default settings were used for reaction conditions and the following parameters were set before selecting primers: primer concentration = 250 nM, primer melting temperature (Tm) range = 58°-60° C, primer optimal Tm = 59° C, maximum primer difference = 2° C, probe does not have 5' G, probe Tm must be 10° C greater than primer Tm, amplicon size 75 bp to 100 bp. The probes and primers selected (see below) were synthesized by Synthegen (Houston, TX, USA). Probes were double purified by HPLC to remove uncoupled dye and evaluated by mass spectroscopy to verify coupling of reporter and quencher dyes to the 5' and 3' ends of the probe, respectively. Their final concentrations were: forward and reverse primers, 900 nM each, and probe, 200nM.
PCR conditions: Normalized RNA from each tissue and each cell line was sported in each well of a 96 well PCR plate (Perkin Elmer Biosystems). PCR cocktails including two probes (a probe specific for the target clone and another gene-specific probe multiplexed with the target probe) were set up using IX TaqMan™ PCR Master Mix for the PE Biosystems 7700, with 5 mM MgC12, dNTPs (dA, G, C, U at 1 : 1 : 1 :2 ratios), 0.25 U/ml AmpliTaq Gold™ (PE Biosystems), and 0.4 U/μl RNase inhibitor, and 0.25 U/μl reverse transcriptase. Reverse transcription was performed at 48° C for 30 minutes followed by amplification/PCR cycles as follows: 95° C IO min, then 40 cycles of 95° C for 15 seconds, 60° C for 1 minute. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) using a log scale, with the difference in RNA concentration between a given sample and the sample with the lowest CT value being represented as 2 to the power of delta CT. The percent relative expression is then obtained by taking the reciprocal of this RNA difference and multiplying by 100.
In the results for Panel 1, the following abbreviations are used: ca. = carcinoma,
* = established from metastasis, met = metastasis, s cell var = small cell variant, non-s = non-sm = non-small, squam = squamous, pi. eff = pi effusion = pleural effusion, glio = glioma, astro = astrocytoma, and neuro = neuroblastoma.
Panel 2
The plates for Panel 2 generally include 2 control wells and 94 test samples composed of RNA or cDNA isolated from human tissue procured by surgeons working in close cooperation with the National Cancer Institute's Cooperative Human Tissue Network (CHTN) or the National Disease Research Initiative (NDRI). The tissues are derived from human malignancies and in cases where indicated many malignant tissues have "matched margins" obtained from noncancerous tissue just adjacent to the tumor. These are termed normal adjacent tissues and are denoted "NAT" in the results below. The tumor tissue and the "matched margins" are evaluated by two independent pathologists (the surgical pathologists and again by a pathologists at NDRI or CHTN). This analysis provides a gross histopathological assessment of tumor differentiation grade. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical stage of the patient. These matched margins are taken from the tissue surrounding (i.e. immediately proximal) to the zone of surgery (designated "NAT", for normal adjacent tissue, in Table RR). In addition, RNA and cDNA samples were obtained from various human tissues derived from autopsies performed on elderly people or sudden death victims (accidents, etc.). These tissues were ascertained to be free of disease and were purchased from various commercial sources such as Clontech (Palo Alto, CA), Research Genetics, and Invifrogen. RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s:18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
Panel 3D The plates of Panel 3D are comprised of 94 cDNA samples and two confrol samples. Specifically, 92 of these samples are derived from cultured human cancer cell lines, 2 samples of human primary cerebellar tissue and 2 confrols. The human cell lines are generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: Squamous cell carcinoma ofthe tongue, breast cancer, prostate cancer, melanoma, epidermoid carcinoma, sarcomas, bladder carcinomas, pancreatic cancers, kidney cancers, leukemias/lymphomas, ovarian/uterine/cervical, gastric, colon, lung and CNS cancer cell lines. In addition, there are two independent samples of cerebellum. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. The cell lines in panel 3D and 1.3D are ofthe most common cell lines used in the scientific literature.
RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5:1 28s: 18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
Panel 4
Panel 4 includes samples on a 96 well plate (2 control wells, 94 test samples) composed of RNA (Panel 4r) or cDNA (Panel 4d) isolated from various human cell lines or tissues related to inflammatory conditions. Total RNA from control normal tissues such as colon and lung (Stratagene ,La Jolla, CA) and thymus and kidney (Clontech) were employed. Total RNA from liver tissue from cirrhosis patients and kidney from lupus patients was obtained from BioChain (Biochain Institute, Inc., Hayward, CA). Intestinal tissue for RNA preparation from patients diagnosed as having Crohn's disease and ulcerative colitis was obtained from the National Disease Research Interchange (NDRI) (Philadelphia, PA).
Astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, micro vascular dermal endothelial cells, microvascular lung endothelial cells, human pulmonary aortic endothelial cells, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, MD) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or 12-14 hours, as indicated. The following cytokines were used; IL-1 beta at approximately 1-5 ng/ml, TNF alpha at approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml, IL-4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml, IL-13 at approximately 5-10 ng/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1% serum.
Mononuclear cells were prepared from blood of employees at CuraGen Corporation, using Ficoll. LAK cells were prepared from these cells by culture in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco/Life Technologies, Rockville, MD), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x IO"5 M (Gibco), and 10 mM Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either activated with 10-20 ng/ml PMA and 1-2 μg/ml ionomycin, IL-12 at 5-10 ng/ml, IFN gamma at 20-50 ng/ml and IL-18 at 5-10 ng/ml for 6 hours. In some cases, mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x IO"5 M (Gibco), and 10 mM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5 μg/ml. Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction) samples were obtained by taking blood from two donors, isolating the mononuclear cells using Ficoll and mixing the isolated mononuclear cells 1:1 at a final concenfration of approximately 2xl06 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol (5.5 x IO"5 M) (Gibco), and 10 mM Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging from 1- 7 days for RNA preparation. Monocytes were isolated from mononuclear cells1 using CD 14 Miltenyi Beads, +ve VS selection columns and a Vario Magnet according to the manufacturer's instructions. Monocytes were differentiated into dendritic cells by culture in DMEM 5% fetal calf serum (FCS) (Hyclone, Logan, UT), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x IO"5 M (Gibco), and 10 mM Hepes (Gibco), 50 ng/ml GMCSF and 5 ng ml J -4 for 5-7 days. Macrophages were prepared by culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x IO"5 M (Gibco), 10 mM Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately 50 ng/ml. Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with lipopolysaccharide (LPS) at 100 ng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at 10 μg/ml for 6 and 12-14 hours. CD4 lymphocytes, CD8 lymphocytes and NK cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and a Vario Magnet according to the manufacturer's instructions. CD45RA and CD45RO CD4 lymphocytes were isolated by depleting mononuclear cells of CD8, CD56, CD14 and CD19 cells using CD8, CD56, CD14 and CD19 Miltenyi beads and positive selection. Then CD45RO beads were used to isolate the CD45RO CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes. CD45RA CD4, CD45RO CD4 and CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x IO"5 M (Gibco), and 10 mM Hepes (Gibco) and plated at IO6 cells/ml onto Falcon 6 well tissue culture plates that had been coated overnight with 0.5 μg/ml anti-CD28 (Pharmingen) and 3 ug/ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA preparation. To prepare chronically activated CD8 lymphocytes, we activated the isolated CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and then harvested the cells and expanded them in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyravate (Gibco), mercaptoethanol 5.5 x 10" M (Gibco), and 10 mM Hepes (Gibco) and IL-2. The expanded CD8 cells were then activated again with plate bound anti-CD3 and anti-CD28 for 4 days and expanded as before. RNA was isolated 6 and 24 hours after the second activation and after 4 days ofthe second expansion culture. The isolated NK cells were cultured in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyravate (Gibco), mercaptoethanol 5.5 x IO"5 M (Gibco), and 10 mM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared.
