CZ20014480A3 - Head trauma induced cytoplasmatic calcium binding protein - Google Patents

Abstract

ANIC-BP polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing ANIC-BP polypeptides and polynucleotides in diagnostic assays.

Description

Traumafhlave-induced cytoplasmic calcium-binding proteins

Technical field

The invention relates to human acute neuronal induced calcium binding protein (ANIC-BP), and to polynucleotides that identify and encode this protein. The invention also relates to expression vectors, host cells and antibodies. In addition, the invention relates to methods of producing a protein and to methods of treating or preventing disorders related to protein expression. The invention also relates to inhibiting or activating the action of such polynucleotides and peptide poles.

BACKGROUND OF THE INVENTION

Stroke and acute head trauma, multiple sclerosis and spinal cord injury are diseases for which therapy is far from available. Stroke is the third major cause of mortality and the burden on patients as well as on social systems is huge. In the case of ischemic stroke, which is involved in most strokes, vascular blockage in the brain is the initial event. Head injuries are the leading disease of youth in the Western world. A significantly smaller number of patients are infected with a haemorrhagic stroke due to mechanical trauma and arterial rupture. It is a general phenomenon that approved drugs are difficult to access. Commonly available treatment approaches are based on pathophysiological concepts derived from experimental work on cerebral ischemia. These procedures include pharmacological strategies for arterial recanalization, inhibition of inflammatory processes, and neural protection. Further work with arterial reperfusion strategy continues. Earlier clinical studies with polymorphonuclear leukocite-dependent endothelial adhesion receptor antagonists have been discontinued, but the strategy is yet to be developed - However, any one-step strategy in the ischemic cascade is likely to provide only moderate benefits. The combination of low-dose acetylsalicylic acid (ASA) and modified release of dipyridamole has been shown to be additive in secondary prevention of stroke CTissen et al., Int. J. Clin. Pract. 91, pp. 14-16, 19971. Another combination commonly proposed for clinical trials is t-PA plus an effective neuroprotective agent. The results of these studies are far from highly effective. Thus, there is an urgent need for further research into pathophysiological mechanisms to identify drug targets that could be used to develop new drugs and to generally identify useful treatment regimens for patients suffering from acute nerve damage and disorders such as multiple sclerosis.

The invention is directed to Ca ²⁺ binding proteins, as there is ample knowledge of the role of Ca ²⁺ binding proteins in nerve protection. Although the exact function of Ca ²⁺ binding proteins (CCaBPs) is not definitively known, CaBPs are believed to buffer intracellular Ca ^{2 L} levels. As the excess of Ca ^{2 1} activates biochemical processes leading to proteolysis and mitochondrial function insufficiency may have this buffering capacity of CaBPs protective effect against excitotoxic neuronal injury (Heizmann et al., TINS 15, pp. 259-264, 1992). There is a number of findings supporting the role of CaBP in modulating Ca ²⁺ levels and neuroprotection.

The major Ca ²⁺ binding proteins (CCaBP) expressed in the central nervous system (parvalbumin, calbindin-D28K and calret-inin) have a very unusual and selective expression pattern in various neuronal populations. Neurons expressing CaBP usually express only one type, although a small number of neurons express more than one of the major Ca ²⁺ binding proteins.

It is increasingly clear that the presence or absence of CaBP in particular cell types is subject to a phenomenon known as selective vulnerability. Selective vulnerability is a property of specific types of neurons perishing in response to particular types of central nervous system (CNS) damage. For example, CA1 hyppocampal neurons are selectively vulnerable to general ischemia, Purkin's brain cells are selectively vulnerable to head trauma, stroke and amniotic fluid exposure, and neurons in substantia nigra (black matter) are selectively vulnerable in Parkinson's disease. Efforts have been made to target selective neuronal vulnerability to the expression patterns of various CaBPs, and high levels of CaBPs are reported to be found in neuronal populations that are selectively vulnerable to injury, while high levels of CaBPs in neuronal populations are also reported to be selectively resistant to injury. . For example, neurons expressing high levels of parvalbumin are reported to be selectively vulnerable to AMPA-induced toxicity (Veis et al., Neurol. 40: 1288-1292, 1990), while cultured hippocam neurons expressing high levels are reported. levels of calbindine-D28K, are selectively resistant to glutamate-induced toxicity (Baimbridge et al., TINS 15, 303-308, 1992). Similarly, hippocam neurons expressing high levels of calretinin are resistant to toxic doses of excitotoxin glutamate, NMDA, kainate and quisqualate (Vinsky et al., Novel Calcium-Binding Proteins, 1991, pp. 277-300).

