JP2009159974A - Novel polypeptide hormone phosphatonin - Google Patents

Abstract

The present invention provides means and methods for regulating phosphate metabolism that are particularly effective in the treatment of bone minerals and kidney diseases.
A novel phosphatonin polypeptide derived from human and a polynucleotide encoding phosphatonin are obtained, and further, a vector, a host cell, an antibody, and a gene recombination for producing this polypeptide or polynucleotide Method. It can be used to diagnose and treat bone minerals and kidney diseases. Furthermore, it is also used in screening methods for identifying phosphatonin binding partners.
[Selection] Figure 14

Description

The present invention relates to polypeptides involved in the regulation of phosphate metabolism. More particularly, the present invention relates to a novel polypeptide, metastatic-tumor excreted phosphoric acid element (MEPE), or “phosphatonin”. The present invention also relates to genes and polynucleotides encoding phosphatonin polypeptides, vectors for producing phosphatonin polypeptides, host cells, antibodies to phosphatonin polypeptides, and gene recombination methods. To do. Also provided are diagnostic methods for detecting diseases related to phosphate metabolism and methods for treating such diseases. The present invention further relates to screening methods for identifying agonists and antagonists of phosphatonin activity.

  Numerous references are cited throughout this specification. References cited herein (including details of manufacture, commentary, etc.) are incorporated herein by reference, but no reference is admitted as prior art to the present invention.

BACKGROUND OF THE INVENTION Phosphate plays a central role in various basic cellular processes and bone mineralization. In particular, skeletal mineralization relies on the regulation of phosphate and calcium in the body, and disruption of phosphate-calcium homeostasis can significantly affect bone structural integrity. In the kidney, phosphate is passively lost into the glomerular filtrate and is actively reabsorbed through a sodium-dependent phosphate co-transporter. In the small intestine, phosphate is absorbed from food. A sodium-dependent phosphate co-transporter has been confirmed to exist in the small intestine and has recently been cloned (Hilfiker, PNAS 95 (24), 14564-14569). The liver, skin, and kidneys convert vitamin D3 into calcitriol, an active metabolite that plays an active role in maintaining phosphate balance and bone mineralization.

  Vitamin D deficiency causes rickets in children and osteomalacia in adults. In both cases, this is due to poor calcification of the osteoids that make up the bone matrix. Some rickets occur regardless of dietary conditions, such as X-linked vitamin D resistant hypophosphatemic rickets (HYP), hereditary high calcium low Hereditary hypercalciuria with hypophosphatemic rickets (HHRH), Dent's disease, including certain types of renal Fanconi syndrome (renal Fanconi syndrom), renal 1α-hydroxylase deficiency : VDDR 1), 1,25-dihydroxyvitamin D3 deficiency (end organ resistance, VDDRII), and cancerous hypophosphatemic bone Softening (Oncogenic Hypophosphatemic Osteomalacia: OHO) and the like are included. Thus, a number of familial illnesses are characterized as phosphate intake, vitamin D metabolism, and abnormal bone mineralization. In recent years, genes that have been deficient in patients with X-linked hypophosphatemic rickets (PHEX) have been cloned and investigated (Francis, Nat. Genet. 11 (1995), 130 -136; Rowe, Hum. Genet. 97 (1996), 345-352; Rowe, Hum. Mol. Genet. 6 (1997), 539-549). The PHEX gene is a type II glycoprotein, a family of Zn metalloendopeptidases (M13). PHEX is said to have a function to regulate factors related to phosphate homeostasis and skeletal mineralization (Rowe, Exp. Nephrol. 5 (1997), 355-363; Rowe, Current Opinion in Nephrology & Hypertension 7 (4) (1998), 367-376). Cancerous hypophosphatemic osteomalacia (OHO) shares many similarities with HYP and has many similarities, but the underlying cause is different (Rowe, Exp. Nephrol. 5 ( 1997), 355-363; Rowe, Current Opinion in Nephrology & Hypertension 7 (4) (1998), 367-376; Drezner in Primer on Metabolic Bone Diseases and Disorders of Mineral Metabolism (ed. Favus, MJ) 184-188 ( Am. Soc. Bone and Min. Res., Kelseyville, CA, 1990)). Osteomalacia is an adult disease equivalent to rickets, and the main feature of osteomalacia caused by tumors is that the bones soften. The softened bones are distorted, causing bowed legs and similar deformations, reminiscent of familial rickets. Low plasma phosphate levels and poor vitamin D metabolism are also very similar to HYP. Neoplastic osteomalacia has been reported in many different tumor types, but is rare and the tumor is primarily derived from the mesenchymal system (Rowe, Exp. Nephrol. 5 (1997), 355-363; Francis, Baillieres Clinical Endocrinology and metabolism 11 (1997), 145-163; loakimidis, The J. Rheumatology 21 (6) (1994), 1162-1164; Lyles, Ann. Intern. Med. 93 (1980), 275-278; Rowe , Hum. Genet. 94 (1994), 457-467; Shane, Journal of Bone and Mineral Research 12 (1997), 1502-1511; Weidner, Cancer 59 (1987), 1442-1442). When possible, the tumor is surgically removed so that the disease symptoms disappear and the bone heals, suggesting that circulating hyperphosphatemic factor is the pathogen of the disease . Also, xenotransplantation of human tumors into nude mice (Miyauchi, J. Clin. Endocrinol. Mtab. 67 (1988), 46-53), injection of saline extract into rats and dogs (Aschinberg, J. Paediatr. 91 (1977), 56-60; Popovtzer, Clin. Res. 29 (1981), 418A (Abstract)), the use of cancer medium (Tumor Conditioned Medium: TCM) and human and animal kidney cell line cultures. All indicate that circulating hyperphosphatemic factor is secreted from these cancers.

  Although a fundamental defect in X-chromosome rickets has been identified as a mutation of Zn metalloendopeptidase (PHEX), there is also significant evidence that the circulation of phosphauria factor is involved (Ecarot , J. Bone Miner. Res. 7 (1992), 215-220; Ecarot, J. Bone Miner. Res. 10 (1995), 424-431; Morgan, Arch /. Intern. Med. 134 (1974), 549 -552; Nesbitt, J. Clin. Invest. 89 (1992), 1453-1459; Nesbitt, J. Bone. Miner. Res. 10 (1995), 1327-1333; Nesbitt, Endocrinology 137 (1996), 943-948 ; Qui, Genet. Res., Camb. 62 (1993), 39-43; Lajeunesse, Kidney Int. 50 (1996), 1531-1538; Meyer, J. Bone. Miner. Res. 4 (4) (1989) , 523-532; Meyer, J. Bone. Miner. Res. 4 (1989), 493-500). The pathophysiological agreement between HYP and OHO raised the interesting possibility that this tumor factor was processed by the PHEX gene product in normal individuals. The proteolytic process by PHEX also appears to result in the degradation of this unidentified hyperphosphatemic factor or activation of the phosphate conservation cascade (Carpenter, Pediatric Clinics of North America 44 (1997), 443-466 ; Econs, Am. J. Physiol. 237 (1997), F489-F498; Glorieux, Arch. Pediatr. 4 (1997), 102s-105s; Grieff, Current Opinion in Nephrology & Hypertension 6 (1997), 15-19; Hanna, Current Therapy in Endocrinology & Metabolism 6 (1997), 533-540; Kumar, Nephrol. Dial. Transplant. 12 (1997), 11-13; Takeda, Ryoikibetsu Shokogun Shirizu (1997), 656-659). Thus, by cloning and characterizing neoplastic hyperphosphatemic urinary factor, the association between neoplastic osteomalacia and familial X chromosome-type rickets and other disorders of phosphate metabolism It becomes possible to clarify.

  Rowe et al. (1996) report that candidate 56 and 58 kDa proteins cause renal dysfunction in OHO (Rowe, Bone 18, (1996), 159-169). OHO patients were treated by excision of the tumor, and anti-sera from this patient before and after surgery were used to identify proteins in cancer cultures by Western blotting. However, neither tumor cells nor antisera are provided externally.

  Rowe (1997), in the review of Exp. Nephrol, 5 (1997), 335-363, describes the role of the disease and the PHEX gene (formerly known as the PEX gene). The PHEX gene product was recognized as a Zn metalloproteinase. In symptoms such as familial rickets, phosphatonin is not cleaved by PHEX deficiency, resulting in a decrease in sodium-dependent phosphate co-transport molecules and an increase in renal mitochondrial 24-hydroxylase. However, Rowe (1997) reported nothing about the purification of phosphatonin. Therefore, no source of phosphatonin extraction is publicly available. Furthermore, purification, identification and characterization of phosphatonin has never been possible before.

  Thus, there is a need for polypeptides that regulate phosphate metabolism, but that dysregulation is associated with low and hyperphosphatemia such as osteomalacia, especially osteoporosis and renal failure. Because it may be. In addition, there is a need to identify and characterize polypeptides that serve to detect, prevent, and / or correct such dysfunctions and are effective in diagnosing such dysfunctions.

  The present invention relates to a novel phosphatonin polypeptide and a polynucleotide encoding phosphatonin. The present invention also relates to vectors, host cells, antibodies, and gene recombination methods for producing the present polypeptides and polynucleotides. Furthermore, diagnostic methods for detecting diseases associated with these polypeptides and methods for treating these diseases are also provided. Furthermore, the present invention relates to a screening method for identifying a binding partner of phosphatonin.

Detailed Description of the Invention In view of the need for diagnostic and therapeutic means for the treatment of diseases associated with phosphate metabolism deficiency in the human body, the technical problem in the present invention is particularly effective in the treatment of bone minerals and kidney diseases. It is to provide means and methods for regulating phosphate metabolism.

  The technical problem defined above is solved by the contents specifically described in the claims of the present invention. That is, one aspect of the present invention also relates to an isolated polypeptide having phosphatonin activity.

  Unless otherwise specified, the terms used herein are “A Multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, HGW, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta , CH-4010 Basle, Switzerland, ISBN 3-906 390-13-6. The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

  As used herein, terms such as “treatment”, “treating” and the like generally mean obtaining a pharmacological and / or physiological effect. An effect may be prophylactic in that it completely or partially interferes with the disease or symptom, and / or is therapeutic in that it fully or partially treats the disease and / or the side effects of the disease. May be. The term “treatment” as used herein includes all treatment of diseases in mammals, particularly humans. And further (a) prevention of disease in subjects who are predisposed to the disease but have not yet been diagnosed; (b) inhibition of the disease, ie suppressing progression; or (c) disease alleviation, That is, reducing the disease; etc. are also included in this term. The present invention is directed to the treatment of patients with medical conditions related to phosphate metabolism disorders. Accordingly, treatment according to the present invention includes preventing, blocking and alleviating all medical symptoms associated with phosphate metabolism disorders.

  In the present invention, the term “isolated” refers to a substance that has been removed from its original environment (for example, the natural environment if it occurs naturally) and has been changed “by the hand of man” from its natural state. It is. For example, an isolated polynucleotide is part of a vector or composition that is also “isolated” even if it is contained within a cell. This is because the vector, composition or certain cells are not the natural environment for the polynucleotide.

  The phosphatonin polypeptide isolated according to the present invention is typically about 53-60 kDa, as measured using SDS-PAGE, especially on a 12.5% gel in pH 8.6, Tris-Glycine SDS buffer. See Example 1, which has a molecular weight of about 58-60 kDa. A molecular weight of approximately 200 kDa may be obtained when measured by bis-Tris-SDS-PAGE on a 4-12% gradient gel using MOPS running buffer at pH 7. When such gels are used, molecular weights below 53-60 kDa can also be seen. Polypeptides are generally glycosylated and preferably contain phosphatonin in a sufficiently pure state.

  Surprisingly, phosphatonin was found to be obtained from Saos-2 cells of the European Collection of Cell Culture deposit number ECACC 89050205 by purification according to the method given in Example 1. Therefore, a further aspect of the present invention is the use of Saos-2 cells or HTB-96 cells for the production of phosphatonin. Other transformed or immortalized cell lines, such as transformed osteoblasts and immortalized bone cell lines, may also be able to express phosphatonin in large amounts.

The present invention is also considered to be of a high phosphate urinary factor named phosphatonin or MEPE (Metastatic-tumor Excreted Phosphaturic-Element) derived from the above-mentioned tumor. It also describes the characterization and cloning of the resulting genes. In summary, there is an expression screening (Expression Screening) method for λZAPII-cDNA library consisting of mRNA extracted from OHO tumors using antiserum specific for high phosphate urinary factor in tumor conditioned medium (TCM). Used. The protein was glycosylated and showed two bands on SDS-PAGE electrophoresis (58-60 kDa), indicating that it may be cleaved by splicing or post-translational modification. The cloned cDNA encoded a protein of 430 residues (SEQ ID NO: 2) and was 1655 base pairs in length (SEQ ID NO: 1). The 3 'end of the gene is present but part of the 5' end is deleted. A fusion protein containing 10 residues of β-galactosidase very strongly inhibits Na ⁺ -dependent phosphate cotransport in a human kidney cell line (CL8). Secondary structure predictions confirm that this protein has a small hydrophobic region but is highly hydrophilic and has no cysteine residues. There are many spirals and two separate N-glycosylation motifs at the C-terminus. A key feature is the presence of cell adhesion sequences with the same structural pattern as found in osteopontin. Proteolytic sites adjacent to this motif can change the specificity of the receptor for certain integrins, as seen in osteopontin. Screening of the MEPE sequence in the Trembl database showed sequence homology with Dentin phosphoryn (DPP). In particular, residues of sequences homologous to DPP, Dentin-Matrix Protein-1 (DMA-1) and Osteopontin (OPN) exist at the C-terminus of MEPE. This region of MEPE contains a sequence of repeated aspartic acid and serine residues (DDSSESSDSGSSSESD) and exhibits 80%, 65%, and 62% homology with DSP, DMA-1, and OPN, respectively. Furthermore, considering the physicochemical properties of the residues, this similarity is thought to increase to 93%, suggesting that biological functions are common or related. It is also noteworthy that this structural motif overlaps with the casein kinase II phosphorylation motif in MEPE. In rickets, bone casein kinase II activity is impaired, resulting in a decrease in osteopontin phosphorylation (Rifas, Calcif. Tissue Int. 61 (1997), 256-259). Casein kinase II deficiency has been proposed to act to reduce bone matrix mineralization (Rifas, loc. Cit.).

  Dentin phospholine (DPP) is part of a degradation product derived from dentin sialophosphoprotein (DSSP), the rest of which is known as dentin sialoprotein (DSP) (MacDougall, J. Bios Chem. 272 (1997), 835-842). Of particular interest is that DSSP, DMA-1, OPN and MEPE are RGD-containing phosphorylated glycoproteins with distinct structural similarities and play a major role in bone-tooth mineralization (Linde , Crit. Rev. Oral Biol. Med. 4 (1993), 679-728).

A novel OHO tumor-derived hyperphosphatemic factor named phosphatonin or MEPE described in the present invention regulates Na ⁺ -dependent phosphate cotransport, vitamin D metabolism, and bone mineralization It has the function of maintaining the homeostasis of bone minerals.

  As detailed below, a polynucleotide encoding a polypeptide according to the present invention was isolated; see Example 2. The amino acid and nucleotide sequences of phosphatonin are shown in FIG. 8 (SEQ ID NO: 1 and SEQ ID NO: 2, respectively). Therefore, the polypeptide of the present invention consists of the amino acid sequence of FIG. 8, and can selectively contain mutations and deletions within a range that does not substantially affect its activity. Such mutations include substitution of one or more amino acids (especially substitution with homologues thereof) and addition of one or more amino acids (especially addition at the N-terminus or C-terminus). Deletions include deletions from the N-terminus and C-terminus. The substitution can be a natural amino acid or a synthetic amino acid. Also included are polypeptides modified chemically or enzymatically. In addition, peptide fragments that are chemically synthesized or produced by biological methods, whether modified or not, are also within the scope of the present invention.

Thus, the present invention relates to a phosphatonin polypeptide or a fragment thereof having immunological and / or biological activity, and includes an amino acid sequence that can be encoded by a polynucleotide selected from the following group: .
(a) a polynucleotide encoding at least a mature form of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 (FIG. 8);
(b) a polynucleotide comprising the coding sequence set forth in SEQ ID NO: 1 (FIG. 8) encoding at least the mature form of the polypeptide;
(c) is encoded by polynucleotide (a) or (b) by substitution, deletion and / or addition of one or more amino acids of the amino acid sequence encoded by polynucleotide (a) or (b) A polynucleotide encoding a polypeptide derived from said polypeptide;
(d) a polynucleotide comprising a complementary strand that hybridizes with any one of the polynucleotides (a) to (c);
(e) a polynucleotide encoding a polypeptide having a sequence having 60% or more identity with the amino acid sequence of the polypeptide encoded by any one of the polynucleotides (a) to (d);
(f) a polynucleotide encoding a polypeptide comprising a fragment of a polypeptide encoded by any one of the polynucleotides of (a) to (e) or an antigenic determinant site and having a function of regulating phosphate metabolism;
(g) a polynucleotide encoding the antigenic determinant site of a phosphatonin polypeptide comprising amino acid residues at positions 1 to 40, 141 to 180 and / or 401 to 429 of SEQ ID NO: 2 (FIG. 8);
(h) a polynucleotide comprising at least 15 nucleotides of any one of the polynucleotides (a) to (g) and encoding a polypeptide capable of regulating phosphate metabolism;
(i) A polypeptide encoded by any one of the polynucleotides (a) to (h) contains a cell and / or glycosaminoglycan adhesion motif and / or a bone mineral motif, and is capable of regulating phosphate metabolism. And (j) a polynucleotide in which the nucleotide sequence is degenerated as a result of the genetic code by the nucleotide sequence of any one of the polynucleotides of (a) to (i).

  As used herein, a phosphatonin “polynucleotide” refers to a molecule having the nucleic acid sequence contained in SEQ ID NO: 1, or a molecule encoding a phosphatonin polypeptide of the invention. . For example, a phosphatonin polypeptide includes the full-length nucleotide sequence of a cDNA sequence including 5 'and 3' untranslated sequences, coding regions, fragments of nucleic acid sequences, antigenic determinants, domains, and mutated sequences.

  Further, as used herein, a phosphatonin “polypeptide” refers to a molecule having an amino acid sequence that can be translated from a widely defined polynucleotide.

  The phosphatonin “polynucleotide” further includes a polynucleotide capable of hybridizing to the sequence contained in SEQ ID NO: 1 or a complementary sequence thereof under stringent hybridization conditions. “Stringent hybridization conditions” means 50% formamide, 5 × SSC (sodium chloride 750 mM, sodium citrate 75 mM), sodium phosphate 50 mM, (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate. And incubate overnight at 42 ° C in a solution consisting of 20 μg / ml denatured and shredded salmon sperm, and then wash the filter with a solution of concentration 0.1 x SSC at about 65 ° C. Point to. More suitable hybridization conditions are described in the Examples.

Also expected are nucleic acid molecules that hybridize to phosphatonin polynucleotides at relatively low stringency hybridization conditions. Changing the stringency of hybridization and signal detection can basically be accomplished by changing the formamide concentration (stringency decreases with lower formamide concentration), salt concentration, or temperature. For example, lower stringency conditions include 6 × SSPE (20 × SSPE = 3M NaCl; 0.2M NaH ₂ PO ₄ ; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / Incubate overnight in a 37 ° C solution consisting of ml salmon sperm-derived blocking DNA and wash with 1X SSPE, 0.1% SDS, 50 ° C solution. For even less stringent conditions, wash with a high salt solution (eg, 5 × SSC) after stringent hybridization. It should be noted that variations on the above conditions can also be achieved by including or replacing other blocking reagents used to suppress background in hybridization experiments. . Typical blocking reagents also include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available special reagents. Due to compatibility issues, it may be necessary to modify the hybridization conditions described above in order to use certain blocking reagents. Of course, a poly A ⁺ sequence (such as the poly A + sequence at the 3 ′ end of the cDNA shown in the sequence listing), or a contiguous sequence of T (or U) residues complementary thereto, It is not included in the “polynucleotide” as defined herein because it will hybridize to any nucleic acid molecule containing its complementary sequence (eg, virtually all double-stranded cDNA clones).

  The phosphatonin polynucleotide can comprise any polyribonucleotide or polydeoxyribonucleotide, such as unmodified RNA or DNA, or modified RNA or DNA. For example, phosphatonin polynucleotides are single and double stranded DNA, single and double stranded DNA, single and double stranded RNA, single and double stranded It is also possible to be composed of a hybrid molecule composed of RNA, a single strand or more typically a double strand, or a DNA and RNA in which a single strand and a double strand region are mixed. Furthermore, phosphatonin polynucleotides can also be composed of RNA or DNA, or triple-stranded regions of RNA and DNA. The phosphatonin polynucleotide may comprise one or more degenerate bases or a DNA or RNA backbone that has been modified for stability or otherwise. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA. Thus, “polynucleotide” takes a chemically, enzymatically or metabolically converted form.

A phosphatonin polypeptide is composed of amino acids linked together by peptide bonds or degenerate peptide bonds or peptide isosteres, and contains amino acids other than the 20 amino acids encoded by the gene. You can also The phosphatonin polypeptide may be converted either by natural processing, such as post-translational processing, or by chemical conversion using techniques well known in the art. Such transformations are described in basic texts, more detailed research papers, and various research literature. Conversion may occur anywhere in the phosphatonin peptide, such as the peptide backbone, amino acid side chains, and amino- or carboxy-terminus. It will be appreciated that for the same type of conversion, it can occur to different places in a given phosphatonin polypeptide to the same or different degrees. Furthermore, a given phosphatonin polypeptide may contain various types of transformations. The phosphatonin polypeptide may be branched by, for example, ubiquitination, or may be cyclic with or without a branched structure. Cyclic, branched, and branched cyclic phosphatonin polypeptides can be by natural post-translational processing or by synthetic methods. Conversion includes acetylation, acylation, ADP-ribosylation, amidation, covalent addition of flavins, covalent addition of heme structures, covalent addition of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives Addition, covalent addition of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent bridge formation, cysteine formation, pyroglutamate formation, formulation, γ-carboxylation, glycosylation, GPI Anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic cleavage, phosphorylation, prenylation, racemization, selenylation, sulfation, arginine addition and ubiquitin To proteins mediated by transfer RNA such as addition Include addition of amino acids; for example, PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, 2 nd Ed, TE Coreighton, WH Freeman and Company, New York (1993);.. POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEIN, bc Johnson, Ed, Academic Press, New York (1983), pages. 1-2; Seifter, Meth. Enzymol. 182 (1990); 626-646, Rattan, Ann, NY Acad. Sci. 663 (1992); For example, it is possible that phosphatonin is expressed as a preproprotein, and after processing of the presequence or possibly the prosequence, two or more fragments are not dissociated, for example by the formation of hydrogen bonds. . Processing and / or cleavage of the prepro form or mature phosphatonin polypeptide may involve the deletion of one or more amino acids at the cleavage site. It should be understood that all such types of phosphatonin proteins fall within the vocabulary of “phosphatonin polypeptide”, “polypeptide” or “protein”.

  “SEQ ID NO: 1” represents the phosphatonin polynucleotide sequence, and similarly “SEQ ID NO: 2” represents the sequence of the phosphatonin polypeptide.

  A “biologically active” phosphatonin polypeptide is not necessarily consistent with the activity of a phosphatonin polypeptide in a specific biological assay described below, regardless of whether it is dose-dependent or not. It may mean a polypeptide that exhibits similar activity. Where there is a dose dependency, it does not necessarily match the dose dependency of the phosphatonin polypeptide, but it should have a fairly similar dose dependent activity compared to the phosphatonin polypeptide. Certain (i.e., candidate polypeptides exhibit higher activity or less than about 25-fold, preferably less than about 10-fold, more preferably less than about 3-fold lower activity than phosphatonin polypeptides. Show).