To obtain B cells, tonsils were procured from NDRI. The tonsil was cut up with sterile dissecting scissors and then passed through a sieve. Tonsil cells were then spun down and resupended at IO6 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyravate (Gibco), mercaptoethanol 5.5 x 10"5 M (Gibco), and 10 mM Hepes (Gibco). To activate the cells, we used PWM at 5 μg/ml or anti-CD40 (Pharmingen) at approximately 10 μg/ml and LL-4 at 5-10 ng/ml. Cells were harvested for RNA preparation at 24,48 and 72 hours.
To prepare the primary and secondary Thl/Th2 and Trl cells, six-well Falcon plates were coated overnight with 10 μg/ml anti-CD28 (Pharmingen) and 2 μg/ml OKT3 (ATCC), and then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic Systems,
5 6
German Town, MD) were cultured at 10 -10 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10" 5 M (Gibco), 10 mM Hepes (Gibco) and IL-2 (4 ng/ml). IL-12 (5 ng/ml) and anti-IL4 (1 Dg/ml) were used to direct to Thl, while IL-4 (5 ng/ml) and anti-IFN gamma (1 Dg/ml) were used to direct to Th2 and IL-10 at 5 ng/ml was used to direct to Trl. After 4-5 days, the activated Thl, Th2 and Trl lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyravate (Gibco), mercaptoethanol 5.5 x IO"5 M (Gibco), 10 mM Hepes (Gibco) and IL-2 (1 ng/ml). Following this, the activated Thl, Th2 and Trl lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti- CD95L (1 Dg/ml) to prevent apoptosis. After 4-5 days, the Thl, Th2 and Trl lymphocytes were washed and then expanded again with IL-2 for 4-7 days. Activated Thl and Th2 lymphocytes were maintained in this way for a maximum of three cycles. RNA was prepared from primary and secondary Thl, Th2 and Trl after 6 and 24 hours following the second and third activations with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures in Interleukin 2.
The following leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated by culture in 0.1 mM dbcAMP at 5 xlO5 cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to 5 xlO5 cells/ml. For the culture ofthese cells, we used DMEM or RPMI (as recommended by the ATCC), with the addition of 5%> FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyravate (Gibco), mercaptoethanol 5.5 x IO"5 M (Gibco), 10 mM Hepes (Gibco). RNA was either prepared from resting cells or cells activated with PMA at 10 ng/ml and ionomycin at 1 μg/ml for 6 and 14 hours. Keratinocyte line CCD106 and an airway epithelial tumor line NCI-H292 were also obtained from the ATCC. Both were cultured in
DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x IO"5 M (Gibco), and 10 mM Hepes (Gibco). CCD1106 cells were activated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and 1 ng/ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL-13 and 25 ng/ml IFN gamma.
For these cell lines and blood cells, RNA was prepared by lysing approximately IO7 cells/ml using Trizol (Gibco BRL). Briefly, 1/10 volume of bromochloropropane (Molecular Research Corporation) was added to the RNA sample, vortexed and after 10 minutes at room temperature, the tubes were spun at 14,000 rpm in a Sorvall SS34 rotor. The aqueous phase was removed and placed in a 15 ml Falcon Tube. An equal volume of isopropanol was added and left at -20 degrees C overnight. The precipitated RNA was spun down at 9,000 rpm for 15 min in a Sorvall SS34 rotdr and washed in 70% ethanol. The pellet was redissolved in 300 μl of RNAse-free water and 35 μl buffer (Promega) 5 μl DTT, 7 μl RNAsin and 8 μl DNAse were added. The tube was incubated at 37 degrees C for 30 minutes to remove contaminating genomic DNA, extracted once with phenol chloroform and re-precipitated with 1/10 volume of 3 M sodium acetate and 2 volumes of 100% ethanol. The RNA was spun down and placed in RNAse free water. RNA was stored at -80 degrees C.
Panel CNSD.01
The plates for Panel CNSD.01 include two confrol wells and 94 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center. Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology. Disease diagnoses are taken from patient records. The panel contains two brains from each ofthe following diagnoses: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supernuclear Palsy, Depression, and "Normal controls". Within each of these brains, the following regions are represented: cingulate gyrus, temporal pole, globus palladus, substantia nigra, Brodman Area 4 (primary motor strip), Brodman Area 7 (parietal cortex), Brodman Area 9 (prefrontal cortex), and Brodman area 17 (occipital cortex). Not all brain regions are represented in all cases; e.g., Huntington's disease is characterized in part by neurodegeneration in the globus palladus, thus this region is impossible to obtain from confirmed Huntington's cases. Likewise Parkinson's disease is characterized by degeneration ofthe substantia nigra making this region more difficult to obtain. Normal confrol brains were examined for neuropathology and found to be free of any pathology consistent with neurodegeneration.
RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2: 1 to 2.5: 1 28s: 18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon. In the labels employed to identify tissues in the CNS panel, the following abbreviations are used:
PSP = Progressive supranuclear palsy
Sub Nigra = Substantia nigra
Glob Palladus= Globus palladus Temp Pole = Temporal pole
Cing Gyr = Cingulate gyrus
BA 4 = Brodman Area 4
The AC068339_A gene encodes a G protein-coupled receptor (GPCR), a type of cell surface receptor involved in signal transduction. The AC068339_A gene product is most similar to members ofthe odorant receptor subfamily of GPCRs. Based on analogy to other odorant receptor genes, we predict that expression ofthe AC068339_A gene may be highest in nasal epithelium, a sample not represented on these panels.
NOVl
Expression of gene AC013554_dal was assessed using the primer-probe set Ag2025, described in Table 11. Results from RTQ-PCR runs are shown in Table 12.
Table 11. Probe Name Ag2025
Figure imgf000198_0001
Figure imgf000199_0001
Table 12. Panel 1.3D
Figure imgf000199_0002
Figure imgf000200_0001
Panel 1.3D Summary: Ag2025 Expression ofthe NOVl gene is detected exclusively in testis among the samples on this panel and the transcript is present at moderate levels (CT = 29.6). Therefore, this gene could be used to distinguish testis from the other tissues on this panel. The NOVl gene encodes a protein with homology to thioredoxins, small ubiquitous proteins that participate in different cellular processes via redox-mediated reactions. An example of a thioredoxin that is expressed exclusively in spermatozoa has recently been reported (ref. 1). Therefore, the NOVl gene product may also play a role in spermiogenesis and/or fertility.
Panel 2.2 Summary: Ag2025 Expression ofthe NOVl gene is low/undetectable (CT values > 35) across the samples on this panel (data not shown).
Panel 4D Summary: Ag2025 Expression ofthe NOVl gene is low/undetectable (CT values > 35) across the samples on this panel (data not shown).
References: 1. Miranda- Vizuete A., Ljung J., Damdimopoulos A.E., Gustafsson J.A., Oko R., Pelto-Huikko M., Spyrou G. (2001) Characterization of Sptrx, a novel member ofthe thioredoxin family specifically expressed in human spermatozoa. J. Biol. Chem. Jun 8 [epub ahead of print].