It has also been shown that CaBP has altered expression in various disease states, but again. the results are inconsistent as to whether CaBP expression is related to selective vulnerability or selective resistance to injury. Calbindin-D28K expressing neurons have been reported to be selectively vulnerable to Alzheimer's disease (Iacopino et al., PNAS 87: 4078-4082, Hof et al., Exp. Neurol. 111, pp. 293-301, 1990; 1991) and in Huntingston's disease (Kiyama et al., Brain Res. 526, atr. 303-307, 1990), although calbindin-D28K-expressing neurons in the substania nigra are not selectively vulnerable in Parkinson's disease (Yamada et al., Brain Res., 526: 303-307 (1990). In the gerbil model of global ischemia, the presence of parvalbumin in some types of hippocam cells has been shown to be positively related to survival (Tortoša et al., Neurose! 1: 33-43, 1993), although another study suggests that parvalbumin, expressing hippocampal interneurons, are selectively vulnerable to Alzeheimer's disease (Brady et al., 80: 1113-11112 (1997)).

Calbindin gene knockout mice exhibit functional deficiency (e.g., ataxia) that indicates a strong dysfunction in neurons normally expressing this CaBP (e.g., brain Purkinje cells) despite the fact that these neurons appear morphologically normal. This finding suggests that CaBPs are vital to the pattern of cellular activity (Fliraksinen et al., PNAS 94, pp. 1488-1493, 1997). Retroviral infections of locomotor neurons with calbindin-D28k have also been found to have a neuroprotective action against IgG-induced toxicity from patients with amyotrophic lateral sclerosis (Ho et al., PNAS 93, pp. 6796-6801, 1996) and it has been shown that transfection of calbindine- D28k protects PC12 cells from toxicity due to serum removal, exposure to glutamate and MPP + neurotoxin (McMahon et al., Molec. Brain Res. 526, pp. 303-307, 1998).

Finally, the role of calcium-binding proteins in neurodegeneration is important. Certainly, some CaBPs provide protection and others cause selective vulnerability. However, it is not yet clear whether the expression of certain CaBPs within different neuronal populations results in different functional responses of a given CaBP. Another finding is that the severity of damage to different types of the central nervous system of the CNS may affect the apparent neuroprotective efficacy of CaBP, for example, one CaBP may cause a damage model with moderate damage but may not be able to buffer Ca ²⁺ increases in more severe

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CNS damage. Thus, it is sufficiently clear that CaBP confers protection as well as a vulnerability in CNS damage, but the mechanism for this development and response regulation is not yet known.

Recently, a family of genes containing a functionally unidentified gene (M025) of a mouse derived from a cDNA library has been isolated (Miyamoto et al., Mo. Reprod. Dev. 34, pp. 1-7, 1993). . The library was constructed from RNA isolated from the older mouse embria. The predicted amino acid sequence for M025 showed that the M025 gene may have structural homology to Ca ²⁺ binding proteins and missing membrane joining domains, suggesting that the protein may have been involved in the cytosolic development of the unfertilized egg. However, the real function of this protein remains unknown. Another gene, similar to M025, was cloned from the Drosophila CNozaki et al., DNA Cell Biol. 15: 505-509 (1996). The deduced amino acid sequence of the Mo25 cDNA is 69.3% identical to the murine Mo25 homolog. A homologue in Saccharomyces cerevisiae encodes in the open reading frame next to the subunit gene calcineurin Β. Recently, another gene has been isolated from the mutant Aspergillus hym A (Karos et al., Mol. Gen Genet. 260, pp. 510-521, 1999) and has been shown to correspond to homologues in yeast, fly plants, worms, fish, mice and humans. The cellular function of the Hym protein has not yet been defined in any of the cited organisms. - Like other proteins, where the functional contribution is only partially understood, the drug discovery process is constantly subject to fundamental revolution as it involves functional genomes, a high genome or genome-based biology. the approach as a means of identifying genes and gene products as therapeutic targets rapidly outperforms prior art approaches based on positional cloning. The phenotype that is the biological function of the genetic disease should be identified and this would then be traced back to the responsible gene based on the position in the genetic map.

Functional genomics relies heavily on high-throughput DNA sequencing technologies and various bioinformatics tools to identify gene sequences of potential interest from many of the currently available molecular biology databases. There is a continuing need to identify and characterize other genes and their related polypeptides / proteins as drug discovery targets.

SUMMARY OF THE INVENTION

The present invention provides an isolated polypeptide selected from the group consisting of (a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID No = 1,

Cb) an isolated polypeptide comprising a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID No: 2.

Cc) an isolated polypeptide having at least 95% identity to the polypeptide sequence of SEQ ID No: 2,

Cd) the polypeptide sequence of SEQ ID No: 2,

Ce) fragments and variants of such polypeptides according to Ca) to Cd).