  “Immunologically active” or “immunological activity” means that the essential immunological properties of fragments, analogs and derivatives of the phosphatonin polypeptide of the present invention are not affected. At least one or more epitopes capable of specifically reacting with an antibody specific for the phosphatonin protein encoded by the polynucleotide. It is meant to include all nucleotide sequences that encode a protein or peptide having some primary and / or secondary structure. Preferably, the peptide or protein encoded by the polynucleotide of the present invention has about 20 to 30, 100 to 130, 145 to 160, 300 to 310, 320 to 340, or 380 to 430 amino acid residues of SEQ ID NO: 2. It is preferably recognized by an antibody that specifically reacts with an antigenic determinant of a phosphatonin polypeptide consisting of, or an antigenic determinant of a phosphatonin polypeptide described below. Peptides / antibodies with residues 380-430 are particularly useful for investigating the mineralization process, and peptides / antibodies with residues 145-160 are useful for studying receptor ligand interactions (integrins, etc.) And the residues at positions 20-30 and 100-130 are parts that seem to be particularly important for the study of regulation of phosphate metabolism.

  Preferably, the phosphatonin peptide fragments, analogs and derivatives of the phosphatonin peptides of the present invention that are immunologically active should be able to elicit an immune response in mammals, preferably mice or rats.

  In a preferred embodiment of the present invention, the phosphatonin polypeptide is biologically active, can control or alter phosphate metabolism, and preferably has “phosphatonin activity”.

Phosphatonin activity As used herein, the expression “can control or alter phosphate metabolism” refers to the presence or absence of a phosphatonin polypeptide of the invention, ie, a phosphatonin polypeptide in a subject. Means altering sodium-dependent phosphate cotransport, vitamin D metabolism and / or bone mineralization. Depending on whether the aforementioned activity is up-regulated or down-regulated by the polypeptide of the present invention, the expression “can control or alter phosphate metabolism” is referred to as “phosphatonin activity” and “anti-antigen”, respectively. It means “phosphatonin activity”.

  The phosphatonin activity is in particular the down-regulation activity of sodium-dependent phosphate co-transport and / or the up-regulation activity of renal 25-hydroxyvitamin D3-24-hydroxylase and / or renal 25-hydroxy D-1α- The down-regulation activity of hydroxylase can be measured by a usual assay method. In each case, the control of the relevant enzyme activity can be directly or indirectly influenced by phosphatonin. For example, it is measured by measuring sodium-dependent radioactive phosphate uptake. These activities can be assayed using an appropriate renal cell line such as CL8 or OK (deposited as ECACC 91021202 in the European Collection of Cell Cultures). A suitable assay method can be found in Rowe et al. (1996). The phosphatonin activity may further be measured by the mineralization promoting activity mediated by osteoblasts in tissue culture. Reference examples Santibanez, Br. J. Cancer 74 (1996), 418-422; Stringa, Bone 16 (1995), 663-670; Aronow, J. Cell Physiol. 143 (1990), 213-221, or accompanying implementation It is also described in the examples.

  As a further aspect, a polypeptide comprising a biologically active fragment of the above polypeptide is also included in the present invention. While not intending to be limited to this theory, phosphatonin has a polyhormonal function that is broken down in vivo into one or more fragments, at least some of which are broken down. It is thought that it may have biological activity such as hormone activity. In vivo, it is believed that phosphatonin may be proteolytically cleaved by, for example, the PHEX gene product to produce at least one functional fragment. In a preferred embodiment, the polypeptide comprising a biologically active fragment is phosphorylated, for example, by having the phosphatonin activity discussed above, or by having the activity opposite to that described in detail below. Metabolism can be regulated. The biologically active fragment may be N-terminal, C-terminal, or an intermediate fragment. Polypeptides containing biologically active fragments further include those with additional amino acid sequences to which the activity of the biologically active fragment is not significantly affected.

  In a preferred case, the biologically active fragment preferably has a cell adhesion motif comprising RGD. As discussed in detail later, this motif may also be involved in receptor and / or bone mineral matrix interactions. In a preferred case, the bioactive fragment has a glycosaminoglycan adhesion motif, preferably comprising SGDG (SEQ ID NO: 3). Since it adheres to glycosaminoglycan, it is considered that the fragment has properties similar to proteoglycan. Proteoglycans are known to be involved in bone biological activity, particularly cell signaling. These motifs are described in further detail below.

  In one embodiment of the invention, the polypeptide comprising a fragment with biological activity has phosphatonin activity. While not intending to be limited to this theory, such activities include phosphatonin that is not cleaved by PHEX metalloproteinases, and cleavage occurs between VD residues (residues 235 and 236) It is thought to be due to a biologically active fragment having a PHEX metalloproteinase cleavage site such as ADAVDVS (SEQ ID NO: 4). A biologically active fragment may contain at least the first 236 residues of the amino acid sequence of FIG. 8, and this PHEX metalloproteinase degradation site may be present in a portion of the fragment. Such polypeptides having phosphatonin activity and fragments thereof are considered useful for the treatment of hyperphosphatemic conditions.

Related Proteins As a result of further research on the present invention, phosphatonin (MEPE), Dentin Matrix Protein-1 (DMP-1), Dentin Sialo Phosphoprotein (DSSP); Dentin phosphoryn (C-terminal), bone sialoprotein (BSP), and osteopontin (OPN) revealed several high similarities. . In particular, all the matrix proteins mentioned above have an RGD motif, are glycosylated, and contain a large amount of aspartic acid and serine. All of these possess the phosphorylation motif of casein kinase II, and highly homologous regions exist between these proteins. Lanview-sim analysis Swiss plot software (Duret. LALNVIEW: a graphical viewer for pairwise sequence alignments. Comput. Biosci. 12 (1996), 507-510) is a dot-matrix comparison of high homology regions between phosphatonin and DSSP. It is shown in the figure. At the dentine phospholine (DP) site of DSSP, the motif is repeated five times (FIG. 12a), and this motif is 80% homologous to the C-terminal residue of phosphatonin. According to physicochemical variables, it is inferred that there is 93% homology, and this sequence homology is also found in other bone / dentin molecules with 60-65% sequence similarity. Also in the same region is a sequence homologous to the contiguous residues between DMA-1 and phosphatonin, as seen in Table 2 and the sequence comparison below.
408 SSRRRDDSSESSDSGSSSESDG 429 MEPE (SEQ ID NO: 5)
443 SSRSKEDSN-STESKSSSEEDG 463 DMA-1 (SEQ ID NO: 6)

  Dentinsialophosphoprotein (DSSP) is a large RGD-containing glycoprotein that is degraded in vivo to produce two proteins, dentinsialoprotein (DSP) and dentinphosphorin (DP), respectively (MacDougall, J. Biol. Chem. 272 (1997), 853-842). DSP was an N-terminal peptide and DP was a C-terminal peptide, both of which were initially thought to be different gene products. FIG. 13 shows a statistical dot matrix comparison between phosphatonin and DSSP under high stringency conditions and low stringency conditions. The repetitive nature of the “homology motif (DSSESSDSGSSSES (SEQ ID NO: 7))” in DSSP and its significant homology are both clearly shown in the figure. In MEPE, the motif appears only once at the C-terminus. Moreover, overall low level sequence similarity with the C-terminal site (or DP site) of DSSP is clearly evident. Thus, it is believed that a new “unique” property has been found that appears to function as a bone mineral interaction in the bone-tooth matrix class.

  In conclusion, all of the proteins discussed herein appear to have a significant correlation with bone minerals and the extracellular matrix of teeth, and their interactions are thought to be mediated by the integrin / RGD relationship. . In addition, new partial motifs (rich in serine and aspartate) are ideal for phosphate-calcium interactions. This therefore supports the hypothesis that the C-terminus of phosphatonin plays a role in maintaining bone mineral homeostasis and the N-terminus is responsible for renal phosphate regulation.

In summary, the common properties of these proteins include the following:
1． Has an RGD motif in a similar basic skeleton.
2． It is a glycoprotein.
3． High in serine and aspartic acid.
Four. Casein kinase and protein kinase motif.
Five. Clearly MEPE motif rich in aspartic acid and serine (repeated in DPP).
6． Has many phosphorylation motifs and myristoylation motifs.
7． Evidence for cutting and / or alternative splicing.
8． All are related to the extracellular matrix of bones and teeth.

  Thus, in a preferred embodiment of the invention, the phosphatonin polypeptide comprises a bone mineral motif as described above, preferably the amino acid sequence of SEQ ID NO: 5 or 7, or DMP-1, DSSP, BSP, OPN, Alternatively, it has an amino acid sequence corresponding to the amino acid sequence of DMA-1 protein.

Biologically active fragment In another embodiment of the invention, the polypeptide comprising a biologically active fragment is considered to be suitable for the treatment of hypophosphatemia because it has the opposite activity to phosphatonin. It is done. In this embodiment, the polypeptide may directly or indirectly up-regulate sodium-dependent phosphate cotransport molecules and / or down-regulate 25-hydroxyvitamin D3-24-hydroxylase, and / or kidney 25-hydroxy. Has an up-regulation function of D-1-hydroxylase. The activity already mentioned is also referred to herein as “anti-phosphatonin” activity. However, the use of the term “anti-phosphatonin” activity does not exclude the possibility that said activity is a significant activity of real phosphatonin in phosphate metabolism. Their “anti-phosphatonin” activity has also been demonstrated by Rowe et al. (See Table 8) by assays using appropriate renal cell lines such as CL8 and OK (deposited as the European Collection of Cell Cultures ECACC91021202). 1996) can be easily measured. See above and accompanying examples for the method. Thus, the phosphatonin polypeptide of the present invention can be easily examined for phosphatonin or “anti-phosphatonin” activity by the above-described method or the method described later, for example, as in the appended Examples. Preferably, the phosphatonin fragment is obtained by proteolytically digesting phosphatonin with PHEX metallopeptidase. The PHEX gene has been cloned and found to encode a Zn metallopeptidase, as discussed in Rowe (1997). Again, although not intended to be limited to this theory, structurally, a biologically active fragment with such activity lacks at least a portion of the N- or C-terminus of the amino acid sequence of FIG. Preferably, it is considered to lack from the C-terminal to a site presumed to be a PHEX metalloproteinase degradation site of at least 235/236 residues. Thus, the polypeptide preferably comprises at most about 235 residues or less of the first amino acid sequence of the amino acid sequence of FIG.

  As described in Example 4, the phosphatonin polypeptides of the present invention were cloned using an expression library that links the target cDNA to a portion of the β-galactosidase enzyme. The cDNA thus obtained contained no N-terminal methionine. However, pure phosphatonin would be expected to have an N-terminal methionine in its amino acid sequence. Therefore, as one embodiment of the phosphatonin polypeptide of the present invention, the amino acid sequence of the polypeptide includes an amino acid and methionine at the N-terminus.

  In another embodiment, the polypeptides of the present invention are part of a fusion protein. This aspect will be discussed later.

  In addition, polynucleotides encoding the phosphatonin polypeptides described herein are also provided by the present invention. Such polynucleotides may be DNA, such as cDNA, or RNA, such as mRNA, or other forms of nucleic acids, such as synthetic or converted derivatives, and multiple sequences interrupted by contiguous or inserted sequences. It may be a polypeptide that encodes Whatever form it is, the polynucleotide is an isolated polynucleotide, which is separated from the naturally occurring state. This aspect of the invention is based on the cloning of the gene for human phosphatonin. In a preferred embodiment, the polynucleotide comprises the nucleotide sequence of FIG. 8, optionally with one or more mutations or deletions, as long as they do not substantially affect the activity of the polypeptide encoded by the polynucleotide. Can be included. Such mutations include mutations that occur due to the degeneracy of the genetic code, and mutations that cause mutations or deletions of any of the amino acids described above. That is, by using techniques routinely used by those in the field of molecular biology, the nucleotide sequence shown in FIG. 8 can be used to produce a polynucleotide sequence suitable for encoding a polypeptide useful in the present invention. As already mentioned herein, the present invention also encompasses phosphatonin polynucleotides, which are nucleotide sequences lacking consecutive nucleotides from either the C-terminus or N-terminus, as detailed below. To do.

Extension of Polynucleotide Sequences of the Invention As discussed in Example 4, the phosphatonin polynucleotide obtained by this expression library may not contain the full length of the 5 ′ end. The polynucleotide sequence encoding the phosphatonin polypeptide is extended using various methods known in the art to find partial nucleotide sequences and upstream sequences such as promoters and regulatory elements. It may be. Gobinda (PCR Methods Applic. 2 (1993), 318-322) has proposed a “restriction enzyme site” polymerase chain reaction (PCR) as a direct method using universal primers to locate unknown sequences adjacent to a known site. It is clear. First, genomic DNA is amplified in the presence of a primer for a linker sequence and a primer specific for a known region. The amplified sequence is subjected to a second round of PCR with a primer for the same linker and another specific primer within the first primer. The product of each round of PCR is transcribed by an appropriate RNA polymerase and sequenced by reverse transcriptase.

  Inverse PCR can be used to amplify or extend sequences with primers in the reverse direction based on known regions (Triglia, Nucleic Acids Res. 16 (1998), 8186). Such primers are preferably 22-30 nucleotides in length, preferably more than 50%, by OLIGO® 4.06 Promer Analysis Software (1992; National Biosciences Inc, Plymouth MN), or other appropriate program. The GC can be designed to be annealed to the target sequence, preferably at a temperature of about 68-72 ° C. This method uses several restriction enzymes to make a suitable fragment in a known region of the gene. The fragment is further reused by intramolecular ligation and used as a PCR template.

  Search PCR (Capture PCR) (Lagerstorm, PCR Methods Applic. 1 (1991), 111-119) is a method of amplifying a DNA fragment adjacent to a known sequence, such as human yeast artificial chromosome DNA, by PCR. Search PCR (Capture PCR) also requires digestion and ligation with multiple restriction enzymes to place a double-stranded sequence that has been genetically manipulated before PCR into an unknown part of the DNA molecule.

Another method for searching for an unknown sequence is Parker (Nucleic Acids Res. 19 (1991), 3055-3060). In addition, PCR, nested primers, and promoter finder libraries can be used to penetrate genomic DNA (PromoterFinder®) Clontech (Palo Alto CA)). This process eliminates the need to screen the library and helps find intron and exon junctions. A preferred library for screening full-length cDNAs is one that has been sorted by size to contain larger cDNAs. Further, the randomly primed library is preferable in that it contains more sequences including the 5 ′ region and upstream region of the gene. Randomly primed libraries are particularly useful when the oligo d (T) library does not yield a full-length cDNA. Furthermore, direct sequencing of primer extension products may be performed. Genomic libraries are useful for extension to the 5 'untranslated regulatory region. Capillary electrophoresis may be used to analyze or confirm the size of nucleotide sequences in sequencing or PCR products. For example, see Sambrook above. Systems for rapid sequencing are available from Perkin, Elmer, Beckman Instruments (Fullerton CA) and other companies.

Computer identification of phosphatonin polypeptides and the genes encoding them Basic Local Alignment Search Tool (Altschul, Nucleic Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol. Evol 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410), BLAST2, can be used to search for local sequence alignments. . BLAST can generate alignments of nucleotide and amino acid sequences to find sequence similarities. Due to the local nature of the alignment, BLAST is particularly useful for determining perfectly matching sequences and identifying homologues. The basic unit of BLAST algorithm output is a high-scoring segment pair (HSP). An HSP consists of two fragment sequences, optionally but of the same length, whose alignment is locally maximal and whose alignment score meets or exceeds a threshold or cut-off score set by the user. It is surpassed. The BLAST approach searches the HSP between the target sequence and the sequence in the database. If a match is found, its statistical significance is examined, and the significance threshold set by the user is met. Report only those matches. Variable E sets a statistically significant threshold for sequences reported to match the database. E indicates the upper boundary of the frequency at which an HSP (or set of HSPs) is expected to appear within the overall database search. Any database sequence that satisfies E is reported in the program output.

たとえば、積スコアが40であれば1〜2％の誤差の範囲内で適合し、積スコアが70であれば完全に適合する。相同の分子は通常、15〜40の範囲の積スコアを示す分子を選択することにより同定される。もっと低いスコアであれば関連する分子が同定される。 Similar computer techniques using BLAST (Altschul, 1997, 1993, 1990, supra) are used to search for identical or related molecules in nucleotide databases such as Genbank and EMBL . This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of computer searches can be varied to classify whether any given match is completely identical or homologous. The basis for this search is a product score, which can be expressed as: and considers both the similarity between the two sequences and the length of the matched sequences.

For example, if the product score is 40, it fits within an error of 1-2%, and if the product score is 70, it fits perfectly. Homologous molecules are usually identified by selecting molecules that exhibit product scores in the range of 15-40. A lower score identifies the relevant molecule.

  Various applicable examples of phosphatonin polynucleotides and polypeptides according to the invention and molecules derived therefrom are described in detail below.

Phosphatonin polynucleotides and polypeptide phosphatonin are derived from a cDNA library made from mRNA extracted from meningeal phosphateuria-mesenchymal-tumor resected from patients with neoplastic hypophosphatemic osteomalacia. Released; see Example 4.

  The phosphatonin nucleotide sequence set forth in SEQ ID NO: 1 was assembled from partially homologous ("overlapping") sequences obtained from related DNA clones. The duplicated sequence is incorporated into a single-stranded multiple-overlapping sequence (usually 3-5 times per nucleotide site), resulting in the sequence in SEQ ID NO: 1 It was. Thus, SEQ ID NO: 1 and translated SEQ ID NO: 2 are sufficiently accurate and suitable for various applications well known in the art or detailed below. For example, SEQ ID NO: 1 is useful for designing a nucleic acid hybridization probe that detects the nucleic acid sequence contained in SEQ ID NO: 1. These probes also hybridize with nucleic acid molecules in biological samples, and therefore the present invention can be used as various forensic and diagnostic methods. Similarly, the polypeptide identified from SEQ ID NO: 2 is also used to produce antibodies that specifically bind to phosphatonin. However, the DNA sequence obtained by the sequencing reaction may contain a missequence. This missequence exists as a misidentified nucleotide or as a nucleotide insertion or deletion in the resulting DNA sequence. Improper nucleotide insertions or deletions can cause frameshifts within the reading frame of the predicted amino acid sequence. In these cases, the deduced amino acid sequence is different from the actual amino acid sequence (for example, 1 base in an open reading frame of more than 1000 bases) even though the produced DNA sequence shows 99.9% or more identity with the actual DNA sequence. Is inserted or deleted).

  Therefore, for applications where the accuracy of the nucleotide sequence or amino acid sequence is required, the present invention is limited to the nucleotide sequence provided as SEQ ID NO: 1 or the predicted translated amino acid sequence shown as SEQ ID NO: 2. And means for cloning cDNA and genomic DNA corresponding to the nucleotide sequence of SEQ ID NO: 1. The base sequence of the phosphatonin clone thus obtained can be easily determined by determining the sequence of the clone according to a known method. The putative phosphatonin amino acid sequence can be confirmed by such cDNA or genomic clones. In addition, the amino acid sequence of the protein encoded by the clone so obtained is sequenced according to the methods described herein by expressing the protein by peptide sequencing or by expressing the protein in a suitable host cell and recovering the protein. And can be determined directly by measuring function.

  The invention further relates to the phosphatonin gene corresponding to SEQ ID NO: 1. The phosphatonin gene can be isolated according to a known method using the sequence information disclosed herein. Such methods include preparing probes and primers from the disclosed sequences and identifying or amplifying phosphatonin genes from sources suitable as genetic material. The present invention further provides phosphatonin homologues. Homologues can be isolated and identified by creating appropriate probes and primers from the sequences described herein and screening appropriate nucleic acid sources for the desired homologue.

  Thus, by the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2, the same or similar nucleic acid molecule encoding phosphatonin protein can be obtained from other species or organisms. In particular, the orthologous phosphatonin gene of mammals other than humans can be isolated. As used herein, the term “ortholog” means a homologous sequence in different species derived from a common ancestral gene during species differentiation. Orthologous genes may or may not cause similar functions; see Glossary in Trends Guide to Bioinformatics, Trends Supplement 1998, Elsevier Science.

  The phosphatonin polypeptide can be prepared using a suitable method. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides made by combining these methods. included. Methods for preparing such polypeptides are well known in the art.

  The phosphatonin polypeptide is preferably in isolated form, preferably substantially pure. Recombinantly produced phosphatonin polypeptides, including secreted polypeptides, can be substantially purified by the one-step method described in Smith and Johnson, Gene 67 (1998), 31-40. The phosphatonin polypeptide can also be purified from natural or recombinant sources using the antibodies of the present invention raised against phosphatonin protein by methods well known in the art.

Polynucleotides and polypeptide variants "mutants" are different from phosphatonin polynucleotides or polypeptides, but are essential for their immunological activity, particularly biological activity as described above. It means a polynucleotide or polypeptide that retains the proper properties. In general, variants are generally very similar and are identical to phosphatonin polynucleotides or polypeptides in many regions.

  Such polynucleotides encode fragments, analogs or derivatives and in particular orthologs of the phosphatonin proteins described above, and for example amino acid and / or nucleotide deletions, insertions, substitutions, additions and / or sets. Including a polynucleotide that differs from the above amino acid sequence or the nucleotide sequence from which it is based, either by substitution or by any one or combination of any other modifications known in the art. Methods for introducing such modifications of nucleic acid molecules according to the present invention are well known to those skilled in the art. All such fragments, analogs, derivatives and the like of the proteins of the present invention are subject to the present invention unless the immunological and / or biological properties, which are essential properties as described above, are essentially affected. Included within the scope of the invention.

  The term “variant” is used herein to refer to nucleotides and the amino acid sequences they encode, each differing from one or more nucleotides by the phosphatonin polynucleotides and polypeptides described above. Means having high homology with the nucleic acid molecule. Homology means sequence identity of at least 40%, preferably 50%, more preferably 60%, even more preferably 70%, in particular identity of at least 80%, preferably more than 90%, and more preferably It is understood to refer to sequences that are greater than 95%. Such nucleic acid molecule sequence changes can occur, for example, as a result of nucleotide substitutions, deletions, additions, insertions, and / or recombination; see above. Homology also means that the respective nucleic acid molecule or the protein encoded thereby is functionally and / or structurally equivalent. Examples of nucleic acid molecules that are homologous to the nucleic acid molecule and that are derivatives of the nucleic acid molecule include, for example, modified products having the same biological function, particularly modifications that encode proteins having the same or substantially the same biological function. There are nucleic acid molecule variants that represent things. They can be naturally occurring variants, such as sequences and variants from other mammals. These mutations can occur naturally or can be obtained by mutagenesis techniques. Allelic variants include naturally occurring allelic variants and synthetic or genetic engineering variants; see above.

  A polynucleotide having a nucleotide sequence that is at least 95% “identical” to the nucleotide sequence of the present invention may include up to 5 point mutations per 100 nucleotides of a reference nucleotide sequence encoding a phosphatonin polypeptide. Except that, it means that the nucleotide sequence of the polynucleotide is identical to the reference sequence. In other words, to obtain a polynucleotide having a base sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with other nucleotides, or a reference Nucleotides representing up to 5% of the total nucleotides of the sequence can be inserted into the reference sequence. The sequence of interest is the entire sequence shown in SEQ ID NO: 1, its ORF (open reading frame), or any fragment specified herein.