Thioredoxins (Trx) are small ubiquitous proteins that participate in different cellular processes via redox-mediated reactions. We report here the identification and characterization of a novel member ofthe thioredoxin family in human, named Sptrx (sperm-specific trx), the first with a tissue-specific distribution, located exclusively in spermatozoa. Sptrx ORF encodes for a protein of 486 amino acids comprised of two clear domains: an N-terminal domain consisting of 23 highly conserved repetitions of a 15-residue motif and a C-terminal domain typical of thioredoxins. Northern analysis and in situ hybridization shows that Sptrx RNA is only expressed in human testis, specifically in round and elongating spermatids. hnmunostaining of human testis sections identified Sptrx protein in spermatids while immunofluorescence and immunogold electron microscopy analysis demonstrated Sptrx localization in the cytoplasmic droplet of ejaculated sperm. Sptrx appears to have a multimeric structure in native conditions and is able to reduce insulin disulfide bonds in the presence of NADPH and thioredoxin reductase. During mammalian spermiogenesis in testis seminiferous tubules and later maturation in epididymis, extensive reorganization of disulfide bonds is required to stabilize cytoskeletal sperm structures. However, the molecular mechanisms that control these processes are not known. The identification of Sptrx with an expression pattern restricted to the post meiotic phase of spermatogenesis, when sperm tail is organized, suggests that Sptrx might be an important factor in regulating critical steps of human spermiogenesis.
NOV2a
Expression ofthe NOV2a gene was assessed using the primer-probe set Ag850, described in Table 13. Results from RTQ-PCR runs are shown in Tables 14 and 15.
Table 13. Probe Name Ag850 (please note that there is single base mismatch within the forward primer that is not expected to disrupt primer binding)
Figure imgf000202_0001
Table 14. Panel 2.2
Figure imgf000202_0002
Figure imgf000203_0001
Figure imgf000204_0001
Table 15. Panel 4.1D
Figure imgf000204_0002
Figure imgf000205_0001
Figure imgf000206_0001
Panel 2.2 Summary: Ag850 The NOV2a gene is most highly expressed in a sample derived from normal kidney tissue adjacent to a kidney cancer (CT = 28.4), among the samples on this panel. Furthermore, in 7 out of 9 cases this gene is expressed more highly in normal kidney tissues adjacent to kidney cancers, hi addition, there also appears to be over expression ofthe NOV2a gene in lung cancers when compared to their normal adjacent tissue. Thus, the expression ofthe NOV2a gene could be used to distinguish normal kidney tissue from kidney cancer or lung cancer tissue from normal lung tissue. In addition, therapeutic modulation ofthe protein encoded by this gene may be of use in the treatment of kidney cancer or lung cancer.
Panel 4.1D Summary: Ag850 The NOV2a gene is expressed in normal kidney, thymus, a dermal fibroblast cell line (CCD1070) and in asfrocytes, among the samples on this panel. Expression of this gene is highest in kidney (CT = 28.1), consistent with what is observed in Panel 2.2. The NOV2a gene encodes a protein that has homology to retinoic acid- responsive protein, which is known to be expressed at blood organ barriers and may function in transport (ref. 1). Asfrocytes contribute to the blood brain barrier (ref. 2), so based on the homology of this gene to Stra6 and its expression in asfrocytes, the NOV2a gene product may be important in maintaining the blood brain barrier and perhaps in the fransport of small molecules across this barrier. Therefore, regulation of NOV2a gene product with small molecule therapeutics could allow the passage of specific therapeutic molecules into the brain usually blocked by tight junctions. Furthermore, modulation ofthe function of this protein may also be important in the treatment of autism, since it has been recently shown that autism may be linked to pathology associated with infection and this particular gene is found within a chromosomal locus associated with autism (ref. 3).
References:
1. Bouillet P., Sapin V, Chazaud C, Messaddeq N., Decimo D., Dolle P., Chambon P.
(1997) Developmental expression pattern of Stra6, a retinoic acid-responsive gene encoding a new type of membrane protein. Mech. Dev. 63: 173-186.
Retinoic acid plays important roles in development, growth and differentiation by regulating the expression of target genes. A new retinoic acid-inducible gene, Stra6, has been identified in PI 9 embryonal carcinoma cells using a subtractive hybridization cDNA cloning technique. Sfra6 codes for a very hydrophobic membrane protein of a new type, which does not display similarities with previously characterized integral membrane proteins. Stra6, which exhibits a specific pattern of expression during development and in the adult, is strongly expressed at the level of blood-organ barriers. Interestingly, in testis Sertoli cells, Stra6 has a spermatogenic cycle-dependent expression which is lost in testes of RAR alpha null mutants where Stra6 is expressed in all tubules. We suggest that the Stra6 protein may be a component of an as yet unidentified transport machinery.
PMID: 9203140
2. Pardridge W.M. (1999) Blood-brain barrier biology and methodology. J. Neurovirol. 5: 556-69.
The blood-brain barrier (BBB) is formed by epithelial-like high resistance tight junctions within the endothelium of capillaries perfusing the vertebrate brain. Because ofthe presence ofthe BBB, circulating molecules gain access to brain cells only via one of two processes: (i) lipid-mediated transport of small molecules through the BBB by free diffusion, or (ii) catalyzed transport. The latter includes carrier-mediated transport processes for low molecular weight nutrients and water soluble vitamins or receptor-mediated transport for circulating peptides (e.g., insulin), plasma proteins (e.g., transferrin), or viruses. While BBB permeability, per se, is controlled by the biochemical properties ofthe plasma membranes of the capillary endothelial cells, overall brain microvascular biology is a function ofthe paracrine interactions between the capillary endothelium and the other two major cells comprising the microcirculation of brain, i.e., the capillary pericyte, which shares the basement membrane with the endothelial cell, and the asfrocyte foot process, which invests 99%) ofthe abluminal surface ofthe capillary basement membrane in brain. Microvascular functions frequently ascribed to the capillary endothelium are actually executed by either the capillary pericyte or the capillary asfrocyte foot process. With respect to BBB methodology, there are a variety of in vivo methods for studying biological transport across this important membrane. The classical physiologic techniques may now be correlated with modern biochemical and molecular biological approaches using freshly isolated animal or human brain capillaries. Isolated brain capillary endothelial cells can also be grown in tissue culture to form an 'in vitro BBB' model. However, BBB research cannot be performed using only the in vitro BBB model, but rather it is necessary to correlate observations made with the in vitro BBB model with in vivo studies.
PMID: 10602397
3. Hornig M., Weissenbock H., Horscroft N, Lipkin W.I. (1999) An infection-based model of neurodevelopmental damage. Proc. Natl. Acad. Sci. U S A 96: 12102-12107.
Perinatal exposure to infectious agents and toxins is linked to the pathogenesis of neuropsychiatric disorders, but the mechanisms by which environmental triggers interact with developing immune and neural elements to create neurodevelopmental disturbances are poorly understood. We describe a model for investigating disorders of central nervous system development based on neonatal rat infection with Borna disease virus, a neurotropic noncytolytic RNA virus. Infection results in abnormal righting reflexes, hyperactivity, inhibition of open-field exploration, and stereotypic behaviors. Architecture is markedly disrupted in hippocampus and cerebellum, with reduction in granule and Purkinje cell numbers. Neurons are lost predominantly by apoptosis, as supported by increased mRNA levels for pro-apoptotic products (Fas, caspase-1), decreased mRNA levels for the anti- apoptotic bcl-x, and in situ labeling of fragmented DNA. Although inflammatory infiltrates are observed transiently in frontal cortex, glial activation (microgliosis > asfrocytosis) is prominent throughout the brain and persists for several weeks in concert with increased levels of proinflammatory cytokine mRNAs (interleukins 1 alpha, lbeta, and 6 and tumor necrosis factor alpha) and progressive hippocampal and cerebellar damage. The resemblance ofthese functional and neuropathologic abnormalities to human neurodevelopmental disorders suggests the utility of this model for defining cellular, biochemical, histologic, and functional outcomes of interactions of environmental influences with the developing central nervous system.