Thus, the invention relates to ANIC-BP, in particular ANIC-BP polypeptides and ANIC-BP polynucleotides, recombinant materials, and methods of making the same. Such polypeptides and polynucleotides are important for the treatment of certain diseases including, but not limited to, stroke and acute head trauma, multiple sclerosis, and spinal cord injury, hereinafter referred to as the diseases of the invention. The invention also relates to methods for identifying agonists and antagonists (e.g. inhibitors) using the materials of the invention, and further relates to treatment conditions associated with the disruption of equilibrium identified by the compounds. Further, the invention relates to diagnostic assays for detecting diseases associated with inappropriate ANIC-BP activity or level.

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According to the invention, the isolated polypeptide is selected from the group consisting of:

Ca) an isolated polypeptide encoded by the polynucleotide of SEQ ID NO: 1.

Cb) an isolated polypeptide with a polypeptide sequence having at least 95%, 96%, 97%, 98% or 99% identity to the polypeptide sequence of SEQ ID No = 2,

Cc) an isolated polypeptide having the polypeptide sequence of SEQ ID No: 2,

Cd) an isolated polypeptide having at least 95%, 96%, 97%, 98% or 99% identity to the polypeptide sequence of SEQ ID No: 2,

Ce) fragments and variants of polypeptides according to Ca) to Cd),

Cf) an isolated polypeptide having or comprising a polypeptide sequence having an identity index of 0.95, 0.96, 0.97, 0.98 or 0.99 as compared to the polypeptide sequence of SEQ ID No: 2,

Cg) fragments or variants of polypeptides according to Ca) to Cf).

Polypeptides of the invention are believed to be members of the calcium-calcium binding protein family. They focus on them as they could serve as new targeted drugs.

The biological properties of ANIC-BP are referred to herein as ANIC-BP biological activity or ANIC-BP activity. The polypeptide of the invention exhibits at least one biological activity of ANIC-BP.

As polypeptides, the invention also includes variants of the above polypeptides, including allelic forms and cleavage variants. Such polypeptides differ from reference polypeptides by insertions, deletions, or substitutions, which may be conservative or non-conservative, or in any combination. Particularly preferred variants are those in which amino acids are substantially incorporated, substituted or omitted from 50 to 30, from 30 to 20, from 20 to 10 from 10 to 5, from 5 to 3, from 3 to 5 2 from 2 to 1 in any combination.

Preferred fragments of the polypeptides of the invention include an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID No = 2, or an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids truncated or deleted. amino acid sequence SEQ ID No = 2. Preferred are biologically active fragments that mediate the biological activity of ANIC-BP, including fragments having similar or improved activity or reduced undesirable activity. Also preferred are fragments that are antigenic or immunogenic in animals and especially in humans.

Fragments of the polypeptides of the invention can be used to produce corresponding full-length polypeptides by peptide synthesis: therefore, these variants can be used as intermediates to produce the full-length polypeptides of the invention. The polypeptides of the invention may be in the form of mature proteins or may be part of a larger protein such as a precursor or fusion protein. It is often advantageous to include an amino acid sequence that contains secretion or leader sequences, pro-sequences, sequences that aid in purification, for example, numerous histidine residues, e.g.

or an additional sequence for stability during recombinant production.

The polypeptides of the invention may be prepared by any suitable method, for example by isolation from naturally occurring sources, genetically engineered host cells displaying expression systems (broad infra), or by chemical synthesis, or a combination of such methods. Means for preparing such polypeptides are well known in the art.

• fc fc fc fc fc fc fc

9 9 fc · fc fc fc ··· 99 ··· 9999 99 999

The invention further relates to an ANIC-BP polypeptide. Such polypeptides include i = (a) an isolated polynucleotide comprising a polynucleotide sequence identical to 95%, 96%, 97%, 98% or 99% to the polynucleotide sequence of SEQ ID No-1,

Cb) an isolated polynucleotide comprising the polynucleotide of SEQ ID No: 1,

Cc) an isolated polynucleotide having a polynucleotide sequence identical to 95%, 96%, 97%, 98% or 99% to the polynucleotide of SEQ ID No = 1,

Cd) the isolated polynucleotide of SEQ ID No = 1,

Ce) an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide sequence identical to 95%, 96%, 97%, 98% or 99% to the polypeptide sequence of SEQ ID No ^; 2.