  In fact, certain nucleic acid molecules and polynucleotides are at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the present invention. Whether it matches the nucleotide sequence can be determined using a commonly used computer program. A preferred method for determining the best overall match between the sequence of interest (the sequence of the invention) and the sequence being tested, also referred to as global sequence alignment, is similar to Brutlag et al. (Comp. App. Biosci. 6). (1990), 237-245) and can be determined using the FASTDB computer program. Both sequential alignments of the subject sequence and the sequence to be tested are DNA sequences. RNA sequences can be compared by substituting U for T. The result of the above-mentioned global sequence alignment is expressed as a match rate. Preferred variables used in FASTDB alignment of DNA sequences for calculating the match rate are Matrix = Unitary, k-tuple = 4, Mismatch Penalty = 1, Joining Penalty = 30, Randomization Group Length = 0, Cutoff Score = 1, Gap Penalty = 5, Gap Size Penalty = 0.05, Window Size = 500, or the length of the nucleotide sequence to be tested is the shorter one.

  If the test sequence is shorter than the sequence of the invention due to a 5 'or 3' end deletion rather than an internal deletion, the experimental results must be manually corrected. This is because the FASTDB program does not take into account deletions at the 5 'or 3' end of the test sequence when calculating the percent identity. For test sequences that have 5 'or 3' ends truncated relative to the query sequence, the match rate is the number of matched / unaligned query sequence bases 5 'and 3' of the test sequence, It is corrected by calculating as a percentage of all bases in the query sequence. Whether a nucleotide is matched / aligned is determined by the results of FASTDB sequence alignment. This percentage is subtracted from the sequence identity and calculated by the FASTDB program described above using specific variables to produce the final sequence identity score. This corrected score is what is used for the purposes of the present invention. Only those bases outside the 5 ′ and 3 ′ bases of the test sequence that do not match / align with the query sequence are calculated for the purpose of manually adjusting the sequence match score as indicated by the FASTDB alignment.

  For example, a 90 base test sequence is aligned with a 100 base query sequence to determine sequence identity. A deletion has occurred at the 5 ′ end of the test sequence, so the FASTDB alignment does not show a match / alignment for the first 10 bases at the 5 ′ end. The 10 unsigned bases are represented as 10% of the sequence (number of mismatched bases at the 5 'and 3' ends / total number of bases in the query sequence), so 10% is the match calculated by the FASDB program Subtract from rate score. If the remaining 90 bases are perfectly matched, the final sequence identity is 90%. In another example, a 90 base test sequence is compared to a 100 base query sequence. In this case, since the deletion is an internal deletion, there is no base located at the 5 ′ end or 3 ′ end of the test sequence that does not match / allanate with the query sequence. In this case, the match rate calculated by FASTDB is not manually corrected. Again, only the 5 ′ and 3 ′ bases of the test sequence that do not match / align with the query sequence are manually corrected. Otherwise, manual correction is not performed for the purposes of the present invention.

  A query amino acid sequence of the invention and a polypeptide having at least, for example, 95% “identical” amino acid sequence, except that the test polypeptide sequence contains up to 5 amino acid changes per 100 amino acids of the query amino acid sequence. It means that the amino acid sequence of the polypeptide is identical to the query sequence. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence, up to 5% of amino acid residues in the test sequence are inserted, deleted, added, or replaced with another amino acid. be able to. These changes in the reference sequence are individually interspersed with residues of the reference sequence, either at the amino-terminal or carboxy-terminal site of the reference amino acid sequence, or anywhere between these terminal sites, or the reference sequence May occur in one or more consecutive residues.

  As a practical matter, a particular polypeptide may have at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, for example, in the amino acid sequence shown in SEQ ID NO: 2. , 98%, or 99% can be routinely determined using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the invention) and a test sequence, also referred to as global sequence alignment, is referred to as Brutlag et al. (Comp. App. Biosci. 6 (1990), 237-245). ) Using the FASTDB computer program based on the algorithm. In a sequence alignment, the query and test sequences are both nucleotide sequences or both are amino acid sequences. The result of the global sequence alignment is expressed as a match rate. The preferred parameters used for FASTDB amino acid alignment are: Matrix = PAM0, k-tuple = 2, Mismatch Penalty = 1, Joining Penalty = 20, Randomization Group Length = 0, Cutoff Score = 1, Window Size = Sequence length, Gap Penalty = 5, Gap Size Penalty = 0.05, Window Size = 500, or any length of test amino acid sequence.

  If the test sequence is shorter than the query sequence due to N-terminal or C-terminal deletions rather than internal deletions, manual corrections must be made to the results. This is because the FASTDB program does not consider N-terminal and C-terminal truncations of the test sequence when calculating the overall percent identity. For test sequences shortened at the N- and C-termini compared to the query sequence, the number of residues in the query sequence at the N- and C-termini of the test sequence that do not match / align with the corresponding test residue The percent identity is corrected by calculating as a percentage of all bases. Whether a residue is matched / aligned is determined by the results of FASTDB sequence alignment. This percentage is subtracted from the match rate and calculated by the FASTDB program described above using special variables to produce the final match score. This final match score is the score used for the purposes of the present invention. Only residues for the N-terminus and C-terminus of the test sequence that do not match / align with the query sequence are taken into account to manually correct the percent identity score. That is, only the query residues are located outside the farthest N-terminal and C-terminal residues of the test sequence.

  For example, a 90 amino acid residue test sequence is aligned with a 100 residue query sequence to determine percent identity. A deletion has occurred at the N-terminus of the test sequence, so the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. Ten unmatched residues are present in 10% of the sequence (number of non-matching residues at the N- and C-termini / total number of residues in the query sequence), so 10% is the match calculated by the RASTDB program Subtract from rate score. If the remaining 90 residues are perfectly matched, the final match is 90%. In another example, a 90 residue test sequence is compared to a 100 residue query sequence. If the deletion is an internal deletion at this time, there is no residue that does not match / align with the query sequence at the N-terminus or C-terminus of the test sequence. In this case, the matching rate calculated by FASTDB is not manually corrected. Again, as indicated in the FASTDB alignment, only residues located outside the N-terminus and C-terminus of the test sequence that do not match / align with the query sequence are manually corrected. Otherwise, manual correction is not made for the purposes of the present invention.

  The phosphatonin variant may contain changes in the coding region, non-coding region, or both. Particularly preferred are polynucleotide variants that contain changes that do not alter the properties or activities of the encoded polypeptide but result in silent substitutions, additions, or deletions. Nucleotide variants generated by silent substitution due to the degeneracy of the genetic code are preferred. Furthermore, a mutant in which amino acids at positions 5 to 10, 1 to 5, or 1 to 2 are substituted, deleted, or added in any combination is also preferable. Phosphatonin polypeptide variants can be generated for a variety of reasons, for example, to optimize codon expression for a particular host (by changing the codons of human mRNA to those preferred for bacterial hosts such as E. coli). it can.

  Naturally occurring phosphatonin variants, called “allelic variants”, refer to one of several alternative types in which a gene occupying a given locus on an organism's chromosome is altered. (Genes II, Lewin, B., John Wiley & Sons, New York (1985) and latest edition.) These allelic variants can vary at either the polynucleotide and / or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis or direct synthesis.

  Using known methods of protein engineering and recombinant DNA technology, variants can be produced to improve or alter the properties of phosphatonin polypeptides. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the protein as long as the biological function is not substantially impaired. The author of Ron, J. Biol. Chem. 268 (1993), 2984-2988 reports a KGF mutein with heparin binding activity even after deletion of 3, 8 or 27 amino-terminal amino acid residues. is doing. Similarly, interferon gamma was up to 10 times more active after deleting 8-10 amino acid residues from the carboxy terminus of the protein. (Dobeli, J. Biotechnology 7 (1988), 199-216.)

  Furthermore, extensive evidence shows that mutants often retain biological activity similar to that of natural proteins. For example, Gayle et al. Performed extensive mutational analysis of the human cytokine IL-1A (J. Biol. Chem. 268 (1993); 22105-22111). They used random mutagenesis to generate more than 3,500 different IL-1a variants with an average of 2.5 amino acids changing over the full length of the molecule per variant. Multiple mutations were examined at all possible amino acid sites. Researchers have found that "the majority of molecules can change with little or no effect on either [binding or biological activity]"; see summary. In fact, only 23 unique amino acid sequences out of the over 3,500 nucleotide sequences investigated produced proteins that differed significantly in activity from the wild type.

  In addition, other biological activities remain retained even if one or more biological functions are altered or lost as a result of deletion of one or more amino acids from the N-terminus or C-terminus of the polypeptide. May remain. For example, the ability of a deletion mutant to induce and / or bind to a protein-recognizing antibody can be retained if the major protein residue has not been removed from the N-terminus or C-terminus High nature. Certain polypeptides that do not have N-terminal or C-terminal residues of proteins that retain such immunological activity are readily determined by routine methods described herein or methods known in the art. it can. In addition, the PESTFIND program (Rogers, Science 234 (1986), 364-368) can be used to identify PEST sequences (rich in proline, glutamate, serine, and threonine) that are characteristically present in unstable proteins it can. Such sequences can be removed from the phosphatonin protein to increase protein stability and optionally activity. Methods for introducing such modifications into the nucleic acid molecules according to the present invention are well known to those skilled in the art.

  Accordingly, the present invention further includes phosphatonin polypeptide variants that exhibit substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as to have little effect on activity. For example, guidance on how to make phenotypic silent amino acid substitutions is provided in Bowie (Bowie, Science 247 (1990), 1306-1310) where authors can study resistance to amino acid changes. It shows that there are two main methods.

  The first method takes advantage of resistance to amino acid substitution by natural selection during the evolution process. By comparing amino acid sequences in different species, conserved amino acids are identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid sites whose substitutions are allowed by natural selection indicate that these sites are not important for protein function. Therefore, even if the site where the amino acid substitution is allowed is modified, the biological activity of the protein is still maintained.

  The second method uses genetic engineering that introduces amino acid changes at specific sites in the cloned gene to identify regions important for protein function. For example, site-directed mutagenesis or alanine scanning mutagenesis (introducing a single alanine mutation at every residue in the molecule) can be used. (Cunningham and Wells, Science 244 (1989), 1081-1085.) The resulting mutant molecules can be tested for biological activity.

  As the authors state, these two methods revealed that proteins surprisingly tolerate amino acid substitutions. The authors further show that amino acid changes are likely to occur at certain amino acid sites in the protein. For example, the most buried amino acid residues (within the tertiary structure of the protein) require nonpolar side chains, whereas surface side chain features are generally largely conserved. In addition, acceptable conservative amino acid substitutions include substitution of aliphatic hydrophobic amino acids Ala, Val, Leu and Ile; substitution of hydroxyl groups Ser and Thr; substitution of acidic residues Asp and Glu; amide residues Asn and Gln Substitution of the basic residues Lys, Arg, and His; substitution of the aromatic residues Phe, Tyr, and Trp; and substitution of the smaller amino acids Ala, Ser, Thr, Met, and Gly.

  In addition to conservative amino acid substitutions, variations of phosphatonin include the following: (i) substitution of one or more non-conservative amino acid residues, where the substituted amino acid residue is a gene A substitution that may or may not be encoded by the code, or (ii) a substitution with one or more amino acid residues having a substituent (iii) a compound that increases the stability and / or solubility of the polypeptide Fusion of a mature polypeptide with another compound such as polyethylene glycol, or (iv) a polypeptide with another amino acid, such as an IgG Fc fusion region peptide, or a leader sequence, secretory sequence, or an easily purified sequence Fusion. Such mutant polypeptides are deemed to be within the scope of those skilled in the art from the disclosure herein. For example, phosphatonin polypeptide variants that include substitution of charged amino acids with other charged or neutral amino acids produce proteins with improved characteristics, such as forming smaller aggregates. Aggregation of pharmaceutical formulations reduces activity and increases loss due to the immunological activity of the aggregate; for example, Pinckard, Clin. Exp. Immunol. 2 (1967), 331-340; Robbins, Diabetes 36 (1987 ), 838-845; Cleland, Crit. Rev. Therapeutic Drug Carrier Systems 10 (1993), 307-377.

Polynucleotide and polypeptide fragment In the present invention, “polynucleotide fragment” means a short polynucleotide having the nucleic acid sequence contained in SEQ ID NO: 1. Short nucleotide fragments are preferably at least about 15 nucleotides in length, more preferably at least about 20 nucleotides, even more preferably at least about 30 nucleotides, and even more preferably at least 40 nucleotides. A fragment of “at least 20 nucleotides in length” means, for example, that it contains 20 or more consecutive bases of the cDNA sequence contained in the nucleotide sequence shown in SEQ ID NO: 1. These nucleotide fragments are useful as diagnostic probes and primers as described herein. Of course, larger fragments (eg, 50, 150, 500, 600, 1000 nucleotides) are preferred.

  Further, representative examples of phosphatonin polynucleotide fragments include, for example, about 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 250, 251 to 300, 301 to SEQ ID NO: 1. 350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, Fragments having a nucleotide sequence at positions 1001 to 1050, 1051 to 1100, 1101 to 1150, 1151 to 1200, 1201 to 1250, 1251 to 1300, and 1301 to 1350 are included. In this context, “about” means more than a specific range, or some nucleotides (5, 4, 3, 2, or 1) located at either end or both ends. Or include less. Preferably, these fragments encode polypeptides having biological activity. More preferably, these polynucleotides can be used as probes or primers as described herein.

  In the present invention, the “polypeptide fragment” means a short amino acid sequence contained in SEQ ID NO: 2. A protein fragment may be “present alone” or contained within a larger polypeptide as a fragment or region that forms part of a larger polypeptide, most preferably as a single contiguous region. Also good. Representative examples of polypeptide fragments according to the present invention include fragments up to the end of the coding region, such as about 1 to 20, 21 to 40, 41 to 60, 61 to 80, 81 to 100, 102 to 120, 121. ~ 140,141 ~ 160,161 ~ 180,181 ~ 200,201 ~ 220,221 ~ 240,241 ~ 260,261 ~ 280,281 ~ 300, 301 ~ 320, 321 ~ 340, 341 ~ 360, 361 ~ 380 , 381-400, and fragments derived from amino acids 401-421. Further, the polypeptide fragment can be an amino chain of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 in length. In this context, “about” is more due to a specific range or some amino acids (5, 4, 3, 2, or 1) located at either or both ends. Including those that are less or less.

  Preferred polypeptide fragments include phosphatonin proteins having a contiguous sequence of residues deleted from the amino chain terminus or the carboxy terminus, or both. For example, any number of amino acids from 1 to 60 may be deleted from the amino terminus of the phosphatonin polypeptide. Similarly, any number of amino acids from 1 to 30 may be deleted from the carboxy terminus of the phosphatonin protein. Furthermore, any combination of the amino and carboxy terminal deletions described above is preferred. Likewise preferred are polynucleotide fragments encoding these phosphatonin polypeptide fragments.

  In particular, the N-terminal deletion of a phosphatonin polypeptide can be represented by the general formula m-430, where m is an integer from 2 to 416, and m is the amino acid residue shown in SEQ ID NO: 2. Corresponds to the position of the group.

  Also preferred are phosphatonin polypeptides and polynucleotide fragments characterized by structural or functional domains. Preferred embodiments of the present invention include an alpha helix, an alpha helix structure region (“alpha region”), a beta sheet, a beta sheet structure region (“beta region”), a rotation, a rotation structure region (“rotation region”), a coil, a coil Structural region (“coil region”), hydrophilic region, hydrophobic region, alpha amphiphilic region, beta amphiphilic region, flexible region, surface structure region, substrate binding region, and high antigenicity index A fragment containing the region is included. As shown in the figure, such preferred regions include Garnier-Robson alpha region, beta region, rotation region, coil region, Chou-Fasman alpha region, beta region, rotation region, Kyte-Doolittle hydrophilic region, hydrophobic Regions, Eisenberg alpha, beta amphipathic regions, Krplus-Schulz flexible regions, Emini surface structure regions, Jameson-Wolf high antigenicity index regions, and the like. A polypeptide fragment of SEQ ID NO: 2 that aligns within the conserved region is specifically contemplated by the present invention and is shown in the figure. In addition, polynucleotide fragments encoding these domains can be expected. In addition, polynucleotides encoding these domains are also contemplated.

  Another preferred fragment is a biologically active phosphatonin fragment. A biologically active fragment need not be exactly the same as the activity of a phosphatonin polypeptide, but is a fragment that exhibits similar activity. The biological activity of the fragment includes an improved desired activity or a reduced undesirable activity.

  However, many polynucleotide sequences, such as EST sequences, are publicly available and are available through sequence databases. Some of these sequences are related to SEQ ID NO: 1, and some may be publicly available prior to the concept of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Enumerating all related sequences is cumbersome.

  Accordingly, the nucleotide sequence represented by the general formula ab (where a is an integer from 1 to 1655 of SEQ ID NO: 1, b is an integer from 15 to 1655, and a and b are both sequences. One or more polynucleotides comprising the position of the nucleotide residue shown in number 1 and including b greater than or equal to a + 14 are preferably excluded from the present invention.

Antigenic determinant & antibody In the present invention, "antigenic determinant" refers to the body of an animal such as a rat, rabbit, human, mouse (including a transgenic mouse having a human immunoglobulin gene and producing a human antibody molecule). Means a phosphatonin polypeptide fragment having antigenic or immunological activity. A preferred embodiment of the present invention relates to a phosphatonin polypeptide fragment comprising an antigenic determinant, as well as a polynucleotide encoding this fragment. The region of a protein molecule to which an antibody can bind is defined as “antigenic antigenic determinant”. In contrast, an “immunoantigenic determinant” is defined as the portion of a protein that elicits an antibody response; see, eg, Geysen, Proc. Natl. Acad. Sci. USA 81 (1983); 3998-4002 . Fragments made yesterday as antigenic determinants can be made by any conventional means; see for example Houghten, Proc. Natl. Acad. Sci. USA 82 (1985), 5131-5135, see US Pat. No. 4,634,211 for details. .

  In the present invention, the antigenic antigenic determinant preferably comprises a sequence of at least 7, more preferably at least 9, most preferably from about 15 to about 30 amino acids. Antigenic antigenic determinants are particularly useful for generating antibodies, including monoclonal antibodies, that bind to antigenic determinants; see eg Wilson, Cell 37 (1984), 767-778; Sutcliffe, Science 219 (1983), See 660-666.

  Similarly, immunogenic determinants can be used to elicit antibodies according to methods well known in the art; for example, Sutcliffe, supra; Wilson, supra; Chow, Proc. Natl. Acad. Sci. USA 82 (1985), 910-914; and Bittle, J. Gen. Virol. 66 (1985); 2347-2354. Preferred immune antigenic determinants include soluble proteins. The immune antigenic determinant is presented with a carrier protein such as albumin in animal systems (such as rabbits and mice) or without a carrier protein if it is of sufficient length (at least about 25 amino acids). sell. However, it has been shown that an immune antigenic determinant having only 8-10 amino acids can produce sufficient antibodies that can bind at least to the linear antigenic determinant of a degenerate polypeptide (eg, (For Western blotting)

  GCG-peptide structure provided by The Human Genome Resource Center (http://www.hgmp.mrc.ac.uk/homepage.html) (Rice, Program Manual for the EGCG package, Cambridge, CB10 1RQ England: Hinxton Hall; 1995) was found to be antigenic at the amino acid residues in the region shown in FIG. Therefore, these regions can be used as antigenic determinants that produce antibodies against the protein encoded by SEQ ID NO: 1.

As used herein, “antibody (Ab)” or “monoclonal antibody (MAb)” refers to complete molecules and antibody fragments (eg, Fab and F (ab ′) ₂ ) capable of specifically binding to a protein. A fragment, etc.). Fab and F (ab ′) ₂ fragments lack Fc fragments of intact antibodies, disappear more rapidly from the circulation, and have less non-specific tissue binding than intact antibodies; for example, Wahl , J. Nucl. Med. 24 (1983), 316-325. Thus, these fragments are preferred, as are FAB products or immunoglobulin expression libraries. Furthermore, the antibodies of the invention include phage display, transgenic mice carrying human immunoglobulin genes and / or human chromosomes, immune cells isolated from the human body, in vitro or ex vivo immunity of human immune cells, or any Chimeric antibodies, single chain antibodies, humanized antibodies, or human antibodies obtained or obtained from other methods are included.

  In another embodiment, the present invention hybridizes with a complementary strand of a phosphatonin polynucleotide of the present invention and encodes a variant of the above protein that has lost its immunological activity, preferably biological activity. Relates to nucleic acid molecules. This embodiment proves useful, for example, for generating allelic dominant variants of the phosphatonin protein described above. The mutant is preferably produced by substituting, deleting, and / or adding an amino acid residue at positions 1 to 5 or 5 to 10 in the amino acid sequence of the wild type protein.

Production of vectors, host cells and proteins The present invention also relates to vectors comprising phosphatonin polynucleotides, host cells and the production of polypeptides by recombinant techniques. The vector can be, for example, a phage, plasmid, virus, retrovirus vector. Retroviral vectors may be replication competent or replication defective. In the latter case, virus propagation generally occurs only in complementary host cells.

  The phosphatonin polynucleotide may be linked to a vector containing a selectable marker for growth in the host. In general, plasmid vectors are introduced as a precipitate, such as a calcium phosphate precipitate, or as a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and subsequently transduced into host cells.

  Phosphatonin polynucleotide inserts are suitable, such as the phage lambda PL promoter, the E. coli lac, trp, phoA, and tac promoters, the SV40 early and late promoters, and the retroviral LTR promoter, to name just a few. Should be operably linked to a promoter. Other suitable promoters will be known to those skilled in the art. The expression construct further includes a transcription initiation site, termination site, and transcription region, a ribosome binding site for translation. The coding portion of the transcript expressed by the construct preferably includes a translation initiation codon present at the start position and a termination codon (UAA, UGA, or UAG) located appropriately at the end of the polypeptide to be translated.

  As indicated, the expression vector preferably includes at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin, or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of suitable hosts include E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9; CHO, COS, 293 and Bowes melanoma cells, etc. Animal cells; as well as plant cells. Suitable culture media and conditions for the above host cells are known in the art. Preferred vectors for use in bacteria include: pQE70, pQE60, and pQE-9 available from QIAGEN, Inc .; from Stratagene Cloning Systems, Inc. Available pBluescript vector, phage script vector, pNH8A, pNH16a, pNH18A, pNH46A; and ptrc99a, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Preferred eukaryotic cell vectors are pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

  Further, for example, using a mammalian cell that already contains a nucleic acid molecule encoding the above phosphatonin polypeptide in its genome, but does not express it, for example due to a weak promoter, or in an appropriate manner, To induce expression of a phosphatonin polypeptide, expression control sequences such as a strong promoter that is very proximal to the endogenous nucleic acid molecule encoding it can be introduced into mammalian cells.

  In this context, the term “expression control sequence” refers to a nucleic acid that can be used to increase the expression of a phosphatonin polypeptide because it integrates into the cellular genome very proximal to the phosphatonin-encoding gene. Represents a molecule. Such regulatory sequences induce the expression of promoters, enhancers, inactivated silencer intron sequences, 3′UTR and / or 5′UTR coding regions, proteins and / or RNA stabilizing elements such as phosphatonin genes Or a nucleic acid molecule encoding a regulatory protein, such as a transcription factor that can be triggered or other gene expression regulatory elements known to activate gene expression and / or increase the amount of gene product included. By introducing the expression regulatory sequence, the expression of phosphatonin polypeptide is increased and / or induced, resulting in an increase in the amount of phosphatonin polypeptide in the cell. Therefore, the present invention aims to newly express and / or increase expression of phosphatonin polypeptides.

  Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals such as Davis, Basic Methods In Molecular Biology (1986). It is specifically contemplated that the phosphatonin polypeptide is expressed by a host cell that does not actually have a recombinant vector.

  Phosphatonin polypeptides can be used in ammonium sulfide or ethanol precipitation, acid extraction, cation or anion exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectins. It can be recovered and purified from recombinant cultured cells by well-known methods including chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification.