PMID: 10518583
NOV3a
Expression ofthe NOV3a gene was assessed using the primer-probe set Agl402, described in Table 16. Results from RTQ-PCR runs are shown in Tables 17, 18, 19, and 20.
Table 16. Probe Name Agl402
Figure imgf000209_0001
Table 17. Panel 1.2
Relative Expression(%)
1.2tml660f_ 1.2tml691f_
Tissue Name ag!402* ag!402*
Endothelial cells 15.3 15.8
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000212_0001
Table 18. Panel 2.2
Figure imgf000212_0002
Figure imgf000213_0001
Figure imgf000214_0001
Table 19. Panel 3D
Figure imgf000214_0002
Figure imgf000215_0001
Figure imgf000216_0001
Table 20. Panel 4D
Figure imgf000216_0002
Figure imgf000217_0001
Figure imgf000218_0001
Figure imgf000219_0001
Panel 1.2 Summary: Agl402 Results from two experiments using the same probe/primer are in good agreement. The NOV3a gene is expressed at highest levels in heart (CT = 22-23), prostate, and ovarian tissue and is expressed to a lesser degree across a number of samples in Panel 1.2. Of note is the observation that this gene is expressed largely in normal tissues when compared to cultured cell lines. Thus, expression ofthe NOV3a gene could be used to distinguish heart, prostate and ovarian tissue from other tissues.
Among other metabolically relevant tissues, the NOV3a gene is moderately expressed in thyroid, pituitary gland, and pancreas, and is highly expressed in adrenal gland, skeletal muscle and fetal/adult liver. High expression of this gene in insulin-responsive tissues such as skeletal muscle and liver suggests that the NOV3a gene product might be an effective drug target for the treatment of Type 2 diabetes. High expression in adrenal gland and heart also suggests that this gene may be an antibody target for the treatment of diseases involving these two tissues.
The NOV3a gene encodes a putative leucine-rich-repeat (LRR), GPCR-like protein. In Drosophilia, the LRR region of axon guidance proteins has been shown to be critical for function, especially in axon repulsion (ref. 1). The leucine-rich-repeat protein encoded by the NOV3a gene shows high expression across all brain regions, with expression detected in amygdala, cerebellum, hippocampus, thalamus, cerebral cortex and spinal cord, making it an excellent candidate neuronal guidance protein for axons, dendrites and/or growth cones in general. Therapeutic modulation ofthe levels of this protein, or possible signaling via this protein, may be of utility in enhancing/directing compensatory synaptogenesis and fiber growth in the CNS in response to neuronal death (stroke, head trauma), axon lesion (spinal cord injury), or neurodegeneration (Alzheimer's, Parkinson's, Huntington's, vascular dementia or any neurodegenerative disease). In addition, the NOV3 gene shows highest expression in the substantia nigra, where loss of dopaminergic neurons is a hallmark of Parkinson's disease and progressive supranuclear palsy; thus, therapies based upon this gene may be particularly useful in the treatment ofthese diseases. This gene also shows overexpression in cell lines such as those derived from brain tumors (Cura 438). The NOV3a protein also contains homology to the GPCR family of receptors. Several neurotransmitter receptors are GPCRs, including the dopamine receptor family, the serotonin receptor family, the GABAB receptor, muscarinic acetylcholine receptors, and others; thus this GPCR may represent a novel neurotransmitter receptor. Targeting various neurotransmitter receptors (dopamine, serotonin) has proven to be an effective therapy in psychiatric illnesses such as schizophrenia, bipolar disorder and depression. Furthermore the cerebral cortex and hippocampus are regions ofthe brain that are known to play critical roles in Alzheimer's disease, seizure disorders, and in the normal process of memory formation. Therapeutic modulation of this gene or its protein product may be beneficial in one or more of these diseases, as may stimulation and/or blockade ofthe receptor coded for by the gene. Levels of this gene are high, however, in areas outside ofthe central nervous system (such as the heart, ovaries, and prostate), suggesting the possibility of a wider role in intercellular signaling.
Panel 2.2 Summary: Agl402 Among the samples on this panel, expression ofthe NOV3a gene is highest in a sample of normal ovarian tissue adjacent to an ovarian cancer (CT = 28.5). There is also a predominance of this expression pattern for a number of other matched normal tissue samples including lung, colon and kidney. Furthermore, unmatched normal tissue samples, such as breast, uterus, ovary and prostate also show appreciable expression ofthe NOV3a gene. This gene also shows expression by certain breast, liver and gastric tumors (Cura 438). Thus, in general, this gene might be used to distinguish adjacent normal tissue from corresponding malignant tissue in the above listed tissue types. In addition, the therapeutic modulation ofthe protein encoded by the NOV3 gene might be of use in the treatment ofthe above listed malignancies.
Panel 3D Summary: Agl402 Expression ofthe NOV3a gene is highest in a sample derived from a histiocytic lymphoma cell line (CT = 28.2). In addition, this gene is also highly expressed in several CNS cancer cell lines and cell lines derived from a variety of sarcomas. Thus, the expression ofthe NOV3a gene could be used to distinguish between cell lines derived from sarcomas and CNS cancers vs. other cell lines, hi addition, this expression pattern indicates that the therapeutic modulation of this gene product might be of therapeutic benefit in the treatment of sarcomas or CNS cancers.
Panel 4D Summary: Agl402 Results from two experiments using the same probe/primer set are in excellent agreement. The NOV3a gene is highly expressed in endothelium and fibroblasts and is selectively down regulated in these tissues by treatment with E -lbeta and TNFalpha. Thus, the NOV3a gene encodes a putative LRR-containing GPCR that may be down regulated in response to the proinflammatory cytokines L -lbeta and TNFalpha in endothelium and fibroblasts. One possible hypothesis is that the natural ligand for this GPCR may be induced by treatment with TNF and IL-1 and that subsequent ligand binding to this putative GPCR could result in feedback inhibition. Alternatively, other signaling pathways induced by TNF and IL-1 could reduce the level of transcription ofthe NOV3a gene. Antibodies, small molecule or protein therapeutics which prevent the signalling through this putative GPCR could therefore be important in the treatment of diseases such as asthma, emphysema, psoriasis, and arthritis.
References:
1. Battye R., Stevens A., Perry R.L., Jacobs J.R. (2001) Repellent signaling by Slit requires the trypsin inhibitors. J. Neurosci. 21: 4290-4298.