Cf) an isolated polynucleotide comprising a polynucleotide sequence encoding the polypeptide of SEQ ID No: 2,

Cg) an isolated polynucleotide having a polynucleotide sequence encoding a polypeptide sequence having at least 95%, 96%, 97%, 98% or 99% identity to the polypeptide sequence of SEQ ID No: 2,

Ch) an isolated polynucleotide encoding the polypeptide of SEQ ID No-2, Ci) an isolated polynucleotide having or comprising a polynucleotide sequence having an identity index of 0.95, 0.96, 0.97, 0.98 or 0.99, as compared to the polynucleotide sequence of SEQ. ID No: 1,

Cj) an isolated polynucleotide having or comprising a polynucleotide sequence encoding a polypeptide sequence having an identity index of 0.95, 0.96, 0.97, 0.98 or 0.99, as compared to the polypeptide sequence of SEQ ID No = 2, and polynucleotides that are fragments and variants of the aforementioned polynucleotides or are complementary thereto over their entire length.

· 4 99 9 · 4 99 9 · 99 4 4 · 4 · 9 <999 9

9 9 9 4

9494 44 499 4999 99 944

Preferred polynucleotide fragments of the invention include an isolated polynucleotide comprising a nucleotide sequence having at least 15, 30, 50, or 100 contiguous nucleotides of SEQ ID No: 1, or an isolated polynucleotide comprising a sequence having at least 30, 50, or 100 contiguous nucleotides cut or deleted from SEQ. ID No = 1.

Preferred variants of the polynucleotides of the invention include cleaved variants, allelic variants, and polymorphisms including polynucleotides having one or more single nucleotide polymorphisms (CSNPs).

Polynucleotides of the invention also include polynucleotides encoding polypeptide variants that comprise the amino acid sequences of SEQ ID No: 2 _and in which, for example, 50 to 30, 30 to 20, 20 to 10, 10 to 5, 5 to 3, 3 to 2, 2 to 2 1 or one amino acid residue substituted, deleted or added in any combination-

The invention further relates to polynucleotides that are transcripts of the RNA DNA sequence of the invention. Thus, the invention encompasses an RNA polynucleotide which:

Ca) comprises an RNA transcript of the DNA sequence encoding the polypeptide of SEQ ID NO: 2,

(Cb) the RNA transcript is a DNA sequence encoding the polypeptide of SEQ ID NO: 2,

Cc) comprises an RNA transcript of the DNA sequence of SEQ ID NO: 1, or Cd) is an RNA transcript of the DNA sequence of SEQ ID NO: 1 and polynucleotides complementary thereto.

The polynucleotide sequence of SEQ ID NO: 1 shows homology to Hym A CNozaki et al., DNA cell Biol. 15, pp. 505-509 (1996) and Mo25 (Karos et al., Mol. Gen Genet. 260, pp. 510-521, 1999). The polynucleotide sequence of SEQ ID NO: 1 is the cDNA sequence that encodes the polypeptide of SEQ ID NO: 2- The polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2, may be identical to the polypeptide encoding SEQ ID NO: 1, or may be a sequence other than SEQ ID NO: 1, which, as a result of the excess (degeneration) of the genetic code, also encodes the polypeptide of SEQ ID NO: 2. The polypeptide of SEQ ID NO = 2 is related to other proteins of the lime-binding family, since it has homologous and / or structural similarity to the Hym A and Mo25 proteins.

Preferred polypeptides and polynucleotides of the invention are expected to have, inter alia, similar biological functions / properties to their homologous polypeptides and polynucleotides. In addition, the polypeptides and polynucleotides of the invention have at least ANIC-BP activity.

Polynucleotides of the invention can be obtained using standard cloning and screening techniques from a cDNA library derived from human central nervous system mRNA cells (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, CoId Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). The polynucleotides of the invention may also be obtained from natural sources such as genomic DNA libraries, or may be synthesized using well known and commercially available techniques.

When the polynucleotides of the invention are used to recombinantly produce polypeptides of the invention, the polynucleotides may comprise coding sequences for mature polypeptides or coding sequences for mature polypeptides in reading frame with other coding sequences, such as sequences encoding a leader or secretory sequence, preprotein, proprotein or preproprotein sequences. or other fusion peptide moieties. For example, a marker sequence that facilitates purification of the fused polypeptides may be encoded. In some preferred embodiments of the invention, the marker sequence is a hexa-histidine peptide that is in the vector pQE (Qiagen, Inc) and which is 9 9 9 9 9 9 9 9

9 9 9 9

999,999 is described by Gentz et al. (Proc. Natl. Acad-Sci. USA 86: 821-824, 1989) or in the Ha tag. The polynucleotide may also contain non-coding sequences 5 and 3, such as transcribed, untranslated sequences, cleavage and polyadenylation signals, ribosome binding sites, and sequences that stabilize mRNA.