  Also, phosphatonin polypeptides can be recovered from: products purified from natural sources, including directly isolated or cultured body fluids, tissues and cells; products from chemical synthesis methods And products produced recombinantly from prokaryotic or eukaryotic hosts including, for example, bacteria, yeast, higher plants, insects, and mammalian cells. Depending on the host used in the recombinant production process, the phosphatonin polypeptide may or may not be glycosylated. In addition, the phosphatonin polypeptide can include an initial (modified) methionine residue. In some cases it is the result of a host-mediated process. Thus, it is well known that the N-terminal methionine encoded by the translation initiation codon is generally efficiently removed from any protein after translation in eukaryotic cells. The N-terminal methionine of most proteins is effectively removed in most prokaryotic cells, whereas the prokaryotic removal process functions in some proteins due to the nature of the amino acid to which the N-terminal methionine is covalently bound. do not do.

In a particularly preferred embodiment, the present invention relates to a method for isolating a phosphatonin polypeptide comprising the following steps:
(A) culturing the tumor conditioned medium or bone softening cells in a serum-containing medium (DMEM Eagles / 10% FCS / glutamine / antimycotic (DMFCS)) until confluence;
(B) incubating the cells every other day in serum-free medium DMEM Eagles / glutamine / antifungal antibiotics (DM) for up to 5 hours;
(C) recovering the conditioned serum free medium from the cells and equilibrating the conditioned medium with 0.06M sodium phosphate pH 7.2 and 0.5M sodium chloride (PBS);
(D) applying the medium of (c) to an equilibrated concanavalin A sepharose column;
(E) thoroughly washing the column with PBS;
(F) eluting the concanavalin A column with PBS supplemented with 0.5M α-methyl-D-glucopyranoside;
(G) subjecting the sample eluted in (f) to cation exchange chromatography; and (h) eluting the fraction containing the phosphatonin polypeptide with 0.5 M sodium chloride. The above method is described in Example 1.

  Another object of the present invention is a method for preparing a phosphatonin polypeptide under conditions that allow expression of the polypeptide by the presence of a vector or a polynucleotide according to the present invention or an exogenous expression regulatory sequence. A method comprising culturing a host cell of the present invention capable of expressing the polypeptide and recovering the polypeptide thus produced from the culture medium. It should also be understood that the protein can be expressed in a cell-free system using, for example, in vitro translation assays known in the art.

  Thus, in a further aspect, the present invention relates to a phosphatonin polypeptide, or an immunologically and / or biologically active fragment thereof encoded by a polynucleotide of the present invention or produced by the method described above. About. Similarly, phosphatonin polypeptides obtained by proteolytic degradation of the above phosphatonin polypeptides with PHEX metallopeptidase are within the scope of the present invention.

  It will be apparent to those skilled in the art that the proteins of the invention can be further conjugated to other molecules as described above, eg, for drug targeting and imaging applications. Such binding may be performed chemically at the junction site after expression of the protein, or the binding product may be genetically engineered into the protein of the invention at the DNA level. Thereafter, the DNA is expressed in a suitable host system, and the expressed protein is recovered and reconstituted as necessary.

Regulation of Phosphate Metabolism As already described herein, the phosphatonin polypeptides of the present invention can regulate phosphate metabolism in different ways. Accordingly, in one embodiment, the present invention relates to a phosphatonin polypeptide having phosphatonin activity in that it has at least one of the following activities:
(A) can down-regulate sodium-dependent phosphate co-transport;
(B) renal 25-hydroxyvitamin D3-24-hydroxylase can be up-regulated; and / or (c) renal 25-hydroxy D-1α-hydroxylase can be down-regulated.

In another embodiment, the present invention relates to a phosphatonin polypeptide having anti-phosphatonin activity in that it has at least one of the following activities:
(A) can up-regulate sodium-dependent phosphate co-transport;
(B) renal 25-hydroxyvitamin D3-24-hydroxylase can be down-regulated; and / or (c) renal 25-hydroxy D-1α-hydroxylase can be up-regulated.

  In a particularly preferred embodiment of the present invention, the phosphatonin polypeptide comprises a bone mineral motif as described above and is adjusted to promote bone mineralization.

  In yet another embodiment, the present invention relates to a phosphatonin polypeptide that has lost at least one of the above activities. Such a polypeptide may be a mutant form of the phosphatonin polypeptide of the present invention, and can be used, for example, to study the effect of mutation of a phosphatonin-encoding gene. In particular, such mutants can prove useful in the development of drugs that can compensate for defects caused by losing one of the biological activities of wild-type phosphatonin. Such phosphatonin polypeptide variants may be best studied in the screening methods described in more detail later herein.

Phosphatonin antibody Furthermore, as described above, a phosphatonin-specific antibody can be produced by providing the phosphatonin polypeptide of the present invention. In this regard, hybridoma technology allows the production of cell lines that secrete antibodies against essentially any desired substance that causes an immune response. RNA encoding the light and heavy chains of the immunoglobulin can be obtained from the cytoplasm of the hybridoma. A cDNA can be prepared using the 5 ′ end of mRNA and inserted into an expression vector. The DNA encoding the antibody or its immunoglobulin chain can be preferably expressed in mammalian cells. Depending on the host cell, reversion techniques may be required to obtain the correct placement of the antibody. If necessary, some substitutions can be made in the DNA to optimize binding using conventional cassette mutagenesis or other protein manipulation methods disclosed herein.

  Accordingly, the present invention also relates to an antibody that specifically recognizes the phosphatonin polypeptide of the present invention.

  In a preferred embodiment of the invention, the antibody is a monoclonal antibody, a polyclonal antibody, a single chain antibody, a human or humanized antibody, a primatized antibody, a chimerized antibody, or a Fab, Fv, scFv fragment, etc. Bispecific antibodies, synthetic antibodies, or peptides containing antibody fragments, fragments thereof that specifically bind to polypeptides, or chemically modified derivatives thereof. General methods for producing antibodies are well known, such as Kohler and Milstein, Nature 256 (1975), 494 and JGR Hurrel, ed., “Monoclonal Hybridoma Antibodies: Techniques and Applications”, CRC Press Inc., Boco Raron, FL (1982), and LTMimms et al., Virology 176 (1990), 604-619. Furthermore, antibodies against the aforementioned peptides and fragments thereof can be obtained by methods described in, for example, Harlow and Lane “Antibodies, A Laboratory Manual” and CSH Press, Cold Spring Harbor, 1988.

  For production of antibodies in laboratory animals, various hosts, including goats, rabbits, rats, mice, etc., are injected with a polypeptide of the invention having immunogenic properties, or any fragment, oligopeptide or derivative thereof. Can be immunized. Polyclonal antibody production techniques are well known in the art and are described in Mayer and Walker, “Immunochemical Methods in Cell and Molecular Biology”, Academic Press, London (1987) and the like. Polyclonal antibodies can also be obtained from animals, preferably mammals. Antibody purification is well known in the art and includes, for example, immunoaffinity chromatography. Depending on the host species, various adjuvants or immunological carriers are used to enhance the immunological response. Such adjuvants include complete Freund or incomplete Freund adjuvants, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsifiers, and dinitrophenol. Including, but not limited to. For example, an example of a carrier to which the peptide of the present invention can be bound is keyhole limpet hemocyanin (KLH).

  A method for producing a chimeric antibody is described, for example, in WO89 / 09622. A method for producing a humanized antibody is described in, for example, EP-A10 239 400 and WO90 / 07861. The antibody source utilized in accordance with the present invention is a so-called xenogenic antibody. General principles for producing exogenous antibodies, such as human antibodies in mice, are described, for example, in WO 91/10741, WO 94/02602, WO96 / 34096, WO96 / 33735.

In a preferred aspect, the present invention antibody has an affinity of at least about 10 ^-7 M, preferably at least about 10 ^-8 M, more preferably at least about 10 ^-9 M, and most preferably at least about 10- ¹⁰ M Has affinity. The phosphatonin antibody, on the other hand, has a binding affinity of about 10 ⁵ M ^-1 and is preferably less than 10 ⁷ M-1 if the phosphatonin activity stimulation is as expected, if phosphatonin activity is suppressed. If present, it may preferably have a binding affinity of up to 10 ¹⁰ M ⁻¹ or more.

Use of Phosphatonin Polynucleotides The phosphatonin polynucleotides identified herein can be used in various ways as reagents. The following description should be considered as an example and uses existing technology.

  Currently, there are few chromosomal marker reagents based on actual sequence data (repetitive polymorphisms), so new chromosomal markers need to be identified. Using phosphatonin related polynucleotides (genomic DNA and / or cDNA), Rowe, Hum. Genet. 94: 5 (1994), 457-467; Benham, Genomics 12 (1992), 368-376; Gillett, Ann. Hum. Genet. 60 (3) (1996), 201-211; Rowe, Nucleic Acids Res. 22 (23) (1994), 5135-5136. it can. In particular, the use of microsatellite (Rowe, Hum. Genet. 94: 5 (1994), 457-467; Rowe, Nucleic Acids Res. 22 (23) (1994), 5135-5136; Rowe, Hum. Genet. 93 ( 1994), 291-294; Roowe, Hum. Genet. 91 (1993), 571-575; Rowe, Hum. Genet. 97 (1996), 345-352; Rowe, Hum. Genet. 89 (1992), 539- 542), very information for screening phosphatonin and derivative genetic diseases by isolating information markers using irradiation fusion gene transfer hybrids and ALU-PCR (Benham, Genomics 12 (1992), 368-376) Allows rapid isolation of high volume methods. In particular, the above methods have been successful in mapping and positioning the PHEX gene (MEPE is considered a PHEX substrate), and extensive mutational analysis has revealed the structural sites and motifs necessary for PHEX biological activity (Rowe, HUm. Mol. Genet. 6 (1997), 539-549; Rowe, Exp. Nephrol. 5 (1997), 355-363; Rowe, Current Opinion in Nephrology & Hypertension 7 (4) (1998), 367 Rowe, Clinical and Experimental Nephrology 2 (3) (1998), 183-193), these similar methods are performed on phosphatonin. Recently, a powerful genome-wide dependency on single nucleotide polymorphisms (SNP's) and the combination of gel-based sequencing and high-density mutation detection DNA chips (Wang, Science 280 (1998), 1077-1082) Linkage (linkage encompassing the entire genome) and screening methods have been developed. Recently, SNP data was published on the Internet by the Center for Genome Research at the Whitehead Institute for Biomedical Research (Whitehead-MIT) at the Whitehead Biomedical Research Institute in Cambridge, Massachusetts, USA. (Http://www-genome.wi.mit.edu/SNP/human/index.html). This powerful novel oligonucleotide array-based method will be the future path for molecular expression analysis, polymorphism and genotyping and disease therapy (Wang, Science 280 (1998), 1077-1082; Chee, Science 274 (1996), 610-614; Gentalen, Nucleic Acids Res. 27 (1999), 1485-1491; Hacia, Nucleic Acids Res. 26 (1998), 3865-3866; Lipshutz, Nat. Genet. 21 (1999), 20 -24; Fan, Eur. J. Hum. Genet. 6 (1998), 134). If there is information on the sequence of MEPE in this use, the new methods and techniques described above are used to address the previously mentioned areas. The sequence is located on a particular chromosome or a particular region of the chromosome using well known techniques. These techniques include in situ hybridization to chromosome spreads, flow-sorted chromosome preparations, or artificial chromosome structures such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial P1 structures, Price (Blood Rev. 7 (1993) , 127-134) and Task (Trends Genet. 7 (1991), 149-154). Fluorescence in situ hybridization of chromosomal spreads is discussed in other literature (Verma, (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York NY). Fluorescence in situ hybridization of chromosome spreads and other physical chromosome mapping techniques may correlate with other genetic map data. The wide range of available mapping data can be found at the following Internet sites: Site provided by the Human-Genome-Mapping-Project United Kingdom (HGMP-RC) (http://www.hgmp.mrc.ac.uk/homepage.html), National Institute of Health of Health USA (NIH) "National Collection of Biological Information (NCBI)" (http://www.ncbi.nim.nih.gov/) and Whitehead Biomedical Research in Cambridge, Massachusetts, USA Find the site (http://www-genome.wi.mit.edu/) provided by the Center for Genome Research at the Whitehead Institute for Biomedical Research (Whitehead-MIT) on the Internet be able to. In addition, a wide range of microsatellite maps covering the entire human genome and associated mapping tools are also accessible through Genethon (a database provided by the French government) (http://www.genethon.fr/genethon en.htnl). Potential maps are also published by Science and Nature (see Dib, Nature 380 (1996), 152-154), but you should look up the latest data on internet sites. The correlation between the physical chromosomal map location of the gene encoding the phosphatonin polypeptide of the present invention and specific features such as hypo / hyperphosphatemia is a region of DNA associated with this feature. To help determine. The nucleotide sequences of the present invention can be used to detect differences in the gene sequences of normal and carrier or affected individuals. Furthermore, the methods and means described herein can be used for animal breeding by using markers. Furthermore, the nucleotide sequences of the present invention can also be used to find differences in chromosome positions caused by translocations, inversions, etc. between normal and carrier or affected individuals.

  Although very rare, phosphatonin polynucleotides are molecular weight markers on Southern blot gels, diagnostic probes for the presence or absence of mRNA in specific cell types, in the process of discovering new polynucleotides, Select or produce oligomers that adhere to other supports and use as probes to “draw” known sequences to produce anti-DNA antibodies using DNA immunization techniques, and antigens to elicit immune responses it can.

Use of phosphatonin polypeptides and antibodies The phosphatonin polypeptides and antibodies can be used in a variety of applications. The following description should be considered exemplary and uses known methods.

Phosphatonin polypeptides can be used to analyze protein levels in biological samples using antibody-based techniques. For example, protein expression within a tissue can be examined by typical immunohistological methods. (Example: See Jalkanen, J. Cell. Biol. 101 (1985), 976-985; Jalkanen, J. Cell. Biol. 105 (1987) 3087-3096). Other antibody-based methods for detecting protein gene expression include immunoassay methods such as solid phase enzyme immunoassay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase, iodine ⁽¹²⁵ I, ¹²¹ I), carbon ⁽¹⁴ C), sulfur ⁽³⁵ S), tritium ⁽³ H), indium ( ¹¹² In), radioisotopes such as technetium ( ⁹⁹ mTc), fluorescent labels such as fluorescein, rhodamine and biotin.

  In addition to analyzing protein levels in biological samples, proteins can also be detected by in vivo imaging. Antibody labels and markers for in vivo imaging of proteins include methods detectable by X-radiography, NMR, ESR. In X-radiography, suitable labels include radioactive isotopes such as barium and cesium that emit radioactivity but do not adversely affect the test article. Suitable markers for NMR and ESR include those with characteristic spins that can be detected, such as deuterium, which can be incorporated into antibodies by labeling the relevant hybridoma nutrients.

Specific protein antibodies and antibody fragments labeled with radioisotopes ( ¹³¹ I, ¹²¹ In, ⁹⁹ mTc, etc.), radiopaque materials, and materials detectable by nuclear magnetic resonance are incorporated into mammals (eg parenteral, Subcutaneously or intraperitoneally). It will be appreciated by those skilled in the art that the size of the test object and the imaging system used will determine the amount of imaging portion to produce a diagnostic image. When using radioisotopes in human samples, the amount of radioactivity injected is usually about 5-20 millicuries at 99 mTc. Labeled antibodies or antibody fragments are selectively stored in cells that contain a particular protein. In vivo tumor imaging is described, for example, in Burchiel, “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”, Chapter 13, in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel and Rhodes, Ed. Masson Publishing Inc. (1982). Yes.

  Accordingly, the present invention provides a method for diagnosing a disease, including: (a) expression of a phosphatonin polypeptide in a cell or tissue of an individual, or phosphatonin in a body fluid of an individual. Assay of the level of the active fragment, or antigenic criteria, (b) comparison of the normal gene expression level with the sample gene expression level. In this comparison, the presence or absence of a disorder is suggested depending on whether the phosphatonin polypeptide gene level of the sample is higher or lower than the normal level.

  In addition, phosphatonin polypeptides can be used to treat diseases. For example, increased or decreased plasma phosphate levels and / or impaired bone formation (X-chromosome hyperphosphatemia rickets, neoplastic hypophosphatemia nephromalacia, renal failure, osteoporosis, renal bone In order to promote dysplasia, etc., a phosphatonin polypeptide can be administered to the patient. Then, up-regulation and down-regulation receptors of sodium-dependent phosphate cotransporter expression can be activated or suppressed. Furthermore, the phosphatonin gene promoter and suppressor can be used for gene therapy for the treatment of phosphate metabolism-specific dysfunction, especially X-chromosomal hypophosphatemic rickets. In primary or secondary uncertain changes, inappropriate gene regulation and / or post-translational degeneration of MEPE (eg, postmenopausal women, osteoporosis, aging), bone mineral loss abnormalities However, if hormone administration (and / or administration of receptors or agonists / blockers of hormones) is performed as an additional therapy for hormone replacement therapy, the phosphate and bone mineral balance may be restored. An important feature of MEPE biological activity, and hence disease treatment, is that the N-terminal sequence is phosphate uptake in the kidney and the C-terminus (ie, the region associated with the MEPE motif described above) is normal bone mineral. It is a prediction that it is a necessary condition for development and growth.

  After kidney transplantation, chronic hyperphosphatemia, and in other cases, hypophosphatemia is a major feature that causes major clinical complications. For example, transplantation of a normal kidney in a male HYP patient has been reported to have pathological changes such as “rickets-type” phosphate leakage in a normal transplanted kidney (Morgan, Arch. Intern. Med. 134 (1974), 549-552). The clinical use of N-terminal degradation fragments of MEPE may have an effect on antihypophosphatemic treatment. Conversely, in the case of kidney transplantation that causes hyperphosphatemia, it may be possible to treat whole recombinant MEPE or an active derivative peptide formed at a unique N-terminal residue. Other diseases that may be treated with MEPE, MEPE derivative peptides, receptors, blockers-blockers (the peptide increases the potency and specificity of its action) include renal bone dysplasia, nephrotoxicity, bone paget Disease, autosomal rickets, and certain types of renal Fanconi syndrome. Furthermore, if the receptor is expressed in tissues (eg, intestinal tract) as in the kidney, it may be possible to treat patients with end-stage renal disease (eg, complete loss of renal function).

  Similarly, antibodies against phosphatonin polypeptides can be used for disease treatment. For example, administration of an antibody against a phosphatonin polypeptide binds to the polypeptide and suppresses overproduction. Similarly, administration of an antibody can bind to a polypeptide and activate the polypeptide, such as by degradation into different active forms.

  Although very rare, phosphatonin polypeptides can also be used as molecular weight markers in SDS-PAGE gels or molecular sieve filtration columns using techniques well known in the art. Alternatively, phosphatonin polypeptides can be used to increase antibodies, which can then be used to measure protein expression from recombinant cells as a method of examining host cell transformation. Is possible.

  In addition, phosphatonin polynucleotides and polypeptides can be used to analyze one or more biological activities. If one analysis shows that a phosphatonin polynucleotide or polypeptide is active, phosphatonin may be involved in a disease related to its biological activity. Thus, phosphatonin may be used to treat related diseases.

In another aspect of the regulatory sequence of the phosphatonin gene , the present invention provides a promoter that naturally regulates the expression of the polynucleotide encoding the phosphatonin polypeptide of the present invention or a polynucleotide homologous to the polynucleotide of the present invention. Relates to regulatory sequences. Methods well known in the art can be used to isolate the regulatory sequences of the promoters that naturally regulate the above DNA sequence expression. For example, a person skilled in the art can screen a genomic library consisting of human genomic DNA cloned in a phage or bacterial vector using the nucleic acid molecule as a probe. Such a library is composed of genomic DNA prepared, for example, from human blood cells, fractionated into 5-50 kb fragments and cloned into lambda GEM11 (Promega) phage. Then, the phage hybridized with the probe is purified. From the purified phage, DNA can be extracted and sequenced. For example, a human genome P1 library (Genomic Systems, Inc.) was screened with a labeled cDNA probe as described in Example 11. By isolating the genomic sequence corresponding to the gene encoding the above phosphatonin protein, it is possible to fuse heterologous DNA sequences to their promoters and regulatory sequences by transcription and translation fusion well known to those skilled in the art. Become. In order to identify the regulatory sequences or specific elements of these phosphatonin genes, the upstream fragment of the 5 'end was cloned in front of a marker gene such as luc, gfp, GUS coding region, and the resulting chimeric gene was Introduce into cells or animals to obtain transient or stable expression. The expression pattern in transgenic animals and introduced mammalian cells containing a marker gene under the control of the regulatory sequence of the present invention is compared with the expression pattern of the phosphatonin gene described in Example 10 to clarify the boundary between the promoter and the regulatory sequence. To. Usually, the regulatory sequence is part of a recombinant DNA molecule, such as a vector (see above). The invention further relates to a host cell transformed with a regulatory sequence or a DNA molecule or vector comprising the regulatory sequence of the invention. The host cell is a prokaryotic cell or a eukaryotic cell, see above.

Diagnosis of Phosphate Metabolism Disorder Another object of the present invention is for patients with phosphate metabolism disorders (see, eg, Example 6) for the pharmacogenomics of drugs or prodrugs that can be candidates for drug treatment. ) It is about selection. Therefore, the discovery of the substance of the present invention enables the development of a new drug for drug administration carried out in order to restore the genetically altered phosphatonin protein function to normal. In addition, a gene therapy method can be predicted by the present invention.

Accordingly, the present invention includes the following methods for diagnosing a disorder: (a) quantification of the expression level of a phosphatonin gene found in cells or body fluids of an individual;
(b) Comparison of standard gene expression level with the gene expression level of the sample, depending on whether the quantified phosphatonin polypeptide gene level is higher or lower than the standard level, e.g. kidney Suggests the presence or absence of impaired phosphate metabolism in the bone system, or other tissues.

More specifically, the present invention provides:
(a) determining the presence or absence of mutations in the polynucleotide encoding phosphatonin; and
(b) susceptibility to a pathological condition or pathological condition in a subject associated with a disorder of phosphate metabolism, including diagnosis of a pathological condition or susceptibility to a pathological condition based on the presence or absence of the mutation Related to the diagnosis.

In yet another aspect, the present invention provides:
(a) determining the presence or amount of expression of a phosphatonin polypeptide, or variant, in a biological sample; and
(b) to a pathological condition or pathological condition in a subject associated with a disorder of phosphate metabolism, including a diagnosis of a pathological condition or susceptibility to the pathological condition based on the presence or amount of the polypeptide It relates to the diagnosis of susceptibility.

  It will be appreciated that the nucleic acid probes and antibodies of the present invention are preferably used for the described methods.

  The above diagnostic methods can be used to determine the state of the disease. In the context of the present invention, the term “pathological condition” refers to the gene, mRNA, protein or transcriptional regulatory element such as a promoter / enhancer sequence when compared to a wild-type normal gene product. Including the possibility of having a mutation, deletion, or any other modification that affects the overall activity of the gene. This term includes post-translational modifications of the protein.

  In a more preferred embodiment of the method of the invention, the condition of the test subject exhibits a certain form of disorder in phosphate metabolism. Furthermore, in the method of the invention, it is advantageous to determine the state of the subject in the embryo state or in the neonatal state, for example using an amniocentesis.

  Specific analysis of phosphate metabolism disorder (or potential disorder) status at the embryonic, neonatal or adult stage provides further insights, such as specific disease states for each stage. For example, the etiology of X-chromosomal hypophosphatemic rickets (XHL) and neoplastic hypophosphatemic osteomalacia (OHO) is expected to be revealed by the diagnosis using the present invention. Drugs with new pharmacological activity based on this knowledge will be developed and tested. The method using the present invention can also be used on various animals depending on the purpose of the study. Thus, in a preferred embodiment, the animal is a mouse. This aspect is particularly useful for basic research to understand more clearly the interrelated functions of different proteins that regulate phosphate metabolism. In yet another aspect, the animal is a human. In this aspect, preferably diagnostic or therapeutic use is expected.