Slit is a repellent axon guidance cue produced by the midline glia in Drosophila that is required to regulate the formation of contralateral projections and the lateral position of longitudinal tracts. Four sequence motifs comprise the structure of Slit: a trypsin inhibitor
(LRR), epidermal growth factor-like (EGF) repeats, a laminin-like globular (G)-domain, and a cysteine domain. Here we demonstrate that the LRR is required for repellent signaling and in vitro binding to Robo. Repellent signaling by slit is reduced by point mutations that encode single amino acid changes in the LRR domain. By contrast to the EGF or G-domains, the LRR domain is required in fransgenes to affect axon guidance. Finally, we show that the midline repellent receptor, Robo, binds Slit proteins with internal deletions that also retain repellent activity. However, Robo does not bind Slit protein missing the LRR. Taken together, our data demonstrate that Robo binding and repellent signaling by Slit require the LRR region. NOV7
Expression of gene NOV7 was assessed using the primer-probe sets Agl 631 and
Ag2448, described in Tables 21 and 22. Results from RTQ-PCR runs are shown in Tables 23 and 24.
Table 21. Probe Name Agl631
Figure imgf000222_0001
Table 22. Probe Name Ag2448
Figure imgf000222_0002
Table 23. Panel 2D
Figure imgf000222_0003
Figure imgf000223_0001
Figure imgf000224_0001
Table 24. Panel 4D
Figure imgf000224_0002
Figure imgf000225_0001
Figure imgf000226_0001
Panel 1.3D Summary: Agl631/Ag2448 Expression ofthe NOV7 gene is low/undetectable (CT values > 35) across the samples on this panel (data not shown).
Panel 2D Summary: Ag2448 The expression ofthe NOV7 gene is highest in a sample derived from an ocular melanoma metastasis (CT = 34.3). Other observed expression of this gene on this panel is below the level of reliable detection. Ocular melanoma is a unique form of melanoma, and thus, the expression of this gene could be used to distinguish ocular melanoma metastases from other melanomas and also other tissues in general. In addition, therapeutic modulation ofthe NOV7 gene product, through the use of antibodies or small molecule drags might be of use in the treatment of ocular melanoma.
Panel 2.2 Summary: Agl631 Expression ofthe NOV7 gene is low/undetectable (CT values > 35) across the samples on this panel (data not shown).
Panel 4D Summary: Ag 1631 The NOV7 gene is expressed in B cells treated with poke weed mitogen or with CD40L and IL-4 (CT = 34). The protein encoded for by this gene has homology to thymosin-beta 4, which is also upregulated during B cell activation (ref. 1). Therefore, the NOV7 protein may have functions similar to those of other thymosin-beta 4 homologs, including induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation (ref. 2). Thus, protein therapeutics designed with the protein encoded for by the NOV7 gene could be useful for treating inflammatory diseases such as arthritis, asthma, diabetes, lupus, psorisis, and allergy. Ag2448 Expression ofthe NOV7 gene is low/undetectable (CT values > 35) across the samples on this panel (data not shown).
References:
1. Gondo H., Kudo J., White J.W., Barr C, Selvanayagam P., Saunders G.F. (1987) Differential expression ofthe human thymosin-beta 4 gene in lymphocytes, macrophages, and granulocytes. J. Immunol. 139: 3840-3848.
A cDNA clone encoding human thymosin-beta 4 was isolated from a cDNA library prepared from peripheral blood leukocytes of a patient with acute lymphocytic leukemia. This clone contained the entire coding sequence of 43 amino acid residues of thymosin-beta 4 and had an imtiation codon and two termination codons. The amino acid and nucleotide sequences in the coding region were well conserved between rat and human. Nine of 132 nucleotides were different in the coding sequences (93% homology), but the deduced amino acid sequences were identical. No signal peptide was found in the deduced protein sequence. Human thymosin-beta 4 mRNA, approximately 830 nucleotides in length, was about 30 nucleotides larger than rat thymosin-beta 4 mRNA. Expression ofthe human thymosin-beta 4 gene in various primary myeloid and lymphoid malignant cells and in a few human hemopoietic cell lines was studied. Northern blot analyses of different neoplastic B lymphocytes revealed that steady state levels of thymosin-beta 4 mRNA varied as a function of differentiation stage. Thymosin-beta 4 mRNA levels were decreased in myeloma cells as are class II human leukocyte antigen, Fc receptor, and complement receptor, suggesting a relationship between thymosin-beta 4 and the immune response. Thymosin-beta 4 mRNA was more highly expressed in mature granulocytes than in immature blastic cells. Treatment of THP-1 cells, a human monocytic cell line, with recombinant human interferon-lambda reduced the levels of thymosin-beta 4 mRNA. Its level decreased after differentiation of THP-1 cells into Ia+ macrophages, but increased after differentiation of HL-60 cells into la- macrophages. The pattern of thymosin-beta 4 gene expression suggests that it may play a fundamental role in the host defense mechanism.
PMID: 3500230
2. Huff T., Muller C.S., Otto A.M., Netzker R., Hannappel E. (2001) beta-Thymosins, small acidic peptides with multiple functions. Int. J. Biochem. Cell. Biol. 33: 205-220.
The beta-thymosins are a family of highly conserved polar 5 kDa peptides originally thought to be thymic hormones. About 10 years ago, thymosin beta(4) as well as other members of this ubiquitous peptide family were identified as the main intracellular G-actin sequestering peptides, being present in high concentrations in almost every cell, beta- Thymosins bind monomeric actin in a 1 : 1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression of beta-thymosins appear to be related to the differentiation of cells. Increased expression of beta-thymosins or even the synthesis of a beta- thymosin normally not expressed might promote metastasis possibly by increasing mobility of the cells. Thymosin beta(4) is detected outside of cells in blood plasma or in wound fluid. Several biological effects are attributed to thymosin beta(4), oxidized thymosin beta(4), or to the fragment, acSDKP, possibly generated from thymosin beta(4). Among the effects are induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. However, nothing is known about the molecular mechanisms mediating the effects attributed to extracellular beta- thymosins.
PMID: 11311852
NOV8
Expression of geneNOVδ was assessed using the primer-probe set Ag2447, described in Table 25.
Table 25. Probe Name Ag2447
Figure imgf000229_0001
Expression of this gene is low/undetectable (CT values > 35) across the samples on Panels 1.3D, 2D, and 4D (data not shown).
Example 3. SNP analysis of NOVX clones
SeqCallingTM Technology: cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, cell lines, primary cells or tissue cultured primary cells and cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression for example, growth factors, chemokines, steroids. The cDNA thus derived was then sequenced using CuraGen's proprietary SeqCalling technology. Sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled with themselves and with public ESTs using bioinformatics programs to generate CuraGen's human SeqCalling database of SeqCalling assemblies. Each assembly contains one or more overlapping cDNA sequences derived from one or more human samples. Fragments and ESTs were included as components for an assembly when the extent of identity with another component of the assembly was at least 95% over 50 bp. Each assembly can represent a gene and/or its variants such as splice forms and/or single nucleotide polymorphisms (SNPs) and their combinations.
Variant sequences are included in this application. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA. A SNP can arise in several ways. For example, a SNP may be due to a substitution of one nucleotide for another at the polymorphic site. Such a substitution can be either a transition or a transversion. A SNP can also arise from a deletion of a nucleotide or an insertion of a nucleotide, relative to a reference allele. In this case, the polymorphic site is a site at which one allele bears a gap with respect to a particular nucleotide in another allele. SNPs occurring within genes may result in an alteration ofthe amino acid encoded by the gene at the position ofthe SNP. itragenic SNPs may also be silent, however, in the case that a codon including a SNP encodes the same amino acid as a result ofthe redundancy ofthe genetic code. SNPs occurring outside the region of a gene, or in an intron within a gene, do not result in changes in any amino acid sequence of a protein but may result in altered regulation ofthe expression pattern for example, alteration in temporal expression, physiological response regulation, cell type expression regulation, intensity of expression, stability of transcribed message.