Polynucleotides that are identical or have sufficient identity to the polynucleotide sequences of SEQ ID NO = 1 may serve as hybridization probes for cDNA and genomic DNA, or as primers for a nucleic acid enhancement reaction (e.g., PCR). Such probes or primers may be used to isolate full-length cDNA or genomic clones encoding polypeptides of the invention and to isolate cDNAs and genomic clones of other genes (including genes encoding human and non-human paralogs) having high sequence similarity to SEQ ID NO = 1, typically at least 95% identity. Preferred probes and primers generally comprise at least 15 nucleotides, preferably at least 30 nucleotides, and may have at least 50, if not at least 100 nucleotides. Particularly preferred probes have 30 to 50 nucleotides. Particularly preferred primers have 20 to 25 nucleotides.

A polynucleotide encoding a polypeptide of the invention, including homologues from non-human species, can be obtained by a method comprising the steps of library screening and under stringent hydridization conditions with a labeled probe having the sequence of SEQ ID NO1 or fragment thereof, preferably at least 15 nucleotides; genomic clones comprising said polynucleotide sequence. Such hybridization techniques are well known in the art. Preferred hybridization stringent conditions include overnight incubation at 42 ° C in a solution containing 50% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhards reagent, 10% dextran sulfate and 20 micrograms / ml denatured salmon sperm DNA cut followed by washing

Thus, the invention also includes isolated polynucleotides, preferably having a nucleotide sequence of at least 100, obtained by screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NCB1 or a fragment thereof, preferably at least 15 nucleotides. '

It will be appreciated by those skilled in the art that in many cases the isolated cDNA sequence will be incomplete in that the polypeptide coding region does not extend over the entire end 5 pathway. This is due to reverse transcriptase, an enzyme with an inherently low processivity (which is a measure of the ability of the enzyme to remain attached to the matrix during the polymerization reaction), missing to complete copy of the cRNA matrix DNA during synthesis of the first cDNA strain.

There are a number of methods known to those skilled in the art to obtain full length cDNAs or to stretch short cDNAs, for example, methods based on the Rapid Amplification of RACE cDNAs (e.g., Frohman et al., Proc. Nat. Sci. USA 85 1988, 8998-9002). Recent modifications to the technique, for example with Marathon ™ technology (Clontech Laboratories Inc), have significantly simplified the search for longer cDNAs. By marathon ™ technology, cDNAs were prepared from mRNAs extracted from the selected tissue and adapter sequences bound to each end. Nucleic acid amplification (PCR) is then performed to amplify the missing 5 'end of the cDNA using a combination of gene-specific oligonucleotide primers and adapter. The PCR reaction is then repeated using nested primers, i.e. primers designed for thermal hybridization within the amplified product (typically an adapter-specific primer that anneals an additional 3 in the adapter sequence and a gene-specific primer that anneals an additional 5 in a known gene sequence) . The products of this reaction can then be analyzed by sequencing DNA and full-length cDNAs constructed by either attaching the product directly to an existing cDNA to obtain a full length cDNA. Υ »υ · 9 · · · 9 · · »» »· · · · · · · · · · · ·

Full length PCR using 5 primers.

sequence, or by performing a separate - <

new sequence information for construction

The recombinant polypeptides of the invention can be prepared by methods well known in the art from genetically engineered host cells containing expression systems. The invention further relates to expression systems comprising the polynucleotide and / or polynucleotides of the invention for host cells that are genetically engineered with such expression systems and further relates to. production of the polypeptides of the invention by recombinant techniques. Cells free of translation systems using RNA derived from the DNA constructs of the invention can also be used to produce such proteins.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for the polynucleotides of the invention. Polynucleotides can be introduced into host cells by methods described in many standard laboratory manuals (e.g., Davis et al., Basic Methods of Molecular Biology (1986) and Sambrook et al.). Preferred methods of introducing polynucleotides into host cells include, for example, kaleium phosphate transfection, DEAE-dextran mediated transfection, transection, microinjection, cationic lipid mediated transfection, electroporation, tranduction, diffusion, ballistic delivery, or infection.

Representative examples of suitable host cells are bacterial cells such as Streptococci, Staphylococci, E-coli, Streptomyces, and Bacillus subtilis cells: fungal cells such as yeast cells, and Aspergillus; insect cells such as Drosofila S2 and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells and plant cells.