  In a preferred embodiment of the above-described method, other steps are performed, including treatment of the newborn with an agent to stop or reduce phosphate metabolism disorders. Early diagnosis of phosphate metabolism disorders or susceptibility to the disease is particularly advantageous and very medically important. This preferred mode is applied to the diagnosis of conditions such as coronary villi, i.e. conditions preceding embryo transfer. Furthermore, if the method of the invention is used, the state can be diagnosed through amniocentesis. If diagnosed early according to the application of the diagnostic method of the present invention, disorders of phosphate intake and / or reabsorption can be treated after birth and before clinical symptoms appear.

  Patients with X-chromosome rickets and neoplastic osteomalacia (before or after resection of the tumor) are known as calcitriol or 1,25 dihydroxyvitamin D3 (trade name Rocaltrol®), Roche (Roche): For more information, visit the official website http://www.rochecanada.com/rocaltrolpm/w.html.) Or administer a phosphate supplement (dibasic phosphate) And / or phosphate). Analogs of vitamin D are sometimes used (eg dihydrotaxosterol), and can be used in combination with thiazide diuretics such as hydrochlorothiazide and amiloride (Alon, Paediatrics 75 (1985), 754-763) It has been reported that the excretion of phosphorus and calcium is further suppressed. See (Carpenter, Pediatric Clinics of North America 44 (1997), 443-466) for an intensive review of current treatments. In children's bones, resection of the deformed limbs (osteotomy) is required, and these drugs cause severe vomiting and diarrhea. Growth deficits associated with family rickets are not satisfactory with current treatments.

  Replacing the above agents with phosphatonin and / or phosphatonin peptide derivatives can treat clinical symptoms and normalize growth defects without undesirable side effects and surgical osteotomy There is sex.

  In a preferred embodiment of the above method, the aforementioned method further comprises the introduction of a functional and expressible phosphatonin gene into the cells of a subject having a disorder of phosphate metabolism or sensitivity to the disorder. A “functional” phosphatonin gene in this context and as used herein includes part or all of the primary structural arrangement of a phosphatonin polypeptide having the biological activity described above. It means a gene encoding a protein. By detecting the expression of a phosphatonin variant, it can be concluded that the expression is related to the development or maintenance of a disease in phosphate metabolism. It is also possible to suppress the expression of the mutant phosphatonin to a low level or not to express it at all by performing or adding another step. This can be done, for example, by at least partially removing the expression of the mutant gene by biological means such as using ribozymes, antisense nucleic acid molecules, or intracellular antibodies to mutants of these proteins. In addition, drug products may be developed that reduce the level of expression of the corresponding mutant gene.

In yet another aspect, the invention relates to a method of identifying a binding partner with a phosphatonin polypeptide,
(a) contacting the phosphatonin polypeptide of the present invention with the compound to be screened; and
(b) includes determining whether the compound affects the activity of the polypeptide.

  The phosphatonin polypeptide may be used to screen for proteins that bind to phosphatonin and proteins that bind phosphatonin. The binding of phosphatonin to the molecule activates (agonists), inhibits (antagonists), or decreases the activity of phosphatonin and bound molecules. Examples of such molecules are antibodies, oligonucleotides, proteins (eg receptors) or small molecules.

  Preferably, the molecule is closely related to the natural ligand of phosphatonin, eg, a fragment of the ligand, or a natural substance, a ligand, a structural / functional mimic. (See Coligan, Current Protocols in Immunology (2) (1991); Chapter 5). Similarly, the molecule can be closely related to a natural receptor to which phosphatonin binds, or at least a fragment of the receptor to which phosphatonin can bind (eg, active site). In either case, the molecule is reasonably made with existing technology. (See above)

  Preferably, screening for these molecules includes suitable cells that express phosphatonin as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. The phosphatonin-expressing cell (cell membrane containing the expressed polypeptide) is then preferably contacted with a test compound that may contain the molecule to promote binding of either phosphatonin or molecule, or activity. Observe or inhibit.

  The assay can easily test the binding of the candidate compound to phosphatonin, and the binding can be detected using a label or in an assay involving competition with a labeled competitor. Furthermore, the assay can also test whether the candidate substance produces a signal resulting from the binding of phosphatonin.

  Alternatively, it can be assayed using cell-free preparations, polypeptides / molecules bound to a solid support, chemical libraries, or natural mixtures. This assay includes procedures for mixing phosphatonin-containing solutions with candidate substances, measuring phosphatonin / molecule activity or binding, and comparing standard phosphatonin / molecule activity and binding. It is.

  Preferably, the activity level of a specimen (for example, a biological specimen) can be measured by a solid phase enzyme immunoassay using a monoclonal antibody or a polyclonal antibody. An antibody can measure phosphatonin levels or activity by binding directly or indirectly to phosphatonin, or by competing for phosphatonin for its substance.

  All of the assays described above can be used as diagnostic or prognostic indicators. By activating or inhibiting phosphatonin / molecules, the molecules detected in these assays can be used to treat disease or to give patients specific results (increase blood phosphate levels). it can. Furthermore, these assays can find substances that promote or inhibit the production of phosphatonin from appropriately treated cells and tissues.

Accordingly, the present invention includes a method for identifying a compound that binds to phosphatonin (the following procedure), comprising the following steps:
(a) a candidate culture that binds to phosphatonin; and
(b) Determining whether a join has occurred.

The invention further includes a method of identifying an agonist / antagonist,
(a) cultivation of phosphatonin and a candidate compound,
(b) a biological activity assay as described above, and
(c) determining whether the biological activity of phosphatonin has been altered by binding.

  As already mentioned herein, the polynucleotides and polypeptides of the present invention form the basis for developing mimetics that inhibit or activate phosphatonin and the genes that encode it. It will be appreciated that the present invention also provides cell-based screening methods that allow high-throughput screening (HTS) of compounds that can be candidates for the inhibitor or active agent.

In another aspect, the invention relates to methods for identifying and obtaining drug candidates for the treatment of phosphate metabolism disorders,
(A) in the presence of a component capable of providing a detectable signal in response to phosphate uptake, a cell in which the polypeptide of the present invention or the polypeptide is expressed, under conditions capable of phosphate metabolism Contact with the drug candidate to be screened; and
(B) including the presence or absence of a signal generated by phosphate metabolism or the detection of an increase, where the presence or increase of the signal indicates a putative drug.

For example, renal cell line CL8, primary human kidney cells, using primary human osteoblasts, for example by the method of Rowe, 1996, can be measured radioactive Na ⁺ dependent phosphate uptake and / or vitamin D metabolism.

  In addition, poly A + RNA or total RNA extracted from the cells described in (a), and phosphate transporter genes (such as NPTII), renal 24-hydroxylase, α-hydroxylase, PTH (parathyroid hormone), and osteopontin Oligonucleotide primers complementary to the sequence can be utilized, for example using PCR, to measure the expression of these genes.

Furthermore, the von Kossa staining method is appropriate for measuring the mineralization of human primary osteoblasts. The methods include, for example:
-Cultivation of primary human osteoblasts (available from Clonetics-Biowhitaker) to confluence using medium materials and conditions recommended by Clonetics-Phosphate donation to cells for mineralization experiments Body β-glycerophosphate and hydrocortisone-11-hemisuccinate as a control group,
-Add β-glycerophosphate and MEPE 25ng / ml to the test cells,
-A series of medium changes (AgNO3; silver salt precipitation), cultured for 3 weeks, using the von Kossa staining method as described by Clonetics, and staining osteoblasts involved in bone mineralization;
-Further analysis including the following measurements:
-Conduct rat perfusion experiments to determine the effect of phosphatonin on renal phosphate intake.
To examine the expression of a series of related genes in the human kidney cell line CL8 and the effects of MEPE administration;
Na + -dependent phosphate transporter 24-hydroxylase, 1-α hydroxylase Osteopontin, osteocalcin-co-transfection system of MEPE and PHEX in COS cells,
-Perform a bioassay using a peptide fragment comprising at least one of the above moieties. Therefore, other detection methods include measurement of proteolytic enzyme C, casein degrading enzyme II, tyrosine degrading enzyme, and other signal transduction pathways in cells exposed to phosphatonin and derivative peptides using state of the art technology. Furthermore, the method as described in the appended examples can be easily adapted to the screening.

  The drug candidate may be a single compound or a plurality of compounds. The “plurality of compounds” in the method of the present invention is understood to mean a plurality of substances which are identical or not identical.

  The compound or compounds are chemically synthesized, produced by microorganisms, and / or included in a sample such as a cell extract extracted from plants, animals, microorganisms, and the like. Furthermore, although the compounds may be known in the art, it has not been known so far that they can inhibit or activate phosphatonin polypeptides and other components in phosphate metabolism. The reaction mixture can contain a cell-free extract or cells or tissue. Methods of the method suitable for the present invention are known to those skilled in the art, and are generally described in, for example, Alberts et al., Molecular Biology of the Cell, 3rd edition (1994) and the appended examples. . Multiple compounds are added to, for example, reaction mixtures, culture mediators, etc., injected into cells, or otherwise added to transgenic animals. The cell or tissue used in the experiment of the present invention is preferably a host cell, a mammalian cell, or a non-human transgenic animal of the present invention as described above.

  In the experiments of the present invention, once a sample containing a compound or compounds is identified, it can be isolated from an original sample that has been identified as containing a compound that can inhibit or activate phosphatonin, or For example, when it is composed of a plurality of different components, the original sample can be further subdivided to reduce the number of different components for each sample, and the subdivision of the original sample can be repeated. Depending on the complexity of the sample, the above steps can be repeated many times, preferably until the sample identified according to the present invention consists of only a limited number of components or a single substance. Can do. Preferably the sample comprises substances of similar chemical and / or physical properties, most preferably the components are identical.

  Compounds that can be tested and identified according to the method of the present invention include, for example, cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, mock peptides, PNA, etc. (Milner, Nature Medicine 1 (1995) , 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198, see above). Further, for example, gene targeting vectors known in the art (eg, nucleic acids, mitochondria, cytoplasm, pEF / myc / nuc, pEF / myc / mito, pCMV / myc / mito, pEF / myc / cyto, pCMV / myc / pShooter plasmid series that expresses cytos as a target, or pDISPLAY expression vector that targets recombinant proteins on the surface of mammalian cells), for example, if mutagenesis is inserted, presumably encodes a putative regulator of phosphatonin protein Putative genes that affect upstream or downstream of the gene or protein of the phosphatonin protein may be identified. All of these vectors are available from Invitrogrn (http://www.invitrogen.com/).

Whether a compound can inhibit or activate phosphatonin protein can be analyzed, for example, by observing Na ⁺ -dependent phosphate intake and bone mineralization (see above). Furthermore, it can be determined by monitoring the phenotypic properties of the cells of the invention that have been contacted with the compound and comparing them to wild type cells. In other embodiments, the property can be compared to the property of a cell contacted with a compound known to be capable of inhibiting or activating phosphatonin protein.

Once the aforementioned compound is identified and obtained, it is preferably provided in a therapeutically acceptable form. Accordingly, the present invention provides a therapeutically effective amount of said agent, or (i) a compound obtained or identified in step (b) of the present invention, or an analog or derivative thereof. Synthesis in an amount sufficient to give the patient, and
(Ii) It relates to a method for producing a therapeutic reagent comprising a combination of the compound obtained or identified in step (b) of the present invention or an analogue or derivative thereof with a pharmaceutically acceptable carrier.

  Methods for preparing chemical derivatives and analogs are well known to those skilled in the art, and are also described in Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc. ,, 175 Fifth Avenue, New York, NY 10010 USA, Organic Synthesis, Wiley, New It is also described in York, USA, etc. Furthermore, the derivatives can be synthesized according to techniques known in the art and examined for effects (see also above and below).

  In summary, the present invention provides a method for identifying compounds capable of modulating phosphate metabolism by directly or indirectly activating phosphatonin. Accordingly, compounds as inhibitors or activators of phosphatonin activity discovered according to the present invention are also within the scope of the present invention.

  As is apparent from the above, the present invention generally relates to a component composed of at least one of the aforementioned polynucleotides, nucleic acid molecules, vectors, proteins, regulatory sequences, recombinant DNA molecules, antibodies, or compounds. Preferably, the component is composed of a component such as a buffer or an antifreezing agent, which is not related to the described compound of the present invention in the natural state but exhibits the same effect for a special use.

  Conveniently, the compound can be used as a medicament, diagnostic tool or kit. The pharmaceutical ingredients are described in detail in Examples 6 and 7. In particular, the above-mentioned biologically active fragments may be effective as a medicament for treating phosphate metabolism disorders such as X-chromosome rickets, osteomalacia and other bone mineral metabolism diseases. In addition, phosphatonin and PHEX metallopeptidase are supplied as pharmaceuticals, preparations that can be used simultaneously, separately or in succession. Thus, it is possible that PHEX metallopeptidases can be used to degrade phosphatonin to produce active phosphatonin fragments that can be used to treat phosphate metabolism disorders as described herein. These diseases are particularly serious for humans, but non-human mammals can also be treated according to the present invention.

  The present invention provides for the first time phosphatonin in an essentially isolated and purified form, preferably free of contaminants. Natural phosphatonin and phosphatonin fragments do not contain mixtures such as SDS and / or other interfering proteins, regulate phosphate metabolism and become a pharmaceutical ingredient for the treatment of diseases related to phosphate metabolism disorders. The active ingredient can be supplied.

  Accordingly, the present invention encodes a phosphatonin polypeptide of the present invention, or such a polypeptide or antibody, an activator / agonist, an inhibitor / blocker for the preparation of a therapeutic agent for phosphate metabolism disorders. And the use of the expressed or expressed binding partner of the present invention.

  In particular, the present invention is preferably used for the preparation of a therapeutic agent for hyperphosphatemia, preferably bone dysplasia, hyperphosphatemia before renal dialysis / dialysis, secondary hyperparathyroidism (function) and cyst Phosphatonin polypeptide having phosphatonin activity, or DNA encoding and expressing such a polypeptide or antibody, activator / agent, or the like for the preparation of a therapeutic agent for osteofibrosis Relates to the use of a binding partner of the invention that causes phosphatonin activity when present in a cell.

  In another aspect, the present invention provides for the preparation of a therapeutic agent for hyperphosphatemia, preferably hereditary hypophosphatemic with X-chromosomal hypophosphatemic rickets, hypercalciuria (HHRH). Rickets, bone damage due to low mineralization, stunted growth in childhood, neoplastic hypophosphatemic osteomalacia, phosphate leakage from the kidney, osteoporosis, vitamin D-resistant rickets, Paget's disease, renal failure, A phosphatonin polypeptide having anti-phosphatonin activity for the preparation of a therapeutic agent for tubular acidosis, cystic fibrosis, sprue (insufficient intestinal malabsorption), or the polypeptide or the antibody or nucleic acid of the present invention The molecule relates to the use of DNA capable of encoding and expressing the inhibitors / blockers of the invention.

  In a preferred embodiment of the present invention, a phosphatonin polypeptide having anti-phosphatonin activity, or the polypeptide, the antibody of the present invention, a nucleic acid molecule, or the inhibitor / blocker of the present invention is encoded and expressed. The DNA that can be used is used in the manufacture of a therapeutic agent for bone mineral loss disorders.

  In another preferred embodiment, the phosphatonin poly for the preparation of a preparation for the treatment of phosphate metabolism disorders, for the preparation of a preparation for simultaneous, separate or sequential use. The use of peptides and PHEX metallopeptidases.

  The above uses and (manufacturing) methods are described in more detail in Example 6.

  In another aspect, the invention relates to the use of transformed osteoblasts or bone cell lines capable of overexpression of phosphatonin for the isolation and production of phosphatonin.

  The following examples are provided to provide those skilled in the art in order to fully disclose and explain the methods of carrying out various aspects of the present invention, and they are deemed to be within the scope of the present invention. It is not intended to represent or represent that the experiment described below is all or the only experiment performed. Care was taken to ensure accuracy with respect to numerical values used (eg, amounts, temperature, etc.) but some experimental error and deviation should be considered. Unless otherwise defined, percentages are percentages by weight, molecular weight is average molecular weight, and temperature is in degrees Celsius.

Example 1: Purification of tumor-derived phosphatonin A mesenchymal tumor expressing phosphateuria was removed from a patient and the following samples were obtained:
A: A sample of two large bean pure tumor tissues was placed in a 2 ml vial containing DMEM Eagle / 10% FCS / glutamine / antifungal antibiotic Gibco-BRL.
B: Subdural tumor sample of the same size or smaller if possible. Placed in the same medium as A.
C: Abnormal dura sample: Hard white sample: Place in the same medium as A.
D: Tumor fluid sample.

Sample processing
First day :
The sample was cut into small cubes of 0.5 cm square each with a sterilized knife. Half of each sample was placed in a freezing tube and quickly frozen with N2 (1). Fluid around the tissue (DMEM / 10% FCS, etc.) was also collected and frozen. The remaining half of the sample was left overnight at 37 ° C. in addition to DMEM Eagle / 10% FCS / glutamine / antifungal antibiotics supplemented with 0.2 mg / ml (˜15 ml) collagenase A1.

the 2nd day :
1． When cultured overnight in serum supplemented with DMEM, most of the cells that appeared were erythrocytes and few adherent cells were found. Cells were spun down at room temperature and the supernatant was collected and frozen immediately (˜15 ml).
2． The pellet was resuspended in 10 ml DMEM Eagle plus antibiotic / antifungal (medium flask) and left for another 8 hours 10 minutes.
Serum-free supernatant was collected (˜10 ml) as in 3.1, and cells were resuspended in DMEM EAGs containing 10% FCS and continued to culture. The supernatant was stored at -80 ° C.

Day 6:
1. After the culture from the second day, the cells were centrifuged and sedimented in the same manner as in the first day. A 10% FCS sample was collected and frozen.
2. Similar to the second day, the pellet was resuspended in serum-free DMEM (10 ml) and left for 4 hours this time.
3. The same processing as 3 on the second day was performed.

Day 7:
1． In particular, in subdural and tumor cultures, many cell clumps formed by adhering to the plastic on tissue culture plates. Under the ridges resembling those polyps, a layer of fibroblast-like cells radiated from beneath the structure resembling a tumor. This layer of cells appeared to act like a matrix connected to polyp-like tumors. This was not observed in the dura mater sample, and the dura mater sample was deficient in cells at this stage, and a structure like a fibrous entanglement was seen.
2． The culture was centrifuged to settle and the supernatant was collected (10% FCS). The pellet was then placed on one side.
3． Plates were left for ˜15 minutes with 10 ml trypsin EDTA solution Gibco / BRL diluted 1/10 with PBS. Then the plate was tapped strongly and 5 ml of FCS was added.
Four. The resuspended cells were added to the pellet obtained in 2, resuspended and centrifuged to sediment. The supernatant was discarded.
Five. Cells were plated out (using large slices) in 18 ml of 10% FCS DMEM Eagle medium containing glutamine and antibiotic antifungal.
6． Finally, the cells were cultured in a 37 ° C. CO ₂ environment.

Day 9:
1． Cancer cells and some dural cells appeared to have many small clumps and had the same morphology as the cells before trypsinization. Some small clumps were quite large and visible to the naked eye.
2． The medium with serum added was collected and stored. A large flask was used and 18 ml was added per flask (DMEM 10% FCS antifungal / antibiotic / glutamine).

Day 13:
1． Cells were frozen (˜15 ml) and stored in Falcon as 10% FCS DMEM conditioned medium.
2． Cells were resuspended in serum-free DMEM Eags (˜11 ml) 11.10am and left at 37 ° C. for 6 hours (CO ₂ incubator).
3． Cells were spun down and the supernatant collected (serum-free control medium). 10% FCS DMEM EAG was added to the remaining cells.

Day 16:
The above procedure was repeated and tumor conditioned medium (TCM) was collected over several weeks. Alternatively, TCM can be collected from Saos-2 cells (ECACC 89050205) or U-2 OS cells (ATCC HTB-96).

Purification of phosphatonin Concanavalin A Sepharose affinity chromatography:
1. Prepare 3 ml TCM with 1 M sodium phosphate pH 7.2 and 5 M sodium chloride and finally with a solution of 0.06 M sodium phosphate pH 7.2 and 0.5 M sodium chloride containing 0.01% sodium azide did.
2. Concanavalin A Sepharose (Pharmacia code number: 17-0440-01) in 20% ethanol was first washed repeatedly with the same volume of water as the column and equilibrated in running buffer. Concanavalin A was placed in a small C10 / 10 column (Pharmacia code number: C10 / 10 id 10 mm) until the height became 5.5 cm (approximately 4.3 to 5.0 ml capacity). Equilibrated at a maximum flow rate of 0.5 ml / min.
3． A sample (sodium phosphate adjusted to pH 7.2 / 0.5M sodium chloride / 0.01% sodium azide) was added to the column with a gravity feed and loaded three times. The color of the sample can be visualized as it passes through the column. Unbound material was collected and stored for later reference.
Four. The Waters LC system was connected and the sample was washed with several column volumes of loading buffer.
Five. After loading and washing, elution was carried out using 60 mM sodium phosphate buffer 0.5 M sodium chloride, 0.5 M α-methyl-D-glucopyranoside, 0.01% azide buffer (pH 7.2). See Figure 1a. A single peak was detected and collected.
6． The column was then returned to a maximum of about 40 mL baseline and left overnight.
7． When left overnight in methylglycoside buffer, the second peak eluted at ~ 5 ml (see Figure 1b).
8. The second peak was collected, dialyzed against 0.05M acetic acid and lyophilized. Both concanavalin peak A1 (low affinity) and concanavalin peak A2 (high affinity) strongly inhibit Na + -dependent phosphate cotransporter and vitamin D metabolism in human kidney cell line (CL8) There is. The high affinity fraction, human kidney cell line (CL8), and conditions used for the measurement are described in Rowe et al., 1996. A known renal cell line that is more suitable for this measurement is the OK cell line deposited as ECACC91021202.

Cation exchange chromatography using HiTrap SP cation exchange 1 ml column (code number 17-1151-01; Pharmacia)
1． The lyophilized protein was redissolved in 0.05 M ammonium acetate pH 5 and loaded onto an equilibrated 1 ml HiTrap SP Sepharose cation exchange column.
2． Before adding the sample, the column was equilibrated by washing with water followed by 5 volumes of starting buffer (0.02M ammonium acetate pH 5).
3． The sample was eluted using the following protocol;

  A single sharp peak was obtained, after which the sample was dialyzed against 0.05M acetic acid and lyophilized; see FIG.

After resuspension in 20 μl of 10 mM phosphate buffer pH 7.2, aliquots were analyzed with SDS-PAGE sample buffer (final concentration = 125 mM TRIS-HCL pH 6; 2.5% glycerol; 0.5% w / v SDS; 5% β − When resuspended in mercaptoethanol (0.01% bromophenol blue), boiled (5 minutes), cooled and loaded on SDS PAGE gel 12.5% (see chromatogram), two bands were detected at 55 kD. (See Rowe et al., 1996). Both concanavalin A and the band from the cation exchange column were in condensed form. All fractions containing tumor conditioned medium (1/1000 dilution) have the activity of inhibiting Na ⁺ -dependent phosphate co-transport in human kidney cell lines and altering vitamin D metabolism. For a complete description of how to measure phosphate transport and vitamin D metabolism, see Rowe et al., 1996. All purified preparations were performed on a waters HPLC / FPLC programmed by computer-millennium software. The most active fraction was the concanavalin A1 fraction from the OHO tumor. The patient's anti-operation antisera was used for screening of the purified purified fraction. This fraction also inhibits NaPi and alters vitamin D metabolism in the human kidney cell line (CL8).