Method of novel SNP Identification: SNPs are identified by analyzing sequence assemblies using CuraGen's proprietary SNPTool algorithm. SNPTool identifies variation in assemblies with the following criteria: SNPs are not analyzed within 10 base pairs on both ends of an alignment; Window size (number of bases in a view) is 10; The allowed number of mismatches in a window is 2; Minimum SNP base quality (PHRED score) is 23; Minimum number of changes to score an SNP is 2/assembly position. SNPTool analyzes the assembly and displays SNP positions, associated individual variant sequences in the assembly, the depth ofthe assembly at that given position, the putative assembly allele frequency, and the SNP sequence variation. Sequence fraces are then selected and brought into view for manual validation. The consensus assembly sequence is imported into CuraTools along with variant sequence changes to identify potential amino acid changes resulting from the SNP sequence variation. Comprehensive SNP data analysis is then exported into the SNPCalling database.
Method of novel SNP Confirmation: SNPs are confirmed employing a validated method know as Pyrosequencing (Pyrosequencing, Westborough, MA). Detailed protocols for Pyrosequencing can be found in: Alderborn et al. Determination of Single Nucleotide
Polymorphisms by Real-time Pyrophosphate DNA Sequencing. (2000). Genome Research. 10, Issue 8, August. 1249-1265. In brief, Pyrosequencing is a real time primer extension process of genotyping. This protocol takes double-stranded, biotinylated PCR products from genomic DNA samples and binds them to streptavidin beads. These beads are then denatured producing single sfranded bound DNA. SNPs are characterized utilizing a technique based on an indirect bioluminometric assay of pyrophosphate (PPi) that is released from each dNTP upon DNA chain elongation. Following Klenow polymerase-mediated base incorporation, PPi is released and used as a substrate, together with adenosine 5'-phosphosulfate (APS), for ATP sulfurylase, which results in the formation of ATP. Subsequently, the ATP accomplishes the conversion of luciferin to its oxi-derivative by the action of luciferase. The ensuing light output becomes proportional to the number of added bases, up to about four bases. To allow processivity ofthe method dNTP excess is degraded by apyrase, which is also present in the starting reaction mixture, so that only dNTPs are added to the template during the sequencing. The process has been fully automated and adapted to a 96-well format, which allows rapid screening of large SNP panels. The DNA and protein sequences for the novel single nucleotide polymorphic variants are reported. Variants are reported individually but any combination of all or a select subset of variants are also included. In addition, the positions ofthe variant bases and the variant amino acid residues are underlined.
Results Variants are reported individually but any combination of all or a select subset of variants are also included as contemplated NOVX embodiments ofthe invention. NOVl SNP data:
NOVl has seven SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOs: 1 and 2, respectively. The nucleotide sequence ofthe NOVl variant differs as shown in Table 26.
Figure imgf000232_0001
NO 4 SNP data:
In the following positions, one or more consensus positions (Cons. Pos.) ofthe nucleotide sequence have been identified as SNPs. "Depth" rerepresents the number of clones covering the region ofthe SNP. The Putative Allele Frequency (Putative Allele Freq.) is the fraction of all the clones containing the SNP. A dash ("-"), when shown, means that a base is not present. The sign ">" means "is changed to".
Cons. Pos.: 97 Depth: 118 Change: C > A Putative Allele Freq. 0.034 133000338 (+,i) unrev. Fpos : 81
-> 133000555 (+,i) unrev. Fpos: 69 -> 133001366 (+, i) unrev. Fpos: 68 - > 133001516 (+, i) unrev. Fpos: 61
Cons. Pos.: 97 Depth: 118 Change: C > G Putative Allele Freq.: 0.017 133741176 (+,i) unrev. Fpos: 107
~> 133742288 (-,i) unrev. Fpos: 379
Cons. Pos.: 116 Depth: 220 Change: C > - Putative Allele Freq. 0.009 132999287 (+,i) unrev. Fpos: 67
-> 133012157 (-,i) unrev. Fpos: 387
Cons. Pos.: 122 Depth: 220 Change: T > C Putative Allele Freq.: 0.009 133001338 (+,i) unrev. Fpos: 88
-> 133018706 (-,i) unrev. Fpos: 360
Cons. Pos.: 130 Depth: 220 Change: C > T Putative Allele Freq. 0.009 133001002 (-,i) unrev. Fpos: 334
-> 133018604 (+,i) unrev. Fpos: 117
Cons. Pos.: 203 Depth: 219 Change: G > A Putative Allele Freq.: 0.009 133001421 (+,i) unrev. Fpos: 151 -> 133018747(-,i) unrev. Fpos: 289
Cons. Pos.: 204 Depth: 219 Change: C > T Putative Allele Freq.: 0 . 009 132999373 (+ ,i) unrev. Fpos:: 150 -> 133012199 (-,i) unrev. Fpos: 304
Cons. Pos.: 221 Depth: 219 Change: A > G Putative Allele Freq.: 0.009 133001494 (-,i) unrev. Fpos:: 278 -> 133018759 (+,i) unrev. Fpos: 200
Cons. POS.: 235 Depth: 219 Change: A > G Putative Allele Freq.: 0.009
133001613 (+ ,i) unrev. Fpos : 182
-> 133018773 (-,i) unrev. Fpos: 278
Cons. Pos.: 243 Depth: 219 Change: A > G Putative Allele Freq.: 0.009
133001409 (+,i) unrev. Fpos : 180
-> 133018744 (-,i) unrev. Fpos: 253
Cons. Pos.: 249 Depth: 219 Change: G > A Putative Allele Freq.: 0.009 133001292 (+,i) unrev. Fpos: 195 -> 133018686 (-,i) unrev. Fpos: 246
Cons. Pos.: 255 Depth: 219 Change: C > T Putative Allele Freq.: 0.009 133006504 (-,i) unrev. Fpos: 230 -> 133026853 (+,i) unrev. Fpos: 250
Cons. Pos.: 300 Depth: 273 Change: G > A Putative Allele Freq.: 0.007
133555265 (-, i) unrev. Fpos : 208
-> 13363452 (+,i) unrev. Fpos: 84
Cons. Pos.: 308 Depth: 273 Change: G > A Putative Allele Freq.: 0.007 133001516 (+,i) . unrev. Fpos 254 -> 133018761(-,i) unrev. Fpos: 190
Cons. Pos.: 330 Depth: 273 Change: C > T Putative Allele Freq.: 0.007 133001366 (+,i) unrev. Fpos: 282 -> 133018735'-, i) unrev. Fpos: 171
Cons. Pos.: 339 Depth: 273 Change: T > C Putative Allele Freq.: 0.007 133605071 (-,i) unrev. Fpos: 167 - > 133635377 (+,i) unrev. Fpos: 122
Cons. os.: 354 Depth: 273 Change: A > T Putative Allele Freq.: 0.007 132999419 (-, i) unrev. Fpos: 132 -> 133012245 (+,i) unrev. Fpos: 325
Cons. Pos.: 358 Depth: 273 Change: G > A Putative Allele Freq.: 0.007
133001351 (+,i) unrev. Fpos: 301
-> 133018729(-,i) unrev. Fpos: 151.
NOV5a SNP data:
NOV5a has four SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOs:X and Y, respectively. The nucleotide sequence ofthe NOV5a variant differs as shown in Table 27.