Many expression systems can be used, for example chromo15

somal, episomal and virus-derived systems, for example vectors derived from bacterial plasmids, bacteridophage, transposons, yeast episomes, yeast chromosomal elements incorporated, viruses such as swine hunts, papovaviruses such as SV4C1, vacciniaviruses adenoviruses, smallpox viruses, pseudoraviruses and retroviruses, and vectors derived from combinations thereof, such as plasmid-derived vectors and bacteriophage genetic elements such as cosmids and phagemids. Expression systems may contain control regions that regulate and also produce expression. Generally, any system or vector that is capable of maintaining, extending or expressing a polynucleotide or polypeptide in a host may be used. A suitable polynucleotide sequence can be incorporated into the expression system by any well-known routine technique, such as that described by Sambrook, cited above. Appropriate secretion signals may be incorporated into the polypeptide of interest to allow secretion of the trans-soldered protein into the lumina of the endoplasmic reticulum, the periplasmic space, or the extracellular environment. These signals may be endogenous to the polypeptide or may be heterologous signals.

If the polypeptide of the invention is to be expressed for use in screening studies, it is generally desirable that the polypeptide be formed on the surface of the cell. For this purpose, the cells can be harvested before using the screening assay. When the polypeptide is secreted into the medium, the medium may be recovered to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before recovering the polypeptide

Polypeptides of the invention can be obtained and purified from recombinant cell cultures by known methods such as ammonium sulfate precipitation or ethanol, acid extraction, anion exchange or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is used for purification. A well known method of protein winding can be used to regenerate active conformation in the case of denatured polypeptides during intracellular synthesis, isolation and / or purification.

The polynucleotides of the invention can be used as diagnostic reagents to detect mutations in the associated gene. Detection of a mutated form of the gene, characterized by the polynucleotides of SEQ ID NO: ± in the cDNA or genomic sequence, and which is associated with dysfunction, provides a diagnostic pathway to contribute to or define a diagnosis of a disease or a suspected disease,

That is due to under-expression or over-expression or altered spatial or temporal expression of the gene. Individuals with mutations in genes can be detected at the DNA level by various techniques known in the art.

Nucleic acids for diagnosis may be obtained from cells of a subject such as blood, urine, saliva, tissue biopsy, or autopsy material. Genomic DNA can be used for detection directly, or it can be amplified enzymatically using PCR, preferably RT-PCR or other amplification methods prior to analysis. Similarly, RNA or cDNA can be used. Drain and incorporation can be detected by varying the amount of product amplification compared to the normal genotype. Point mutations can be identified by hybridizing the amplified DNA to the ANIC-BP labeled nucleotide sequences. Perfectly aligned sequences can be distinguished from misaligned duplexes by RNase digestion or differences in melting points. The difference in DNA sequence can also be detected by altering the electrophoretic mobility of DNA fragments in gels with or without denaturing agents, or by direct DNA sequencing (e.g., Myers et al., Science 230, 1242 (1985)). Sequence changes at specific sites can also be detected by nuclease protection assays such as RNase and SI protection by the method *, * 9 9 9 ♦

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Chemical cleavage (Cotton et al., Proc. Natl. Acad. Sci. USA 85: 4397-4401, 1985).

A number of oligonucleotide probes containing the ANIC-BP polynucleotide sequence or fragments thereof can be constructed to introduce efficient screening of, for example, genetic mutations. Such rows are preferably high density rows or grids. Methods of series technology are well known and generally used and can be used to pose a number of questions in molecular genetics, including gene expression, genetic linkage, and genetic variability (e.g., M. Chee et al., Science 274: 610-613, 1996). and references cited therein).

Detection of abnormally increased or decreased expression levels of polypeptides or mRNA can also be used to diagnose and determine a subject's susceptibility to the diseases of the invention. Reduced or overexpressed can be measured by RNA level using any method known in the art for quantifying polynucleotides, such as nucleic acid enhancers such as PCR, RT-PCR, RNase protection, Northern blotting, and other hybridization methods. Assessment techniques that can be used to determine the level of a protein, such as a polypeptide of the invention, in a host-derived sample are well known to those skilled in the art. Such assay methods include radioimmunoassays, competitive binding assays, Western Blot analysis, and ELISA.

The invention also relates to a diagnostic kit comprising:

(a) a polynucleotide of the invention, preferably a nucleotide sequence of SEQ ID No = 1 or a fragment or RNA transcript thereof, (b) a nucleotide sequence complementary to (a), (c) a polypeptide of the invention, preferably a polypeptide of SEQ ID No: 2; a fragment thereof, or (d) an antibody to the polypeptides of the invention, preferably to the poles 18 9 9 4 9 9 9 9 9 9 9 ·

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9 «44 ··· 99«

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99 to 99 of the peptide of SEQ ID NO: 2.

It is believed that in such a kit Ca), (b), Cc), or Cd) may contain a substantial component. Such kits are used in the diagnosis of a disease or riáchylnosati to a disease, particularly in the case of the diseases according to the invention.