Example 2: Tumor culture supernatant (TCM) screening, and pre- / post-purified fractions purified with antisera: and glycoprotein screening . It is described in. Only the preoperative antiserum detected purified fractions and hormones in TCM, which were purified by Western and glycoprotein detection methods using enhanced chemiluminescence. Detection was done. The protein marker was biotinylated and tagged with a streptavidin peroxidase complex. Arrows indicate aggregates and active glycoproteins. No fractions were detected in post-operative antiserum and pre-immune sera of rabbits. Furthermore, only tumors that secrete phosphate factor were positive. Medium and skin control group were negative. ConA1, ConA2, and CA1 samples clearly differed in their ability to inhibit NaPi and their ability to regulate vitamin D metabolism in a human kidney cell line (CL8). All purified fractions tend to agglomerate on SDS-PAGE and have a low migration rate. The purified and TCM active fractions are highly glycosylated. The degree of glycosylation can be confirmed by oxidizing the protein immobilized on the PVDF membrane with periodate hydrochloric acid and then biotinylating the carbohydrate component. Next, it is screened with streptavidin that binds to horseradish peroxidase and has enhanced chemiluminescence. Active forms (such as NaPi inhibition) are associated with the 58-60 kDa fraction. Another powerful protein purification method is to use a neutral (pH 7) SDS-PAGE system using 4-12% Bis-Tris gel with MOPS running buffer. Pre-caste gel can be purchased from Novex.

Example 3: Neutral SDS-PAGE using a 4-12% polyacrylamide gradient and bis-Tris gel MOPS running buffer (Novex Nu-PAGE system): reduced migration rate in hormones On this system : Glycosylated hormones have a reduced migration rate and migrate to ~ 200 kDa. A low molecular weight form can also be seen at 58/60 kDa. The appearance of the ~ 200 kDa protein is thought to be due to the equipotential of the protein (the charge is different at neutral pH) and the interaction between the carbohydrate component and the gel matrix. Furthermore, a low concentration of acrylamide (4-12% gradient) above the gradient gel increases the efficiency of the electroblot for high molecular weight compounds. Electrophoretic fractionation in this system increases molecular purity and homogeneity. The following samples were screened by Western blot using this system (blot screening was performed with pre-operative antiserum by detecting chemiluminescence with increased luminescence). 1． Protein markers, 2. 2. Intracranial tumor cell line OHO, 3. subdural cells adjacent to the tumor; 4. Dura cells adjacent under the dura mater; HTB6 cell line, 7. 7. Control medium defined, Control skin fibroblasts, 9. Linear sebaceous nevus polyp tumor. Nine linear sebaceous nevus polyp tumors were demonstrated that the nevus polyp tumors showed a specific phosphateuria band at ~ 200 kDa on SDS-PAGE neutral gel.

Example 4: Cloning and sequencing of phosphatonin
1. mRNA was extracted by a conventional method from a tumor collected from a patient (BD, Rowe et al., 1996) described in a publication published in the library construction site. MRNA was copied using reverse transcriptase to generate a population of cDNA. It was then subcloned into the bacteriophage vector λ-ZAPII Uni (vector purchased from Strategene Ltd. Unit 140, Cambridge Science Park, Milton Road, Cambridge, CB4 4GF United Kingdom). Cloning was unidirectional, the 5 'end of the gene was next to the T3 promoter and touched the EcoRI site. The 3 ′ end of the cDNA was in contact with the upstream of the Xho-1 site of the bacterial T7 promoter. Briefly, tumors excised from patient BD were cut into 1 mm squares, and poly A + RNA was directly extracted using Streptavidin-Magnesphere paramagnetic particle technology (PolyATract ^R system Promega). Next, a cDNA template was prepared from the purified mRNA using the cDNA synthesis kit of Strategene. A linker primer was attached to the cDNA to produce a 5 ′ end EcoRI compatible cDNA end and an Xhol compatible 3 cDNA end for directed cloning into the λZAPII unibacteriophage vector. Recombinant bacteriophages were plated out and amplified on E. coli XL-1 Blue mrf ′. The total number of primary clones, including the appearance of 6% wild type, was 800,000.

2. Screening with antisera before surgery
The cDNA bacteriophage library was plated out on NZY agar plates and the β-galactosidase operon was induced using IPTG. The expressed fusion protein was then transferred to a high-bond-C membrane (Amersham) and the membrane was screened with patient-preoperative antisera. The antiserum used was described previously (Rowe et al., 1996). The antiserum had previously absorbed E. coli lysate well and reduced signal noise for the whole blood. Rabbit antiserum (Rowe, Bone 18 (1996) 159-169) prepared against preoperative sera of patient BD removes the background of E. coli antibodies and antibodies derived from human sera. E. coli lysate had been absorbed. Briefly, five 80 mm diameter nitrocellulose filters were added to total E. coli lysate (Strategene) and the next five filters were usually immersed in human serum (10 ml). The permeated filter was washed in 250 ml of anti-rabbit pre-serum antiserum diluted 1000-fold in 1% BSA, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl (TBS), 0.02% NaN ₃ at room temperature. The cells were cultured in order for 10 minutes. The pre-treated antiserum (pre-Anti op) was used for screening of the cDNA library. Bacteriophage λZAPII UNI OHO cDNA clones were plated out on E. coli XL-Blue mrf ′ and cultured at 37 ° C. for 3 hours. A high-bond N ⁺ filter pre-cultured with 10 mM IPTG was placed on a developing plaque and further cultured at 42 ° C. for 3 hours. The filter was then removed, washed with TBS (TBST) supplemented with Tween 20, and blocked overnight at 4 ° C. in TBS containing 1% BSA, 0.02% NaN ₃ . Pre-Aanti-op was added to the blocked filter and allowed to stand at room temperature for 1 hour. Subsequently, after washing the filter and culturing with a goat-anti-rabbit alkaline phosphatase conjugate, 5-bromo-4-chloro-3-indolylphosphatase / nitroblue tetrazolium was used, Strategenes picoblue (registered trademark); immunoscreening kit Visualized as described in. When ~ 600,000 clones were screened, nine positive clones were selected and purified by secondary and tertiary screening. Bacteriophage clones were rescued as phagemids by ExAssist helper phage and cloned into E. coli SOLR cells. ExAssist helper phage and SOLR cells were purchased from Strategene Ltd. Sunit 140, Cambridge Science Park, Milton Road, Cambridge, CB4 4GF United Kingdom.

3． Sequencing of clones Phagemids were prepared and DNA was sequenced. The sequence of all nine clones was determined. After screening with sterile hollow quill three times, positive bacteriophage plaques were removed from the agarose medium. The agarose plug containing the lysed plaque is then placed in 0.5 ml SM buffer with 0.02% chloroform and left at 4 ° C. overnight. ExAssist phage was used as described by Strategene, and bacteriophage clones were rescued and transformed into BSCPT SKII ^- phagemid. For purified phagemid, E. coli SOLP cells were used as host cells. Plasmid DNA was prepared in the usual manner (Rowe, Nucleic Acids Res. 22 (1994), 5134-5136) and sequenced using an ABI fluorescent automated sequencer and standard vector specific primers. Six clones were in-frame with the β-galactosidase promoter, giving the expressed protein to flanking / overlapping antigenic determinants and individual overlapping DNA sequences. The longest sequenced clone contained the other five cDNA sequences as shown in FIG. This sequence (amino acid / cDNA) is the complete sequence of phosphatonin. It is a cloned 430 amino acid residue (SEQ ID NO: 2) and a 1655 bp DNA sequence (SEQ ID NO: 1). The predicted secondary structure indicates the presence of a highly hydrophilic protein glycosylated at the COOH terminus and a cell adhesion tripeptide at the amino terminus: see FIG. Proteins are also highly antigenic and have many helical domains (Figure 10). In addition, BLAST was used to screen as many databases as possible, but no statistically meaningful homology was shown with known genes and protein sequences.

4. Purification of recombinant human phosphatonin The isolated cDNA clone is contained in the SOLR E. coli host cell as a rescued phagemid of the Bscpt SKII vector (Stratagene vector). By inducing the β-galactosidase promoter by IPTG, the fusion protein is expressed at a low level. The fusion product of the phosphatonin clone reacts with the preoperative antiserum on the Western blot. When the fusion protein is subcloned into a pCAL-n-EK vector (Stratagene vector), expression and biological activity are increased (see below). The construct containing human phosphatonin is in E. coli (BL21 (DE3) pLys S) cells (purchased from Stratagene). The IPTG induction of the fusion protein is much higher and essentially pure protein can be obtained by calmodulin affinity chromatography of cell lysates. Recombinant phosphatonin with a fusion tag binds to the calmodulin resin in the presence of Ca ²⁺ . Washing with EGTA releases the phosphatonin fusion protein. Treatment with enterokinase removes the fused tag of the microorganism, leaving pure human phosphatonin.

4a Subcloning of phosphatonin into the pCAL-n-EK vector The predicted total cDNA coding sequence of phosphatonin (MEPE) (estimated from the largest cDNA clone, pHO11.1) is the eukaryotic expression vector plasmid pCAL-n-EK ( Stratagene vector) was subcloned, and the construct was transformed into E. coli XLi-Blue mrf 'and E. coli BL21 (DE3) pLysS, respectively (the strain was obtained from Stratagene). Ligation-independent cloning (LIC) was performed using Stratagene Affinity (registered trademark); cloning and protein purification kit (Cat. Nos. 214405 and 214407). Two primers were designed from each of the 5 ′ and 3 ′ ends of the phosphatonin sequence with an attached overhanging linker sequence as follows (the bold sequence indicates the linker).

PCR amplification of phosphatonin is carried out, including a DNA sequence encoding from the first valine residue to the stop codon of phosphatonin (see FIG. 8) (see FIG. 8) and further to the linker sequence. Treatment of the PCR fragment with Pfu polymerase and dATP produces a linker sequence that overhangs the 5 'end. When cells are cultured and IPTG is added, induction of the fusion protein occurs. PCR conditions are as follows: as denaturation pretreatment at 95 ° C for 3 minutes, followed by denaturation at 95 ° C for 45 seconds, annealing at 59 ° C for 60 seconds, 2 cycles at 72 ° C for 20 cycles, then final Elongate at 72 ° C for 7 minutes and cool to 4 ° C. A Perkin Elmer 9600 thermocycler was programmed to perform the PCR. The PCR buffer (PB) used was as follows: 10 mM Tris-HCl pH 8, 50 mM KCl, 1 μM primer, 200 μM dNTPs. The PB buffer were added to the MgCl ₂ of 2 mM. To perform LIC, the amplified product was treated with pfu polymerase and dATP as described by Stratagene and annealed to directly bind the pCAL-n-EK plasmid vector with the complementary linker overhang. This construct was then transformed into E. coli XL-Blue mrf 'competent cells, competent E. coli BL21 (DE3) clones were selected on ampicillin plates, plasmids were prepared and sequenced. An overview of the vector and fusion construct is shown in FIG. The plasmid copy number was high when E. coli XL-Blue mrf 'was used as the host cell, and expression of many recombinant proteins was obtained in E. coli BL21 (DE3).

4b Purification of phosphatonin by calmodulin affinity resin
The method described in Staratagene (cat-214405) can be used. The upstream sequence derived from the phosphatonin specific residue contains a calmodulin binding sequence. Addition of calmodulin resin to the crude cell lysate in the presence of calcium will allow protein binding. The slurry was washed with a buffer containing calcium, and the phosphatonin fusion protein was eluted with a solution of EGTA 2 mM in Tris-buffer (50 mM Tris HCl pH 8). Therefore, removal of the calmodulin binding protein is performed by degradation with the site-specific protease EK to obtain pure recombinant human phosphatonin. Preferably, the method is carried out as follows (see the table below for buffer composition).

1． Cells are cultured and the pCAL-n-EK vector (Cat. No. 214405) is derived using BL23 (DE3) E. coli host cells containing plasmid p1BL21 as in the Stratagene protocol (see FIG. 14).
2． Prepare protein lysate as described in the Starategene protocol, except use CCBB-II (500 ml cell pellet resuspended in 10 ml CCBB-II) as resuspension buffer . It is important to cool with ice for 4 minutes after sonication with a 30 second pulse. During ultrasonic disruption, the tube containing the cells is placed on ice.
3． Supernatant cells are collected after centrifugation of sonicated cells at 10,000 g. Most recombinant MEPE is contained in the supernatant (protein lysate).
Four. The protein lysate is concentrated using a VIVA SCIENCE VIVA SPIN (catalog number: VS1521, 30,000 MWCO PES) concentrator with a cut-off value of 30,000 molecular weight.
Five. The protein lysate prepared from 1901-200 ml cells (˜1.3 ml equivalent protein lysate) was then added to 1 ml of equilibrated calmodulin resin (equilibrated resin as described by Stratagene using CCBBII buffer). Add).
6． Swirl the suspension at 4 ° C overnight.
7． The suspension is spun down (3000 rpm for 2 minutes in an Eppendorf centrifuge) and the supernatant is removed. Resuspend the resin in 1 ml CCBB-II buffer.
8． The resin is centrifuged to settle and the first wash is removed. Repeat two more times (wash 3 times in total in CCBB-II).
9． The resin is then washed with WB-III; care must be taken not to include detergent in the final wash buffer. Cells used in bioassays are extremely sensitive to surfactants, even in very small amounts. WB-III is the same as CCBB-II but does not contain a protease inhibitor.
Ten. Nonspecific proteins are eluted by washing the buffer twice with EB-I (1 ml).
12． Concentrate proteins at 4 ° C using a flowgwn 10K microscope concentrator. In general, 3 ml of MEPE eluate can be concentrated to ˜170 μl in 2 hours.
13. Samples are run on an SDS-PAGE gel to assess purity and quantity, then aliquoted and frozen at -80 ° C. Avoid repeated freeze / thaw cycles.
Buffer:

  In using the buffer, 1 tablet of protease inhibitor (Boheringer Mannheim, protease inhibitor w / oEDTA (catalog number: 1836170)) was added per 10 ml. The final eluate in 1 M NaCl, EGTA (4 mM) buffer contains> 95% pure phosphatonin.

Example 5: Structure of phosphatonin
1． The primary structure and the primary structure of the motif protein and nucleic acid sequence are shown in FIG. The largest of the isolated MEPE cDNA clones was 1655 bp and had all 3 'ends of the gene containing poly A ⁺ tail and a single-stranded polyadenylation sequence (AA [T / U] AAA) (Figure 8). The 430 residue open reading frame was found to overlap across other small isolated MEPE cDNA clones, presumed to be Mγ47.3 kDa and pl7.4. The most consistent site starts at 255 bp of the Kozak start codon (Nucleic Acids Res. 15 (1987), 8125-8148), and the other two methionines are present upstream. Accompanying 5 'sequences can be lost, and previous start codons and / or extended 5' untranslated sequences need to be characterized. The prediction of GCG-secondary structure suggests that this protein is very hydrophilic with three less hydrophobic regions (Figure 9). This protein has glycosylation motifs at residues 382 and 385 (NNST), and 383-386 (NSTR). It also has a glycosaminoglycan adhesion site at 161-164 residues (SGDG). The unglycosylated molecular weight is approximately 54 kDa, which is in good agreement with the purified glycosylated form of 58-60 kDa. There are many phosphorylation site motifs (see Table 1), which are expected to affect the biological activity of hormones and their fragments.

The key structure of this protein is a cell adhesion sequence at residues 152-154 (RGD). Arg-Gly-Asp sequences generally affect receptor interactions, and fibronectin is essential for cell surface receptors that bind certain integrins. Also noteworthy are the types of collagen (bone matrix protein), fibrinogen, vitronectin, von Willebrand factor (VWF), snake integrin, slime mold discoidin with this motif. Is also present. This indicates that this portion of phosphatonin is likely to be involved in receptor and / or bone mineral matrix interactions. Furthermore, these interactions mediate the following actions.
1． Bone mineralization (osteoblasts)
2． Regulation of Na-dependent phosphate cotransporter gene expression
3. Regulation of expression of 24 hydroxylase and / or 1-α-hydroxylase gene (kidney)
Four. Mineral deposition through bone and tooth mineral matrix interactions and nucleation

  The presence of glycosaminoglycan adhesion sequences at residues 161-164 (SGDG) has important implications for bone mineralization adhesion and interrelationship. The role of proteoglycans in bone is well studied, particularly in cellular signaling. This part of the molecule is important in the aforementioned biological activity (points 1 to 4) and is likely to play an important role in bone mineralization mediated by osteoblasts in particular.

  The RGD motif is present in the predicted turn region (Garnier prediction Antheprot) and is adjacent to the two β-sheet regions (residues 134-141, 172-178). The putative sheet structure is adjacent to the two expanded α-helix regions (residues 121-134, 196-201) and is in a turn. In the general structure, the predicted turn and the presence of the RGD cell adhesion sequence is similar to that found in osteopontin. In addition, this protein has many putative phosphorylation motifs in protein kinase C, casein kinase II, tyrosine kinase, and cAMPcGMP-dependent protein kinase. In addition, many N-myristolein oxidation sites have been found in MEPE, which are phosphate glycoproteins (osteopontin, vitronectin, collagen, h-integrin, binding protein, dentinsialophosphate protein, dentin It has been revealed that it has a structure of RGD including matrix protein 1, bone-sialoprotein III, fibronectin). It usually contains high concentrations of aspartic, serine and glutamic acid residues (26%), which is also found in osteopontin (37%). Of particular interest is the absence of any cysteine residues in the MEPE sequence. This indicates that the cysteine-cystine disulfide bond has no effect on the secondary structure of the molecule. Screening the tremble database with MEPE revealed sequence homology with dentin phospholine (DPP). The C-terminal region of MEPE has a sequence of aspartic acid and serine residues (414-427 residues), which is almost identical (80% homology) to the repetitive motif found in DPP (FIGS. 26A and 26B). ). Comparing the MEPE motif (DDSSESSDSGSSSESD) and the DSP motif (SDSSDSSDSSSSSDSS) physicochemically, the homology is 93%. When the MEPE motif starts at the C-terminus in MEPE (residues 141-427), the DSP homologue repeats at DSP residues 686-699, 636-646, and 663-677. Two more related sequences, DSSDSSDSNSSSDS and DSSDSSDSSNSSDS, have 80% homology with the MEPE motif, and this sequence is found in DSP at positions 576-589 and 800-813, respectively. A similar motif with 60% homology (DDSHQSDESHHSDESD) is also found in osteopontin (residues 101-116), and the phosphorylation site of casein kinase II is contained within the same region of the sequence (Figure 12). X chromosome rickets lacks skeletal casein kinase II activity (Rifas, loc. Cit.). Although the osteopontin MEPE motif is in the middle rather than the C-terminus, since osteopontin degradation has been reported in vivo, it is believed that peptides with the MEPE motif present at the C-terminus are produced (Smith, J. Biol. Chem. 271 (1996), 28485-28491). Sequence homology with the C-terminal MEPE motif is also found in DMA-1 residues 408-129 (SSRRRDDSSESSDSGSSSESDG). Partial sequence homology with the MEPE motif in DSSP, DMA-1, and OPN is illustrated graphically in the “llanview” statical plot of FIG. Table 2 also shows sequence similarity in alignment.

  Interestingly, there is a motif repeat in the C-terminal region of the DSSP and dentinphosphorin sites. A dot matrix alignment between MEPE and DSSP is shown in FIG. 13 at high and low stringency. This is a graphic representation of the repeat of MEPE motif rich in aspartic acid and serine in DSSP.

  DPP consists of a larger post-translationally degraded protein, dentin sialophosphate protein (DSSP), which is degraded into DPP and dentin sialoprotein (DSP). MEPE and osteopontin have significant homologous sequences in parts of dentin phospholine (DPP) and dentin sialophosphate protein (DSSP). However, there is no homology in the molecule with the dentin sialoprotein (DSP) site (Figure 13). It should be noted that the sequences of RGD motif, casein kinase II phosphorylation motif, and N-glycosylation site in DPP and MEPE are similar (FIG. 13). Moreover, all the sites of protein kinase C related to DSSP are gathered in the overlapping region with MEPE (dentin phospholine site), but it is not found in the DSP site in the molecule.

2． Secondary structure
The predicted hydrophilicity / hydrophobicity, antigenicity, and flexibility of the GCG peptide structure are shown in FIGS. These figures show a predictive analysis of the GCG structure of the primary amino acid sequence. Hydrophilicity and hydrophobicity are indicated by triangles and ellipses, respectively. The glycosylation motif is shown as a circle with a bar at residues 382-386. In FIG. 6, the state of glycosylation can be seen more clearly. Protein turn is represented by a line indicating the primary structure of the amino acid sequence. The regions of the α helix structure, the coil structure, and the sheet structure are indicated by partially wavy lines (see details in FIG. 7). For computer prediction, GCG software based on the program manual (Cambridge, CB10 1RQ, England: Hinxton Hall) for the HGCG resource center Cambridge (Rice, 1995) Program Manual EGCG package was used. As a prominent structure, there is no cystine residue, it has a high degree of hydrophilicity, and contains minor low hydrophobicity indexes (48-53, 59-70, 82-89, 234-241) It has a special part. From the prediction of secondary structure using antheorplot software, this protein does not have the property of crossing the membrane. The protein is also highly antigenic and flexible (Figures 4 and 5). The overall secondary structure characteristics indicate that it is a protein secreted extracellularly, which is consistent with the suspected molecular function. FIG. 7 shows the phosphatonin helix structure, sheet structure, turn, and coil region. This is based on the results predicted using Garnier analysis of the antheplot v2.5e package. The four lines in each section (top and bottom) indicate the possible primary amino acid sequence helix, coil, sheet, and turn. The lower graph shows the same data in block form. It should be noted that there are many helix structures, especially at the NH ₂ terminus and toward the C terminus, which is considered to be a functional structure.

Example 6: Medical use of phosphatonin and phosphatonin fragments Treatment with a polypeptide according to the invention is suitable for the treatment of a number of diseases.

X chromosome rickets (hypophosphatemia) (HYP)
X chromosome rickets is one of the most common genetic disorders associated with bone mineral metabolism (Rowe, 1997). Phosphatonin bioactive fragments and non-degradable hormones degraded to PHEX are thought to play a major role in the treatment of this disease. The cloned proteins described herein interact with syngeneic receptors in the kidney, resulting in inhibition of renal Na-dependent phosphate cotransporter (NaPi) expression and direct or indirect It is speculated that it will upregulate the expression of normal renal 24 hydroxylase. It is also predicted that down-regulation (direct / indirect) of renal 1α hydroxylase expression will occur. After degradation by PHEX or other post-translational modifications, hormone peptide fragment derivatives are presumed to have the opposite biological activity (NaPi upregulation, 24 hydroxylase downregulation, 1-α-hydroxylase upregulation). The While other peptide derivatives mediate the interaction of receptor ligands with essentially different biological activities, the fragment containing RGD cell adhesion residues (positions 152-54) plays a role in receptor interactions. Conceivable. Furthermore, phosphatonin derivatives have an important role in restoring normal bone damage due to low mineralization. This is presumed to result from changes in osteoblasts that mediate bone mineralization and to restore normal expression / phosphorylation of bone mineral matrix proteins (osteopontin / osteocalcin). RGD cell adhesion sequences and glycosaminoglycan adhesion motifs may also be required for nucleation and crystallization with hydroxyapatite and bone mineral functions.

  Growth failure is a key feature of HYP, but current therapies are not optimal. If phosphatonin-derived fragments are administered instead of inorganic phosphate or vitamin D supplements, it may be possible to restore this to normal.