Table 27. cSNP and Coding Variants for NOV5a
NT Position Wild Type Variant NT Amino Acid Amino Acid of cSNP NT position Change
Figure imgf000234_0001
NOV5b SNP data:
NOV5b has three SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOs:X and Y, respectively. The nucleotide sequence ofthe NOV5b variant differs as shown in Table 28.
Figure imgf000234_0002
NOV5g SNP data: In the following positions, one or more consensus positions (Cons. Pos.) ofthe nucleotide sequence have been identified as SNPs. "Depth" rerepresents the number of clones covering the region ofthe SNP. The Putative Allele Frequency (Putative Allele Freq.) is the fraction of all the clones containing the SNP. A dash ("-"), when shown, means that a base is not present. The sign ">" means "is changed to". Cons.Pos.: 814 Depth: 13 Change: C > A Putative Allele Freq.: 0.231
NOV5h SNP data:
In the following positions, one or more consensus positions (Cons. Pos.) ofthe nucleotide sequence have been identified as SNPs. "Depth" rerepresents the number of clones covering the region ofthe SNP. The Putative Allele Frequency (Putative Allele Freq.) is the fraction of all the clones containing the SNP. A dash ("-"), when shown, means that a base is not present. The sign ">" means "is changed to".
Cons.Pos.: 820 Depth: 8 Change: G > - Putative Allele Freq.: 0.250 NOV8 SNP data:
NOV8 has one SNP variant, whose variant position for its nucleotide and amino acid sequences is numbered according to SEQ ID NOs:X and Y, respectively. The nucleotide sequence ofthe NOV8 variant differs as shown in Table 29.
Figure imgf000235_0002
Example 4: Sage analysis for NOV3
6A: Unigene Cluster Hs. 17270
Figure imgf000235_0001
Library name __ j __mlHionL _ ^jLsSirø Totaitags
" SAGE Duke H1043 ~ 12 " " 77449 gTOAAgA Il — - - fagspetϊ T*8 Ubrarymme eounts Total tags
SAGE Duke BB542 normal
24 1 41450 cerebellum
SAGE Chen LNCaP 15 1 B26B1
SAGE Chen Normal Pr 14 1 B6B87
SAGE Duke H392 17 1 58099
SAGE Pane 91-16113 29 •**• 1 34159
SAGE Pane 96-6252 27 ■***> 1 360B7
SAGE Duke H341 22 * 1 449B3
SAGE OVT-6 23 -#*> 1 43074
SAGE Duke H VEC 2 530B9
SAGE Duke HMVEOVEGF 34 « m- 2 5B499
SAGE DCIS 24 m 1 41540
SAGE Duke 757 50 •<**• 1 19674
SAGE OVT-B 29 1 3409B
SAGE A27B0-9 44 *■» 1 22551
SAGE Duke H247 normal 18 1 6109B
SAGE IOSE29-11 20 * 1 48876
6B. Unigene cluster 274136
UniGene cluster Hs
Figure imgf000236_0001
Hs 274136 : hypothetical protein FLJ11052
SAGE library data and reliable taq summan f
Reliable tags found in SAGE libraries lAGATCCΪ-CTA1 Tags per [ Ta&
1 ' brsry name •> million I counts Totaitags
SAGE Caco 2 16 61998
SAGE Chen LNCaP 15 65206 no-DHT
SAGE Chen Tumor Pr 14 <■ 69202
SAGE Duke GB H1110 14 71138
SAGE CPDR LNCaP-T 22 44370
SAGE NC1 19 50601
SAGE Tu 102 34 2 58190
SAGE SciencePark MCF7 16 60164 estradiol 3h
SAGE HOSE 4 20 48925
SAGE OVP-5 179 2 1 1 118
SAGE OVT-7 18 55476
SAGE normal cerebellum 22 45079
SAGE L10-10 17 57326
SAGE Duke H247 normal 16 61096
SAGE DCIS 2 34 29201
SAGE Duke H 1043 12 77449
Tags per J T I library narnp _ „ , miiliori « counts Total tag
SAGE NHA(5th) 18 53219
53089
T
Figure imgf000236_0002
counts Totaitags
SAGE Chen Normal Pr 29 * 2 66687
SAGE RKO 19 1 52423
SAGE CPDR LNCaP-C 23 1 41848
SAGE normal pooliΕth) 15 1 64136
SAGE Tu 102 17 1 58190
SAGE HOSE 4 20 1 48925
ΪOTCTAA©AA * " a s er J Tag
Ubrar/namβ million i counts Totaitags
SAGE Duke BB542 normal 24 1 41450 cerebellum
SAGE Chen LNCaP 15 1 62681
SAGE Chen Normal Pr 14 1 66687
SAGE Duke H392 17 1 58099
SAGE Pane 91-16113 29 < 1 34159
SAGE Pane 96-6252 27 1 36067
SAGE Duke H341 22 1 44983
SAGE OVT-6 23 1 43074
SAGE Duke HMVEC 37 2 53089
SAGE Duke HMVEC+VEGF 34 2 58499
SAGE DCIS 24 1 41540
SAGE Duke 757 50 •*«• 1 19674
SAGE OVT-B 29 - 1 34096
SAGE A2780-9 44 1 22551
SAGE Duke H247 normal 16 1 61096
SAGE 10SE29-11 20 1 48876 OTHER EMBODIMENTS
Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope ofthe appended claims, which follow. In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope ofthe invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge ofthe embodiments described herein. Other aspects, advantages, and modifications considered to be within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37;
(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% ofthe amino acid residues from the amino acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37; and
(d) a variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15%) of amino acid residues from said amino acid sequence.
2 The polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence selected from the group consisting SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and
37.
3. The polypeptide of claim 2, wherein said allelic variant comprises an amino acid sequence that is the translation of a nucleic acid sequence differing by a single nucleotide from anucleic acid sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36. The polypeptide of claim 1, wherein the amino acid sequence of said variant comprises a conservative amino acid substitution.
An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37;
(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% ofthe amino acid residues from the amino acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37;
(d) a variant of an amino acid sequence selected from the group consisting SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence;
(e) a nucleic acid fragment encoding at least a portion of a polypeptide comprising an amino acid sequence chosen from the group consisting of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37, or a variant of said polypeptide, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence; and
(f) a nucleic acid molecule comprising the complement of (a), (b), (c), (d) or (e).
The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally-occurring allelic nucleic acid variant.
7. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of a naturally-occurring polypeptide variant.
8. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36.
9. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36;
(b) a nucleotide sequence differing by one or more nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, provided that no more than 20% of the nucleotides differ from said nucleotide sequence;
(c) a nucleic acid fragment of (a); and
(d) a nucleic acid fragment of (b).
10. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule hybridizes under stringent conditions to a nucleotide sequence chosen from the group consisting SEQ ID NOS:l, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, or a complement of said nucleotide sequence.
11. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:
(a) a first nucleotide sequence comprising a coding sequence differing by one or more nucleotide sequences from a coding sequence encoding said amino acid sequence, provided that no more than 20%) ofthe nucleotides in the coding sequence in said first nucleotide sequence differ from said coding sequence;
(b) an isolated second polynucleotide that is a complement ofthe first polynucleotide; and (c) a nucleic acid fragment of (a) or (b).