The polynucleotide sequences of the invention are valuable for chromosome location studies. The sequence is specifically site-directed and can hybridize to a particular location of a single human chromosome. Mapping the relevant sequences to chromosomes of the invention is an important first step in correlating such sequences with gene-related diseases. Once the sequence has been mapped to the exact chromosome location, the physical position of that sequence on the chromosomes can be correlated with the genetic map data. Such data are published (for example, the book V-McKusick, Mendelian Inheritance in Man, available online via the John Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region is _of p and identifying linkage Cdědiěnost Analysis of physically adjacent genes). Accurate human chromosomal locations for the genomic sequence (gene fragment) can be determined by Radiation Hybrid (RH) Mapping (Valter M., Spillet-D-, Thopmas P., Veissenbach J. and Goodfellov P., A raethod for constructing radiation hybrid maps of whole genomes, Nature Genetics 7, pp. 22-28 (1994). A series of RH panels can be obtained from Reseach Genetics (Huntsville, AL., USA), for example, the RH-panel GeneBridge 4 (Hum Mol Genet, March 5 (3), pp. 339-346, 1996; A radiation hybrid map of the human). Gyapay G., Schmitt K., Fizames C., Jones H-, Vega-Czarny N., Spillett D., Muselet D., Prud Homme JF, Dib C., Auffray C., Moissette J., Veissenbach J -, Goodfell PN). To determine the chromosome location of the gene using this panel, 93 PCR was performed using primers determined from the respective gene on RH DNA. Each of these DNAs contains random

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human genomic fragments contained in the hamster environment (human / hamster hybrid cell lines). These PCRs yield 93 results indicating the presence or absence of the PC.R product of the gene of interest. These results are compared with those generated by the PCR product from genomic sequences of known position. This comparison is done at http://www.genome.wi.mit.edu.

The polynucleotide sequences of the invention are also valuable tools for tissue expression studies. Such studies make it possible to determine expression patterns of polynucleotides of the invention that can provide an indication as expression patterns of encoded polypeptides in tissues by detecting mRNAs encoding them. Methods used are well known in the art and include in-situ hybridization techniques to grid-aligned clones, such as cDNA microarray hybridization (Schena et al., Science 270, pp. 467-470, 1995 and Shalon et al., Genome Res 6, 639-645 (1996)) and nucleotide enhancement techniques such as PCR. The preferred method uses TAQMAN ™ technology (Perkin Elmer's commercial product). The results of these studies may provide an indication of the normal function of polypeptides in the body. In addition, comparative studies of a normal mRNA expression pattern with mRNA encoded by an alternative form of the same gene (e.g. having a change in polypeptide coding potential or regulatory mutation) can provide valuable insight into the role of the polypeptides of the invention or their overexpression in disease. temporal, spatial or simply quantitative in nature. The polypeptides of the invention are expressed in the human brain.

The invention further relates to antibodies. Polypeptides of the invention, or fragments thereof or cells expressing them, can be used as immunogens to generate antibodies that are immunospecific for the polypeptides of the invention. The term immunospecific means that the antibodies have substantially greater affinity for the polypeptide. and the house of the invention than their affinity for os20

other related polypeptides known in the art.

Against the substance generated against the polypeptide. of the invention, can be obtained by administering polypeptides or epitope-bearing fragments or cells to animals, preferably not humans, according to routine protocols. Any method that provides antibodies produced by continuous cell line cultures can be used to prepare monoclonal antibodies. Examples are the hybridoma technique (Kohler G. and Milstein C., Nature 256, 495-497, 1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4, 72, 1983) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R., Lis, Inc., 1985).

The techniques for producing single chain antibodies (described in U.S. Patent No. 4,941,778) can also be adapted to produce single chain antibodies to the polypeptides of the invention. Transgenic mice or other organisms, including other mammals, can also be used to express humanized antibodies.

The above-described antibodies can be used to isolate or identify expression clones. or to purify the polypeptides by affinity chromatography. Antibodies against polypeptides of the invention can also be used to treat the diseases of the invention.

The polypeptides and polynucleotides of the invention may also be used as a vaccine. Accordingly, the invention relates to a method of inducing an immunological response in a mammal comprising immunizing the mammal with a polypeptide of the invention at a dose appropriate to generate an antibody and / or immune response of T cells, including, for example, cytokine producing T cells or cytotoxic T cells to protect the mammal from disease, whether or not the individual has the disease.