As described above, effective uses of the phosphatonin peptide fragment include the following.
1． Normalization of hypophosphatemia (NaPi, kidney preferred)
2. Normalization of 24 hydroxylase and 1-α-hydroxylase activity (kidney)
3． Bone mineralization and bone repair (normalization / rickety prevention)
Four. Completely eliminate bone pain symptoms
Five. Normalization of growth disorder

Tumor hypophosphatemic osteomalacia (OHO)
The clinical properties of OHO are similar to HYP. Phosphorus leakage from the kidneys, low levels of 1.25 dihydroxyvitamin D3 (calcitriol) circulation, elevated alkaline phosphatase, bone mineralization in adults, bone softening (osteomalacia) and serum phosphate Generally appears as a decline. Pathophysically there is a clear overlap between HYP and OHO. The defect in rickets is due to the nonfunctional PHWX gene. However, this is due to the circulation of phosphatonin, which is not processed by OHO. Tumors can be difficult to find and are extremely difficult and dangerous to remove. When tumor resection cannot proceed, regulation of phosphate metabolism and bone mineralization is important. It is dangerous to administer PHEX to a patient to break down hormones, because other circulating hormones and proteins can be broken down and affected. In that respect, phosphatonin fragments can be designed to have high receptor affinity and biological activity, effectively competing with processed circulating tumor-derived hormones.

Other rickets or hypophosphatemic symptoms:
There are many other causes of rickets besides HYP and OHO. Among them, vitamin D abnormalities are the most common, but there are other factors such as hypophosphatemia, tubular acidosis, intake of certain drugs, sprue, and pancreatic cystic fibrosis. It is considered that these diseases can be treated by using a phosphorotonin fragment or phosphofrotonin itself. Some of these diseases are briefly discussed below (diseases that cause hypophosphatemia may be treated by overall hormonal use).

Renal transplantation and renal osteodystrophy (bone dysplasia):
Chronic symptoms due to kidney transplantation are those in which phosphate leaks from the kidney (hypophosphatemia) and abnormal bone mineralization. The phosphatonin fragment has the effect of treating without causing the side effects associated with currently used drugs.

  Bone dysplasia (complex bone disease) is usually caused by chronic renal failure (renal disease). Renal failure is thought to result in death if dialysis is not applied (the end stage of kidney disease). However, patients with osteogenesis are usually receiving dialysis treatment. This bone disease, also referred to as “renal bone dysplasia”, generally involves subjecting a patient to long-term hemodialysis. Chronic renal failure in most patients results in secondary hyperparathyroidism and histological appearance of pancreatic cystic fibrosis. In children, especially at very young ages, this disorder is characterized by growth disorders and severe bone deformities. The pathogenesis of renal bone dysplasia is related to phosphate retention (hyperphosphatemia) and affects calcium and calcitriol metabolism. Furthermore, abnormal bone formation is caused by metabolic acidosis, cytokines, and parathyroid hormone degradation. Treatment is with diets that refrain from phosphate intake, phosphate conjugates, and vitamin D metabolic activators. In the present invention, an unprocessed hormone is an effective means of controlling blood phosphate levels and is considered to lead to the healing of bone diseases. If phosphatonin receptivity develops in the kidney or tissue, there may be a cure for patients with end-stage renal disease (ie, complete loss of kidney function).

Osteoporosis / Bone Mineral Loss Postmenopausal women tend to be deficient in bone minerals and as a result tend to damage the normal skeleton. The cause is not clear, but is thought to be due to the interrelation between genetic and environmental factors. Current research focuses on organizing statistical models to analyze multifactorial diseases such as osteoporosis.

  The use of phosphatonin derivative fragments could reverse bone mineral loss and treat this disease. In addition, biologically active peptides can be modified to more likely and more specific forms of action.

Paget's disease of bone:
Paget's disease occurs because bone remodeling is asynchronous. Bone mineralization (mediated by osteoblasts) and bone resorption (mediated by osteoclasts) become stepless. Excess resorption activity by osteoclasts occurs (especially in the early resorption phase), and the bone marrow is replaced by fibrous tissue and unorganized trabeculae. Although the cause cannot be identified, it is thought that this disease can be treated by administering a peptide / derivative of phosphatonin.

Diseases related to NaPi disorders in tissues other than the kidney:
Sodium-dependent phosphate cotransporter (NaPi) is expressed in various tissues in addition to the kidney. There are three types of NaPi, and types I, II, and III are described much earlier in this document. All three types are said to be expressed in the kidney. Type III is said to be ubiquitinated and expressed in tissues other than the kidney (Murer, Eur. J. Physiol. 433 (1997) 379-389; Kavanaugh, Kidney Int. 49 (1996) 956-963). Has been confirmed to be expressed in the liver and brain in addition to the kidney (Hilfiker, PNAS 95 (1998), 14564-14569). On the other hand, type II is thought to be expressed only in the proximal tubule of the kidney.

  Proximal tubules have been shown to express all three types, but it is generally believed that type II plays the largest role in phosphate reabsorption at this site. This was demonstrated in experiments with knockout mice that inactivated the gene encoding Type II NaPi (referred to as Np + 2). Homozygous mutant (Np + 2-/-) is associated with increased urinary phosphate excretion, hypophosphatemia, elevated serum 1,25-dihydroxyvitamin D levels, and other hypercalciuria (HHRH) It showed typical symptoms of hereditary hypophosphatemic rickets (Beck, PNAS 95 (1998), 5372-5377). In mammals, the regulation of phosphate homeostasis takes place in the kidney, so the results of this experiment indicate that type II of the three types plays the most important role in phosphate homeostasis throughout the body. In the examples, combined with the CL8 cell line results, it is suggested that NaPi regulated by phosphatonin in the kidney is clearly type II.

  One of the biggest clinical problems of patients with renal failure is hyperphosphatemia. If such serum phosphate excess can be controlled, it will be very clinically significant. Therefore, phosphatonin, its fragments or derivatives that can down-regulate NaPi and lower serum phosphate levels have great potential. In patients with advanced renal failure (so-called end-stage renal disease = before ESRD), it is important that phosphatonin down-regulates NaPi expression in the kidney.

  However, if the main renal function is lost at the ESRD stage, phosphate is not excreted from the glomeruli, and phosphatonin loses its site of action in the kidney. In such a disease stage, controlling the absorption of phosphate from the gastrointestinal tract by meals would be effective. The gastrointestinal tract, especially the intestinal tract, is the only organ in which phosphate ingested by the diet is taken into the body's blood circulatory system. Thus, the next major target is to regulate the phosphate that is taken into the blood circulation even after losing kidney function.

  A type II NaPi subtype, type IIb, has been reported to be cloned in the mouse intestine. (Hilfiker, PNAS 95 (1998), 14564-14569). Although it is already known that phosphatonin acts on intestinal type IIb NaPi, it is also expected that this type IIb NaPi plays a major role in the absorption of phosphate from the diet in the intestinal tract. Tonin has the potential to be an important factor in its up-regulation and down-regulation.

Example 7: Pharmaceutical Composition For a pharmaceutical composition, a polypeptide according to the present invention and, where appropriate, a pharmaceutically acceptable excipient, diluent or carrier is formulated. The exact properties and dosage of each compound in such compositions may be determined empirically and should be determined by the route of administration of the composition. Routes of administration to patients include oral, buccal, sublingual, topical (including transocular), rectal, vaginal, nasal, parenteral (including intravenous, intraarterial, intramuscular, subcutaneous and intraarticular) and so on. Parenteral administration is also desirable to avoid unwanted proteolysis.

  Appropriate doses of the molecules of the invention will vary depending on factors such as the disease or disorder being treated, the route of administration, the age and weight of the patient being treated. As an example of parenteral administration, administration of a molecule of the present invention at a dose of 0.1 μg to 1.5 mg / kg / day would be suitable for the treatment of a typical adult. Furthermore, 1 μg to 150 μg is optimal. Thus, it is believed that the active polypeptide component should be administered as a daily dose of 0.01 to 100 mg, generally 0.1 to 10 mg for a human adult.

  Examples of compositions for parenteral administration usually use a molecular solution, preferably an aqueous carrier, dissolved in a suitable carrier. Aqueous carriers vary widely, including water, buffer, 0.4% saline, 0.3% glycine. These liquids are sterilized beforehand to remove coagulum and other particles. Pharmaceutically acceptable buffers are included to adjust the pH and denature the toxicity of, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The molecular concentrations in these formulations vary over a wide range, for example 0.5% or more by weight and up to 15-20%. This concentration should be correctly selected by one skilled in the art.

Examples of typical pharmaceutical compositions are described in detail in "Remington's pharmaceutical chemistry (Remington's Pharmaceutical Science), 15 th ed., Mack Publishing Company, Easton, Pennsylvania (1980) ." By way of example, a pharmaceutical composition for injection is prepared to contain 1 ml of sterile buffer and 50 mg of molecules. A typical composition for instillation is made to contain 250 ml of Ringer's solution and 150 mg of molecules. Methods of actually preparing this composition are known to those skilled in the art. Formulation and administration methods for potipeptides for pharmaceutical use are known to those skilled in the art, and examples by P. Goddard, Advanced Drug Delivery Reviews, 6 (1991) 103-131 are also discussed. It has been.

Example 8: Characterization of phosphatonin (MEPE) and gene encoding it
Clinical profile of neoplastic osteomalacia patients (BD, ND, EM, and DS):
Patient BD is described in a previously published paper (Rowe, Bone 18 (1996), 159-169), and a report on patient ND has already been published (David, J. Neurosug. 84 (1996) , 288-292). Both were patients with classic neoplastic osteomalacia, with reduced phosphate levels in the blood, radiological osteomalacia, and decreased blood 1.25 vitamin D3. Patient BD (44 years old, female) and Patient ND (66 years old, female) had left nasal cavity tumor (angioepithelioma), intracranial tumor (tumor-like mesenchymal hemangiocytoma), and symptoms Was significantly reduced. Patient ND has undergone three such surgeries over the course of 20 years, and remissions have been seen with each surgery.

Tumor conditioned medium:
Tumor samples from patients BD, ND, and EM were collected immediately after resection. Samples were cut to ˜1 mm and some were frozen in liquid nitrogen. The remaining tumor tissue pieces were subjected to tissue culture as described in the previous chapter (Rowe, Bone 18 (1996), 159-169). Briefly, samples were digested overnight with collagenase and cultured in different cycles using media with and without serum (DME media). For patient ND, samples from around the dura mater and dura mater were collected and treated as described above. Further, from the patient BD, a control skin fibroblast culture was obtained on the same day as the tumor resection, and the same treatment as the tumor sample was performed. Samples collected from patient BD were labeled as follows: 1: Tumor conditioned medium (TCM-BD); 2: Skin conditioned medium (SCM-BD). Samples collected from patient ND were labeled as follows: 1: Tumor conditioned medium (TCM-ND); 2: Subdural conditioned medium (SDCM-ND); 3: Dural conditioned medium (DCM-ND); 4: Intracranial tumor surrounding fluid (FST-ND). In this specification, unless the above abbreviations are specifically marked with “added serum”, all samples are taken from cells grown in DMEM medium without added serum.

Concanavalin A affinity chromatography on TCM:
The tumor conditioned medium (TCM) collected from the patient ND was subjected to concanavalin A affinity chromatography according to Example 1 to separate a high affinity fraction and a low affinity fraction (HCA and LCA, respectively). Both HCA and LCA fractions were eluted with α-methyl-D-glucopyranoside (0.5M) elution buffer. Briefly, partial purification of TCM was performed using concanavalin A affinity chromatography. The method was modified from the procedure described in (Wagner, Gen. Comp. Endocrinol. 63 (1986), 481-491). Concanavalin A Sepharose (Pharmacia code number: 17-0440-01, 14 ml) in 20% ethanol is first washed with a few column volumes of water and then with running buffer (CRB; 0.06M sodium phosphate ph7.2). 0.5M sodium chloride). The equilibrated slurry was then added to a 12 mm x 115 mm Pharmacia screw top column and 3 volumes of running buffer was added at an effluent of 0.4 ml / min (FPLC / HPLC millennium Waters chromatography system). Conditioned medium equilibrated in CRB buffer (10 ml) was added to the column and allowed to bind. Then wash off with several column volumes of CRB loading buffer and wash out sodium phosphate elution buffer (ERB; 60 mM pH 7.2 / 0.5 M NaCl / 0.5 M α-methyl-D-glucopyranoside / 0.01% azide). Bound protein was eluted by adding at an effluent volume of 0.2 ml / min (40 ml). High affinity proteins were eluted by overnight incubation in ERB buffer followed by a second ERB buffer treatment at an effluent rate of 0.2 ml / min. The elution characteristics of the high and low concabanaline A affinity TCM proteins were equal, showing a single symmetrical peak when the column volume was ~ 1.6. The LCA peak represented 1/3 of the entire HCA peak, and 1 μg of HCA material was collected from 10 ml of tumor conditioned medium (TCM) in patient ND.

SDS-PAGE of TCM and Concanavalin A fraction:
Tumor conditioned medium, conditioned medium, and concanavalin A peaks (HCA and LCA) were separated by SDS-PAGE and imaged with Sybr-orange staining. SDS polyacrylamide gel electrophoresis was performed using a Novex NuPAGE® electrophoresis system. This consists of 4-12% bis-trisacrylamide gradient gel (pH 6.4) and MOPS-SDS (50 mM 3- [N-morpholino] propanesulfonic acid; 50 mM Tris base; 3.5 mM SDS; 1.0 mM ETDA; pH 7.7) Consists of running buffer. Electrophoresis was performed at a constant 200 V for 50 minutes. NuPage LDS sample buffer (10% glycerol; 1.7% Tris base; 1.7% Tris HCl; 2% lithium dodecyl sulfate; dithiothreitol 50 mM; 0.015% EDTA; 0.075% Selva Blue G250; 0.025% Phenol red (pH 7.5, final concentration) and denatured at 70 ° C. for 10 minutes. NuPage antioxidants were added above the electrophoresis chamber according to the manufacturer's recommendations. Following electrophoresis, the protein was stained when the gel was incubated in 7.5% acetic acid with Sybr-orange. Proteins were visualized by UV illumination using the “Bio-Red Fluorlmager gel imaging system”. HCA and LCA fractions were stained. Two proteins of 56 kDa and 200 kDa were stained, each showing the same properties. Conditioned medium taken from patient ND's intracranial tumor, subdural (adjacent to patient's tumor), and dura mater material should contain multiple bands spanning ~ 50-80kDa I understood. Looking at the major bands, there was a fragile band at ~ 66 kDa in all specimens, and high molecular weight antibiotics at ~ 200 kDa were observed in tumors and subdural samples. The tumor material had the highest intensity of ~ 200 kDa and was not present in the dura mater. A band diffused to 55-60 kDa was seen in the tumor and subdura, but was not present in the dural conditioned medium (patient ND). No protein staining was seen in the skin conditioned medium and the control medium. The conditioned media of patient BD and EM showed the same properties except that no high molecular weight protein was present at 200 kDa.

  All non-phosphateuria derived tumor patients and skin control groups from patient LA and patient SL showed a 66 kDa band and diffuse staining at 50-60 kDa. Concanavalin A affinity peaks HCA and LCA were rich in a high molecular weight 200 kDa band and further contained proteins in the 50-60 kDa range. The protein properties of the conditioned media from the bone cell lines HTB96 and SaOs2 were similar to the tumor conditioned media ingested from OHO patient ND. The band strength of 200 kDa in SaOs2 was lower than that of TCM and HTB96 from patient ND brain tumor, patient ND subdura.

Immunoblotting and glycoprotein staining of TCM and purified fragments:
To perform Western blots, proteins were transferred to PVDF membrane using submarine electrophoresis (Amersharm). After performing SDS-PAGE electrophoresis, the gel was placed in a transfer buffer (25 mM Tris HCl, 0.38 M glycine, 0.2% SDS (TB)) and equilibrated at room temperature for 1 hour. The PVDF membrane was cut to an appropriate size, rinsed quickly with methanol, washed with sterilized water, and equilibrated in TB. The equilibrated gel and PVDF membrane were sandwiched between filters and placed in a cassette. The cassette was installed in a Hoeffer system submarine electric blotting apparatus containing TB buffer, and cooled by maintaining at 4 ° C with a thermocooler. When the sandwiched PVDF end was placed on the anode and electrophoresis was performed for 0.4 A (45 V) continuously for 45 minutes, protein was transferred. Pre-Anti-op antiserum, post-Anti-op antiserum, or alkaline phosphatase-binding calmodulin solution using the methods described in Enhanced-Chemiluminescence kit (Amersham; ECL +) or calmodulin affinity detection kit (Strategene). Was diluted 1000 times and the blot was screened. Chemiluminescence was detected, photographed using the Bio-Rad fluorescence imaging system, and calmodulin affinity binding was visualized using the colorimetric system (Stratagene) described above for clone detection. Biotinylated molecular weight markers (Amersham) were used as standards for transcription and molecular weight measurements. Streptavidin conjugated with horseradish peroxylase (HRP) was added to the secondary antibody to facilitate visualization of biotinylated markers by chemiluminescence (goat-anti-rabbit IgG bound to HRP) .

  A chemiluminescent band was evident when pre-treated preoperative antiserum was screened from Western blots of phosphateuria tumor conditioned medium (TCM) from OHO patients. All tumors that were not phosphateuria, skin tissue control group, and medium control group were negatively screened with the same pre-treated antisera. In addition, when screened with antisera after surgery, both TCM and conditioned media were all negative.

  Screening of pretreated antisera with TCM protein from patient ND, osteosarcoma cell line HTB96, and SaOS2 reveals distinct immunopositive bands at ~ 54-57kDa and ~ 200kDa did. The subdural tissue adjacent to the tumor sample of patient ND emitted a stronger signal at 54-57 kDa compared to the conditioned medium of the dura-brain sample. In the dural conditioned medium, the 200 kDa band was not stained. Both HCA and LCA concanavalin A fractions showed a strong signal in the 200 kDa band and a weak but visible signal at 54-57 kDa. SaOS2 and HTB96 cell lines were also positive in the same band, but the signal was weaker at 200 kDa in SaOS2 conditioned medium compared to TCM and HTB96.

  Skin conditioned medium and medium control group derived from patient ND and patient BD were negative after screening with antiserum after surgery (Rowe, Bone 18 (1996), 159-169). Recombinant MEPE (rec-MEPE) stained positively with pre-treated preoperative antiserum, which is possible when rec-MEPE is added. The 54-57 kDa band was confirmed to be positive for Sybr-orange stained protein, and pre-treated preoperative antisera were screened for rec-MEPE. This was the same size as the 55-57 kDa band (screened with a pre-treated anti-operative antiserum Western method) and was found in patient ND tumor conditioned media and osteosarcoma cell lines HTB96 and SaOS2. Recombinant MEPE has an additional 45 kDa CBP tag at the N-terminus. This reduces the migration rate, resulting in a clear increase in molecular weight in the SDS-PAGE gel. Thus, tumor-derived protein and rec-MEPE of equivalent size are considered to have undergone post-translational modifications to tumor-derived MEPE (high possibility of glycosylation).

  Western blotting of BD and EM TCM for OHO tumor patients showed a positive band for pre-operative antiserum pretreated at a slightly lower molecular weight (48-52 kDa), and at 55-57 kDa, rec- A band moving with MEPE was seen. Other high molecular weight bands were seen at 61, 75, 80, and 93 kDa (weak signal).

  Screening with pre-treated pre-operative antiserum showed a negative 66 kDa Sybr-orange stained protein band in all samples. Glycoprotein screening in duplicate blots is the same result as pre-treated preoperative antisera screening, with both 54-57 kDa and 200 kDa bands staining positive, indicating that these proteins are glycosylated. It was. Proteins were separated by SDS-PAGE and blotted onto PVDF membrane by the method described above. The glycoprotein was specifically detected using an immunoblot kit (Bio-Rad) for detecting the glycoprotein. The internal control was used with the addition of Amersham biotinylation marker. Briefly, the membrane after transfer was treated with 10 mM sodium periodate in sodium acetate / EDTA buffer to oxidize hydrocarbon molecules. The blot was then washed with PBS, placed in sodium / acetic acid / EDTA buffer and incubated at room temperature for 60 minutes to hybridize. Subsequently, the filter was washed 3 times with TBS (10 minutes). Blocking and detection were performed as described above using Enhanced Chemiluminescence kit (Amarsham), streptavidin, and horseradish peroxidase. Primary antibody and goat-anti-rabbit-HRP secondary antibody were not used.

  As a result, pre-treated pre-operative antiserum specifically detected tumor hypophosphatemic osteomalacia-TCM-derived protein. The major protein was detected at a limited molecular weight of 2-3, 48-52 kDa, 54-57 kDa, 200 kDa. All OHO-TCM samples were positive for a 54-57 kDa protein, and all proteins detected by the pre-treated preoperative antiserum stained positive in a glycoprotein status screen. A tissue control medium that was not an OHO tumor was negatively screened with pre-treated preoperative antiserum.

Example 9: Expression of MEPE fusion protein from pCAL-n-EK vector
All sequences encoding cDNA were subcloned into pCAL-n-EK as described in Example 4. Pre-surgical antiserum and calmodulin conjugated to alkaline phosphatase were screened by Western blot as described above to confirm the fusion construct that produced IPTG introduction of E. coli host BL21 (DE3). A fusion protein (4.5 kDA calmodulin-binding peptide) containing a calmodulin peptide, enterokinase site, thrombin site and having a microbial CBP tag was predicted to be 56 kDa by SDS-PAGE. This is almost consistent with the expected molecular size (~ 48 kDa). Protein purification was performed by calmodulin affinity chromatography as described above. When pre-incubation antisera with a purified fusion structure was screened by TCM Western blot, a weak signal was observed at 55-57 kDa, but no 200 kDa band was observed. The 55-57 kDa signal did not disappear completely because the highly antibodyable glycosylation moiety was present in the incomplete MEPE protein (TCN) and not in the rec-MEPE microbial fusion construct. . The fusion protein was dissolved in an aqueous Tris buffer. Surfactants were not used at any stage of the purification process.