12. A vector comprising the nucleic acid molecule of claim 11.
13. The vector of claim 12, further comprising a promoter operably-linked to said nucleic acid molecule.
14. A cell comprising the vector of claim 12.
15. An antibody that binds immunospecifically to the polypeptide of claim 1.
16. The antibody of claim 15, wherein said antibody is a monoclonal antibody.
17. The antibody of claim 15, wherein the antibody is a humanized antibody.
18. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising:
(a) providing the sample;
(b) contacting the sample with an antibody that binds immunospecifically to the polypeptide; and
(c) determining the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.
19. A method for determining the presence or amount ofthe nucleic acid molecule of claim 5 in a sample, the method comprising:
(a) providing the sample;
(b) contacting the sample with a probe that binds to said nucleic acid molecule; and
(c) detennining the presence or amount ofthe probe bound to said nucleic acid molecule, thereby determining the presence or amount ofthe nucleic acid molecule in said sample.
20. The method of claim 19 wherein presence or amount ofthe nucleic acid molecule is used as a marker for cell or tissue type.
21. The method of claim 20 wherein the cell or tissue type is cancerous.
22. A method of identifying an agent that binds to a polypeptide of claim 1 , the method comprising:
(a) contacting said polypeptide with said agent; and
(b) determining whether said agent binds to said polypeptide.
23. The method of claim 22 wherein the agent is a cellular receptor or a downsfream effector.
24. A method for identifying an agent that modulates the expression or activity ofthe polypeptide of claim 1, the method comprising:
(a) providing a cell expressing said polypeptide;
(b) contacting the cell with said agent, and
(c) determining whether the agent modulates expression or activity of said polypeptide, whereby an alteration in expression or activity of said peptide indicates said agent modulates expression or activity of said polypeptide.
25. A method for modulating the activity of the polypeptide of claim 1 , the method comprising contacting a cell sample expressing the polypeptide of said claim with a compound that binds to said polypeptide in an amount sufficient to modulate the activity ofthe polypeptide.
26. A method of treating or preventing a NOVX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the polypeptide of claim 1 in an amount sufficient to treat or prevent said NOVX-associated disorder in said subject.
27. The method of claim 26 wherein the disorder is selected from the group consisting of cardiomyopathy and atherosclerosis.
28. The method of claim 26 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
29. The method of claim 26, wherein said subject is a human.
30. A method of treating or preventing a NOVX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the nucleic acid of claim 5 in an amount sufficient to treat or prevent said NOVX-associated disorder in said subject.
31. The method of claim 30 wherein the disorder is selected from the group consisting of cardiomyopathy and atherosclerosis.
32. The method of claim 30 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
33. The method of claim 30, wherein said subject is a human.
34. A method of treating or preventing a NOVX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the antibody of claim 15 in an amount sufficient to treat or prevent said NOVX-associated disorder in said subject.
35. The method of claim 34 wherein the disorder is diabetes.
36. The method of claim 34 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
37. The method of claim 34, wherein the subject is a human.
38. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically-acceptable carrier.
39. A pharmaceutical composition comprising the nucleic acid molecule of claim 5 and a pharmaceutically-acceptable carrier.
40. A pharmaceutical composition comprising the antibody of claim 15 and a pharmaceutically-acceptable carrier.
41. A kit comprising in one or more containers, the pharmaceutical composition of claim 38.
42. A kit comprising in one or more containers, the pharmaceutical composition of claim 39.
43. A kit comprising in one or more containers, the pharmaceutical composition of claim 40.
44. A method for determining the presence of or predisposition to a disease associated with altered levels ofthe polypeptide of claim 1 in a first mammalian subject, the method comprising:
(a) measuring the level of expression ofthe polypeptide in a sample from the first mammalian subject; and
(b) comparing the amount of said polypeptide in the sample of step (a) to the amount ofthe polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, said disease; wherein an alteration in the expression level ofthe polypeptide in the first subject as compared to the confrol sample indicates the presence of or predisposition to said disease.
45. The method of claim 44 wherein the predisposition is to cancers.
46. A method for determining the presence of or predisposition to a disease associated with altered levels ofthe nucleic acid molecule of claim 5 in a first mammalian subject, the method comprising:
(a) measuring the amount ofthe nucleic acid in a sample from the first mammalian subject; and
(b) comparing the amount of said nucleic acid in the sample of step (a) to the amount ofthe nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level ofthe nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.
47. The method of claim 46 wherein the predisposition is to a cancer.
48. A method of treating a pathological state in a mammal, the method comprising administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide comprising an amino acid sequence of at least one of SEQ ID NOS:2, 4, 6, 8, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 37, or a biologically active fragment thereof.
49. A method of treating a pathological state in a mammal, the method comprising administering to the mammal the antibody of claim 15 in an amount sufficient to alleviate the pathological state.
PCT/US2001/026518 2000-08-25 2001-08-27 Novel proteins and nucleic acids encoding same WO2002016600A2 (en)

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EP01964417A EP1332212A2 (en) 2000-08-25 2001-08-27 Proteins and nucleic acids encoding same
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US60/228,324 2000-08-25
US60/227,800 2000-08-25
US22918500P 2000-08-30 2000-08-30
US22899700P 2000-08-30 2000-08-30
US60/228,997 2000-08-30
US60/229,185 2000-08-30
US22978000P 2000-09-01 2000-09-01
US22985000P 2000-09-01 2000-09-01
US22984800P 2000-09-01 2000-09-01
US60/229,850 2000-09-01
US60/229,848 2000-09-01
US60/229,780 2000-09-01
US26333701P 2001-01-22 2001-01-22
US60/263,337 2001-01-22
US26551801P 2001-01-31 2001-01-31
US60/265,518 2001-01-31
US27645101P 2001-03-15 2001-03-15
US60/276,451 2001-03-15
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US09/393,398 2001-08-24

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1776134A4 (en) * 2004-06-17 2009-08-26 Amgen Mountain View Inc C-met kinase binding proteins
US9758554B2 (en) 2012-01-31 2017-09-12 Technische Universitaet Muenchen Muteins of α1m lipocalin and method of production therefor

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SE8903003D0 (en) * 1989-09-12 1989-09-12 Astra Ab NOVEL MEDICAL USE
US5831049A (en) * 1997-01-03 1998-11-03 Incyte Pharmaceuticals, Inc. Human thioredoxin
ATE440865T1 (en) * 1998-03-26 2009-09-15 Univ Leland Stanford Junior NEW G-PROTEIN-COUPLED MAMMAL RECEPTORS WITH EXTRACELLULAR LEUCINE-RICH REGION
WO2001009328A1 (en) * 1999-08-03 2001-02-08 Millennium Pharmaceuticals, Inc. 15571, a novel gpcr-like molecule of the secretin-like family and uses thereof

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1776134A4 (en) * 2004-06-17 2009-08-26 Amgen Mountain View Inc C-met kinase binding proteins
EP2151453A1 (en) * 2004-06-17 2010-02-10 Amgen Mountain View Inc. C-met kinase binding proteins
US7803907B2 (en) 2004-06-17 2010-09-28 Amgen Mountain View, Inc. c-MET kinase binding proteins
US7968681B2 (en) 2004-06-17 2011-06-28 Amgen Mountain View, Inc. c-MET kinase binding proteins
AU2005265150B2 (en) * 2004-06-17 2012-04-12 Amgen Mountain View Inc. C-MET kinase binding proteins
US9758554B2 (en) 2012-01-31 2017-09-12 Technische Universitaet Muenchen Muteins of α1m lipocalin and method of production therefor
US10738092B2 (en) 2012-01-31 2020-08-11 Technische Universitaet Muenchen Muteins of a1m lipocalin and method of production therefor

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