Example 10: Tissue expression (RT / PCR and Northern blot analysis)
We screened MEPE cDNA on Northern blots containing poly A + RNA, but hybridization to stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland, bone marrow, heart, brain, lung, liver, skeletal muscle, kidney, pancreas Not detected (Clontech MTN Blot I and III). To perform Northern analysis, two blots (MTN®; and MTN®; III) were obtained from Clontech, containing the following 1-15 polyA + RNA. 1． Heart, 2. Brain, 3. Placenta, 4; Lung, 5; Liver, 6; Skeletal muscle, 7. Kidney, 8; Pancreas, 9. Stomach, 10; Thyroid, 11. Spinal cord, 12. Lymph nodes, 13. Trachea, 14. Adrenal glands, 15. Bone marrow. They were screened using MEPE cDNA amplified with specific internal primers (Pho433-111F and PHO877-111R). The primer sequences of Pho433-111F and PHO877-111R are highlighted in FIG. 8 (the positions of the nucleic acids are positions 433 to 456 (SEQ ID NO: 24) and 877 to 900 (SEQ ID NO: 25), respectively) . The PCR conditions used are as follows. Denaturation pretreatment: 3 minutes at 95 ° C; followed by denaturation at 95 ° C for 45 seconds, annealing at 65 ° C for 30 seconds, polymerization at 72 ° C for 45 seconds for 30 cycles; final extension at 72 ° C for 7 minutes; Cool to 4 ° C. The PCR buffer (PB) used was MgCl ₂ with a final concentration of 2 mM. Next, the MEPE cDNA amplified to 444 bp was separated by submarine electrophoresis using agarose, visualized by ethidium bromide staining, and purified using glass beads (Gene-clean II kit; Bio 101 INC). The purified DNA was radiolabeled using αP ³² dCTP (3000 ci / mmol) coupled with Amersham Mega Prime labeling kit. Specific activity of 5 × 10 ⁹ cpm / μg was repeatedly obtained. Hybridization (60 ° C.) and pre-hybridization (60 ° C.) in the blot were performed using a method already published (Rowe, Hum. Genet. 97 (1996), 354-352). It was then washed with the following stringency. 1; Wash in 2 x SSC 0.1% SDS twice at room temperature for 30 minutes, place in 0.1 x SSC 0.1% SDS, and wash twice at 60 ° C for 30 minutes. The filter was exposed to film for 7 days at -80 ° C and the film was developed. For human RNA derived from adrenal glands, brain, duodenum, heart, kidney, liver, lung, skin, spleen, thymus, thyroid, and tonsils, we did not perform amplification using RT / PCR and MEPE specific primers. Expression tests using cDNA templates confirmed low levels of expression in the brain, kidney, liver, and pancreas. For this experiment, total RNA was removed from the following human tissues. 1． Thymus, 2. Brain, 3. Testis, 4. Duodenum, 5; Heart, 6; Skin, 7; Liver, 8. Tonsils, 9. Spleen, 10; Thyroid, 11. Adrenal gland, 12. Lung, 13; Kidney, 14. OHO tumor tissue, 14. Human primary osteoblasts. Total RNA was also extracted from rat primary osteoblasts. Total RNA was copied using reverse transcriptase-PCR and perkin Elmer-Roche RNA PCR kits using the MEPE internal primers (Pho433-111F and PHO877-111R) described above. Briefly, 1 μg of total RNA was added to 10 ml of Tris HCl (pH 8.3), 50 mM KCl, 5 mM MgCl ₂ , 1 mM dNTPs, 1 unit / μl ribonucleic acid inhibitor, 2.54 units / μl MULV. Reverse transcriptase was dissolved in 20 μl of a 0.75 μM downstream primer (PHO877-111R) solution. The mixture was then incubated at 37 ° C. for 10 minutes. Next, upstream primer (Pho433-111F) and dNTPs, MgCl ₂ , and AmpliTaq DNA polymerase were added to a final concentration of 0.15 μM, 200 μM, 2 mM, 2.5 units / 100 μl, respectively, for a total volume of 100 μl. A Perkin Elmer thermocycler (System 9700) was set in the following program and PCR was performed. Denaturation pretreatment at 95 ° C for 3 minutes; followed by 35 cycles of denaturation at 95 ° C for 45 seconds, annealing at 65 ° C for 30 seconds, polymerization at 72 ° C for 45 seconds; final extension at 72 ° C for 7 minutes; and 4 Cool down to ℃. Amplified material was separated using an agarose gel, confirmed by Southern blot, and sequenced. The standard panel of cDNA from non-OHO tumors (breast cancer, lung cancer I, colon adenocarcinoma I, lung cancer II, prostate cancer, colon adenocarcinoma II, ovarian cancer, pancreatic cancer; (Clontech human tumor panel # K1422-1) is , Except for low-level expression in colon adenocarcinoma, ovarian cancer, and prostate cancer (detected after Southern blotting of RT / PCR amplification products with radiolabeled MEPE cDNA) In contrast, PCR was negative, and in contrast, amplified poly A + RNA from 4 patients with BD, DM, EM, and DS was subjected to RT / PCR using MEPE primers and high levels. (Standardized for glyceraldehyde 3-phosphate dehydrogenase and transferrin) Non-phosphateuria tumor, control tissue from OHO patients (skin and material adjacent to tumor), CL8 human kidney cells Strain, primary human osteoblasts (Clontech HO Poly A + RNA (purchased from ST, see material) and poly A + RNA from tumor polyps collected from patients with linear sebaceous nevus syndrome (polyps TCM inhibits phosphate uptake in human kidney cell line CL8) Was not amplified using MEPE-specific primers, derived from heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (human panel I # K1420-1) purchased from Clontech When cDNA was used as a template for MEPE primer PCR, low levels of expression were confirmed in brain, liver, lung, and pancreas, and sequencing of the MEPE primer amplification band indicated that it was completely homologous to MEPE cDNA. Sequencing was also confirmed by Southern screening of amplified bands with MEPE cDNA, and OHO template poly A + RNA from all OHO patients was consistently predicted 480 bp band. The upper band was amplified by sequencing and Southern autoradiography, and the lower band was confirmed to be completely homologous to the MEPE sequence. Southern analysis confirmed that it was a derivative: low-level expression in normal tissues and non-OHO tumors did not reveal this low-band, indicating that alternative splicing plays a role in tumor-derived MEPE All RT / PCR and PCR experiments were normalized to G3PDH and transferrin.

  In summary, high level expression of MEPE (measured at the mRNA level) is only seen in OHO tumor samples, and very low level expression (most likely ectopic) is not in the brain, liver, kidney, and OHO It was found in 3 samples of tumor 11. Eight out of 11 tumors were negative for MEPE mRNA expression (RT / PCR). All results were normalized to GA3PDH and transferrin RT / PCR primer.

Example 11: Southern analysis (genome blot)
Immobilized DNA from the autosomal rickets (Rowe, Hum. Genet. 91 (1993), 571-575) family, as well as immobilized DNA digested to Pstl, EcoRl, Pvull, and Mspl, respectively. Genomic blots containing were screened with MEPE cDNA labeled with radiation as described above. Southern analysis was performed using the above-described genomic digestion of DNA extracted from blood (Rowe, Hum. Genet. 93 (1994), 291-294). Pstl blots revealed an 11 kb band and a 4 kb polymorphic band in one of the 16 family members screened. EcoRl, Pvull, and Mspl blots showed positive single bands of 6 kb, 6.5 kb, and 4 kb, respectively, confirming the human origin of the gene. Due to the lack of genetic information, it was impossible to predict whether the diseases in this autosomal rickets family were separated by genes.

Example 12: Phosphate uptake in human kidney cell line CL8: TCM and MEPE administration Phosphate and glucose uptake experiments were performed in the human kidney cell line (CL8) as previously described (Rowe, Bone.18 ( 1996), 159-169). Briefly, cells were placed in defined media (DM) and mixed or cultured overnight in 24-well flat bottom tissue culture plates (Falcon 3047). The DM was then replaced with fresh DM plus purified fusion protein or concanavalin affinity purified TCM and placed at 37 ° C. overnight. And measured intake P ³² and C ¹⁴ methyl glucose (Rowe, Bone.18 (1996), 159-169).

  When TCM (1/20 dilution) was added to human kidney cell lines, Na + -dependent phosphate uptake was greatly reduced as previously reported (Rowe, Bone.18 (1996), 159-169). As previously reported, this inhibition was prevented with TCM with preoperative antiserum before incubation, but not with postoperative antiserum (Rowe, Bone.18 (1996)). , 159-169). Addition of high and low affinity concanavalin A purified fractions (HCA and LCA, respectively) at a concentration of 40 ng / ml also resulted in inhibition of Na + dependent phosphate uptake (NaPi). In both the TCM and concanavalin A fractions, inhibition occurred only on phosphate intake and had no effect on Na + -dependent α-methylglucose intake. In all cases, the magnitude of the effect was related to dose.

  Similar experiments were performed using MEPE fusion protein purified using calmodulin affinity chromatography. Surprisingly, recombinant MEPE did not inhibit Na + -dependent phosphate cotransport, but phosphate uptake increased with dose (see Table 24). Double phosphate uptake was observed at 1000 ng / ml (p <0.001). These experiments confirmed that the MEPE fusion protein specifically promotes Na + -dependent phosphate cotransport in the human kidney cell line CL8.

  It is to be understood that the present invention has been described with reference to specific embodiments, and that various modifications can be made by those skilled in the art, and that their equivalents do not depart from the scope and spirit of the present invention. Can be replaced. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, step, or the like, to the purpose, spirit, or scope of the invention. Such changes and modifications are intended to be included within the scope of the claims appended hereto.

  All documents (including patents, patent applications, journal articles, laboratory manuals, books, or other disclosures), detailed descriptions, and examples cited herein as background to the present invention are hereby incorporated by reference. Is incorporated into. In addition, the sequence listing is also incorporated herein by reference.

FIGS. 1 (a) and 1 (b) show chromatograms having peaks containing low affinity protein and high affinity protein derived from a concanavalin A column, respectively. Cation exchange chromatogram of fraction derived from concanavalin A column. Computer predicts hydrophilicity and hydrophobicity of phosphatonin. Computerized prediction of phosphatonin antigenicity. Computer predicts the flexibility of phosphatonin. Prediction of secondary surface probability of phosphatonin by computer. Computer predicted secondary structure of phosphatonin. Full length cDNA sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) in the largest isolated MEPE clone (pHO11.1). The other 5 isolated clones are included in this largest clone, all clones are in frame with pBSCPT SKII-, the cloning vehicle. Primers used for PCR are shown in bold and the total number of residues is 430 and 1655 bp, respectively. The prokaryotic expression vector pCal-n-EK contained all the residues from MEPE residue V to the 1291-93 bp MEPE stop codon (TAG) in frame. One polyadenylation sequence AA {T / U} AAA is indicated by double underlining. Parts with local homology to DMA-1, DSSP, and OPN are shown with wavy lines (MEPE-motif C-terminal), RGD residues are surrounded by ellipses, and mucopolysaccharide adhesion sites are surrounded by solid lines The tyrosine kinase site is indicated by a single underline, and the N-glycosylation motif is surrounded by a dotted frame. For a complete list of motifs including casein kinase II, protein kinase C, etc., see Table 1 in the prosite screen. MEPE GCG-peptide structure secondary structure prediction. The main skeleton of the amino acid is shown by the solid line in the middle and curves in the region predicted to be bent. The hydrophilic and hydrophobic regions are shown in oval and diamond shapes, respectively, and the RGD motif is also specified. The N-glycosylation site is shown as an ellipse at the end of the rod (C-terminal side), and the α-helix is shown in the region where the main skeleton is wavy. Bar graphs show phosphate uptake in the presence of different amounts of MEPE (A: 92 ng / ml, B: 300 ng / ml, C: 500 ng / ml, and D: 1000 ng / ml). The choline graph shows the sodium-independent results of the control when substituting choline chloride for NaCl. The error bar is SEM, the P value is the value for the difference between MEPE and the control, and in the C and D experiments, P <0.001. In experiment A (92 ng / ml) P <0.05 and in B (300 ng / ml, P, 0.01). The value of N in A and B is 4, and 5 and 6 in C and D, respectively. Anova analysis followed by Newman-Keuls multiple comparison test was used. Dose curve for MEPE dose and phosphate intake, including SEM error bar. Sequence similarity analysis using “sim” and llanview mathematical and software tools (Duret, Comput. Appl. Biosci. 12 (1996), 507-510). In each calculation, the gap open penalty is set to 12, and the gap extension penalty is set to 4. The comparative matrices are “PAM40” for A and “BLOSUM62” for B and C, respectively (Duret, Comput. Appl. Biosci, 12 (1996), 507-510; Hurang, Comput. Appl. Biosci. 8 ( 1992), 155-165; Huang, Comput. Appl. Biosci (1990) 6, 373-381). The similarity score threshold was 70% for A and 40% for B and C. The bold blocks on each protein scheme are> 80% for A and> 62% for B and C. It should be noted that in MEPE vs. DSSP (A), there are 5 blocks in DSSP that show> 80% homology with a MEPE motif (DSSESSDSGSSSES). Similar sequence homology is clearly seen in DMA-1 and OPN vs. MEPE, which is characteristic of all three proteins. DSSP and MEPE dot matrix (Dot matrix) using Antheprot statistical analysis (Deleague, G. Software for protein analysis: Antheroplot V2.5e. Microsoft group. (7 Passages du Vercours 69-367 Vercors Lyon Cedex 07, 1997)) ) Comparison. In (A), we performed a low-rigidity comparison with the window set to 13 as a screen variable, and in (B) we performed a comparison with the window set to 15. The color shading indicates the identity matrix score shown in the figure. The C-terminal residue of the MEPE motif has> 80% sequence homology, and the motif repeat structure is shown in a striped pattern. P1BL21 and p6XL1 recombinant plasmids containing phosphatonin fusion structural molecules. Lacl: (lac promoter); LIC: (ligation-independent cloning sequence); EK: enterokinase cleavage site; Thrombin (thrombin target sequence); Amp: ampicillin resistance gene: Cal peptide (calmodulin peptide sequence); Phosphatonin (phosphatonin) Tonin coding sequence).

Claims (41)

An isolated or essentially pure form of a polypeptide having phosphatonin activity. 2. The polypeptide of claim 1 having a molecular weight of about 53-60 kDa as measured by SDS-PAGE. 2. The polypeptide of claim 1 having a molecular weight of about 200 kDa as measured by bis-Tris SDS-PAGE at pH 7. 4. A polypeptide according to any one of claims 1-3, which is glycosylated and / or not glycosylated. The polypeptide according to any one of claims 1 to 4, which can be obtained by purification from Saos-2 cells (deposit number: ECACC 89050205). A polypeptide according to any one of claims 1 to 5, or an immunologically and / or biologically active fragment thereof, wherein the polypeptide is selected from the group consisting of the following (a) to (j): A polypeptide or fragment comprising an amino acid sequence encoded by a peptide:
(A) a polynucleotide encoding at least a mature form of the polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 (FIG. 8);
(B) a polynucleotide that encodes at least the mature form of the peptide and comprises the coding sequence set forth in SEQ ID NO: 1 (FIG. 8);
(C) encoded by a polynucleotide of (a) or (b) by substitution, deletion and / or addition of one or several amino acids of the amino acid sequence encoded by the polynucleotide of (a) or (b) A polynucleotide encoding a polypeptide derived from the polypeptide to be produced;
(D) a polynucleotide comprising a complementary strand that hybridizes to any one of the polynucleotides (a) to (c);
(E) a polynucleotide encoding a polypeptide sequence having 60% or more identity with the amino acid sequence of the polypeptide encoded by any one of the polynucleotides of (a) to (d);
(F) a polynucleotide encoding a polypeptide that comprises a polypeptide fragment encoded by any one of the polynucleotides (a) to (e) or an antigenic determinant-containing portion and that can regulate phosphate metabolism ;
(G) a poly encoding an antigenic determinant-containing portion of a phosphatonin polypeptide comprising amino acid residues at positions 1-40, 41-180 and / or 401-429 of SEQ ID NO: 2 (FIG. 8) nucleotide;
(H) a polynucleotide encoding a polypeptide comprising at least 15 nucleotides of any one of polynucleotides (a) to (g) and capable of regulating phosphate metabolism;
(I) a polypeptide comprising a cell encoded by the polynucleotide of any one of (a) to (h) and / or a glycosaminoglycan-binding motif and / or a bone mineral motif and capable of regulating phosphate metabolism A polynucleotide encoding a peptide;
(J) A polynucleotide in which the nucleotide sequence is degenerate as a result of genetic coding of the nucleotide sequence of any of the polynucleotides (a) to (i). The polypeptide according to any one of claims 1 to 6, which is capable of regulating phosphate metabolism. An isolated polynucleotide encoding the polypeptide of any one of claims 1-7. 9. The polynucleotide of claim 8, comprising RNA or DNA. 10. The polynucleotide of claim 8 or 9, comprising a deletion of contiguous nucleotides from either the C-terminus or N-terminus. A variant that hybridizes to the polynucleotide of any one of claims 8 to 10 and that has lost at least a portion of the phosphatonin activity of the polypeptide of any of claims 1 to 7. Polynucleotide. A vector comprising the polynucleotide according to any one of claims 8 to 11. A host cell genetically engineered with the polynucleotide according to any one of claims 8 to 11 or the vector according to claim 12, or the expression of a gene encoding the polypeptide according to any one of claims 1 to 7. A host cell produced by introducing an expression control sequence into an intervening host cell. A method for isolating phosphatonin comprising the following steps:
(A) culturing tumor conditioned medium or osteosarcoma cells in a serum-supplemented culture solution (DMEM Eagle solution / 10% FCS / glutamine / antifungal agent (DMFCS)) until confluence;
(B) culturing cells every other day in serum-free medium DMEM Eagle solution / glutamine / antimycotic agent, antibiotics (DM) for up to 5 hours;
(C) collecting serum-free conditioned medium from the cells and equilibrating the conditioned medium to 0.06 M sodium phosphate and 0.5 M sodium chloride (PBS) at pH 7.2;
(D) A step of subjecting the culture solution obtained in (c) to an equilibrated concanavalin A sepharose column;
(E) thoroughly washing the column with PBS;
(F) eluting the column of concanavalin A with PBS supplemented with 0.5 M α-methyl-D-glucopyranoside;
(G) subjecting the material eluted in (f) to cation exchange chromatography;
(H) eluting the phosphatonin polypeptide containing the fragments with 0.5 M sodium chloride. A biological activity and / or an immunological activity of phosphatonin, comprising culturing the host cell of claim 13 and recovering a polypeptide encoded by a polynucleotide obtained from the culture. A method for producing a polypeptide having 16. A polypeptide obtained by proteolysis of the method of claim 14 or 15 or the phosphatonin polypeptide of any one of claims 1-7, or obtained by the method of claim 14 or 15 with a PHEX metallopeptidase. . The polypeptide according to any one of claims 1 to 7 or claim 16, wherein the polypeptide has at least one of the following activities:
(A) can down-regulate sodium-dependent phosphate cotransport;
(B) can up-regulate renal 25-hydroxyvitamin D3-24-hydroxylase; and / or (c) can down-regulate renal 25-hydroxy D-1-α-hydroxylase. The polypeptide according to any one of claims 1 to 7 or claim 16, wherein the polypeptide has at least one of the following activities:
(A) can up-regulate sodium-dependent phosphate co-transport;
(B) can down-regulate renal 25-hydroxyvitamin D3-24-hydroxylase; and / or (c) can up-regulate renal 25-hydroxy D-1-α-hydroxylase. The polypeptide according to claim 16, wherein the polypeptide has lost at least one of the activities shown in claim 17 or 18. 21. An isolated antibody that specifically binds to the isolated polypeptide of any one of claims 1-7 or 16-19. 12. A nucleic acid molecule having a length of at least 15 nucleotides, which specifically hybridizes with the polynucleotide according to any one of claims 8 to 11 or a complementary strand thereof. An isolated regulatory sequence of a promoter that regulates the expression of a nucleic acid molecule comprising the polynucleotide of any one of claims 8-11. 23. A recombinant DNA molecule comprising the regulatory sequence of claim 22. A therapeutically effective amount of the polypeptide of any one of claims 1-7 or 16-19, the polynucleotide of any one of claims 8-11, the vector of claim 11, or 21. A method of treating a medical condition related to phosphate metabolism deficiency comprising administering the antibody of claim 20 to a mammalian subject. A method of diagnosing a subject's susceptibility to a pathological or pathological condition related to phosphate metabolism failure, comprising the following steps:
(A) a step of determining the presence or absence of a variant of the polynucleotide according to any one of claims 8 to 11; and (b) a pathological symptom based on the presence or absence of the variant or disease The stage of diagnosing sex. A method of diagnosing a subject's pathological symptoms or susceptibility to pathological symptoms related to phosphate metabolism deficiency, including the following steps:
(A) determining the presence or expression level of the polypeptide according to any one of claims 1 to 7 or 16 to 19 in a biological sample; and (b) the presence or expression level of the polypeptide. Diagnosing pathological symptoms based or susceptibility to pathological symptoms. A method for identifying a binding partner of a phosphatonin polypeptide comprising the following steps:
(A) contacting the polypeptide according to any one of claims 1 to 7 or 16 to 19 with a compound used for screening; and (b) whether the compound affects the activity of the polypeptide. The stage to decide. A method of identifying and obtaining a drug candidate for the treatment of phosphate metabolism deficiency comprising the following steps:
(A) in the presence of a component capable of producing a detectable signal in response to phosphate uptake, the polypeptide of any one of claims 16-19 or a cell expressing the polypeptide, Contacting with a drug candidate to be screened under conditions capable of phosphate metabolism;
(B) a step of detecting the presence or absence of a signal generated by phosphate metabolism or an increase in the signal, wherein the presence or absence of the signal or an increase indicates a presumed drug. A method for producing a therapeutic agent comprising the steps of the method according to any one of claims 26 to 28 and the following steps:
(I) synthesizing the compound obtained or identified in step (b) or an analog or derivative thereof in an amount sufficient to provide the subject with a therapeutically effective amount of the agent; and / or (Ii) combining the compound obtained or identified in step (b) or an analogue or derivative thereof with a pharmaceutically acceptable carrier. 29. An activator / agonist or inhibitor / antagonist of phosphate metabolism, or a phosphatonin binding partner obtained by the method of any one of claims 26-28. A polypeptide according to any one of claims 1 to 7 or 16 to 19, a polynucleotide according to any one of claims 8 to 11, a vector according to claim 12, an antibody according to claim 20, 31. A composition comprising the nucleic acid molecule of claim 21 or the active agent / agonist, inhibitor / antagonist, or binding partner of claim 30. 32. The composition of claim 31, wherein the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable excipient, diluent, or carrier. 35. The composition of claim 32, wherein the composition is a diagnostic composition and further comprises a means for detection. The polypeptide according to any one of claims 1 to 7 or 16 to 19, the DNA encoding and expressing the polypeptide, for the preparation of a medicament for treating phosphate metabolism deficiency, claim 30 21. Use of an activator / agonist, binding partner, or antibody according to claim 20. The polypeptide according to any one of claims 1 to 7, 16, 17, or 19 for the preparation of a medicament for treating hyperphosphatemia, DNA encoding and expressing the polypeptide, claim Use of an active agent / agonist according to claim 30, a binding partner, or an antibody according to claim 20. 2. For the preparation of a medicament for the treatment of renal osteogenesis abnormalities, hyperphosphatemia before renal dialysis / renal dialysis, secondary hyperparathyroidism, or cystic fibrotic osteoarthritis 21. A polypeptide according to any one of -7, 16, 17, or 19, a DNA encoding and expressing the polypeptide, an active agent / agonist, a binding partner according to claim 30, or a claim according to claim 20. Use of antibodies. The polypeptide according to any one of claims 1 to 7, 16, 18, or 19, intended for the preparation of a medicament for treating hypophosphatemia, a DNA encoding and expressing the polypeptide, claim Use of the antibody of claim 20, the nucleic acid molecule of claim 21, or the inhibitor / antagonist of claim 30. X-chromosomal hypophosphatemic rickets, hereditary hypophosphatemic rickets with hypercalciuria (HHRH), bone damage due to low mineralization, growth failure in childhood, neoplastic hypophosphatemic osteomalacia , Renal phosphate leakage, renal osteogenesis dysfunction, osteoporosis, vitamin D resistant rickets, terminal tissue resistance, renal fanconi syndrome, autosomal rickets, Paget's disease, renal failure, tubular acidosis, cystic 20. A polypeptide according to any one of claims 1 to 7, 16, 18, or 19 for the preparation of a medicament for treating fibrosis or sprue, a DNA encoding and expressing the polypeptide Use of the antibody of claim 20, the nucleic acid molecule of claim 21, or the inhibitor / antagonist of claim 30. A polypeptide according to any one of claims 1 to 7, 16, 18, or 19, for the manufacture of a medicament for treating bone mineral loss deficiency, a DNA encoding and expressing the polypeptide, Use of the antibody of claim 20, the nucleic acid molecule of claim 21, or the inhibitor / antagonist of claim 30. 20. A polypeptide according to any one of claims 1 to 7, 16, 18, or 19 intended for the manufacture of a combined preparation for use separately, or sequentially, concurrently with the treatment of phosphate metabolism deficiencies. And PHEX metallopeptidase. Use of a transformed osteoblast or bone cell line capable of overexpression of phosphatonin for the production of phosphatonin.

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