Λ · toto toto toto toto toto toto toto toto toto toto to to (((((((((((((((((( · ♦ · · ♦ this · ··, ·· ·· this ·

Thus, an immune response in a mammal can be elicited by a method of delivering a polypeptide of the invention via a vector directed to express the polynucleotide and encoding the polypeptide in vivo to elicit an immune response to generate an antibody to protect the animal from the disease of the invention. One way of administering the vector is to accelerate it in the desired cells as a particle train or otherwise. Such a vector may comprise DNA, RNA and a modified nucleic acid or DNA / RNA hybrid. For use of the vaccine, a polypeptide or nucleic acid vector is normally provided as a vaccine formulation (composition). The formulation may further comprise a suitable carrier. Since the polypeptide may be disrupted in the stomach, it is preferably administered parenterally (e.g., by subcutaneous, intramuscular, intravenous or intradermal injection). Formulations suitable for parenteral administration include aqueous or non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats and solutes to render the formulation isotonic with the blood of the recipient; aqueous or non-aqueous sterile suspensions which may include suspending and thickening agents. The formulations may be administered in unit dose or multidose containers, for example, in sealed ampoules and vials, and may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier just prior to use. Vaccine formulations may also contain adjuvant systems to facilitate the immunogenicity of the formulation, such as oil / water systems and other systems known in the art. The dosage depends on the specific activity of the vaccine and can be determined by routine experimentation.

The polypeptides of the invention have one or more biological functions that are useful in one or more disease states, particularly those mentioned above. It is therefore useful to identify compounds that stimulate or inhibit the function or level of a polypeptide. Thus, the invention further relates to a screening method for identifying compounds that stimulate or inhibit the function or level of a polypeptide. Such methods

4 ·. Identify agonists and antagonists that can be used for therapeutic and prophylatic purposes for such diseases of the invention mentioned above. Compounds can be identified from a variety of sources, such as cells, cell-free preparations, chemical libraries, chemical compound collections, and natural product mixtures. Such agonists or antagonists thus identified may be natural or modified substrates, ligands, receptors and enzymes, whether polypeptides, structural or functional mimetics thereof (Coligan et al., Current Protocols in Immunology 1 (2) Chapter 5, 1991) or small molecule. The screening method may simply measure the binding of the compound of interest to the polypeptide or cells or membranes storing the polypeptide or its fusion protein by labeling directly or indirectly associated with the compound of interest. Alternatively, the screening method may comprise measuring or detecting (qualitatively or quantitatively) a competitively binding compound of interest to a polypeptide against a labeled competitor (e.g., an agonist or antagonist). These screening methods may further test whether the compound contemplated results in a signal generated by activation or inhibition of the polypeptide, using detection systems suitable for the polypeptide-storing cells. Activation inhibitors are generally assayed in the presence of a known agonist, and the effect on agonist activation in the presence of a cadidate compound is observed. Screening methods can simply consist of the steps of mixing the compound of interest with a solution containing the polypeptide of the invention to form a mixture, measuring the activity of the ANIC-BP mixture, and comparing the activity of the ANIC-BF 'mixture with a control mixture containing no compound of interest.

The polypeptide of the invention can be used in conventional low-throughput screening methods and also in high-throughput screening (HTS). Such bulk screening includes not only the established 96-well and more recently 384-well microtiter plates, but also more recent methods

·· • 99 9 • · »· • 9 9 ^: · • 9 '9 9 9 9 • · 9 9 9 • 9 9 9 9 9 9 9 · «·· • 9 999 9999 99 9 9

such as the method described by Schul lek et al. (Anal Biochem. 246, pp. 20-29 (1997)).

Fusion proteins, such as proteins from Fc particles and the above-described ANIC-BP polypeptide, can also be used for bulk screening to identify antagonists for a polypeptide of the invention (D. Bennett et al., J. Mol Recognition 8, pp. 52-52). 58, 1995, and K. Johanson et al., J. Biol Chem 270 (16), 9459-9471, 1995).

Skrinovaci techniky

Polynucleotides, polypeptides, and antibodies to a polypeptide of the invention can also serve to create screening methods to detect the effect of added compounds on mRNA and polypeptide production in cells. For example, an ELISA assay can be designed to measure secreted or cell-associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to detect agents that can inhibit or facilitate the production of a polypeptide (also called antagonists or agonists) from appropriately engineered cells or tissues.

The polypeptides of the invention can be used to identify membrane-bound or optionally soluble receptors by standard receptor binding techniques known in the art. Notwithstanding any limitation, such methods include grand binding and cross-linking assays in which a polypeptide is labeled with a radioisotope (e.g. ¹²⁵ J), chemically modified (e.g. biotinylated) or fused to a peptide sequence suitable for detection or purification and incubated with a source the putative receptor (cells, cell membranes, cellular susceptible ants, tissue extracts, body takutins). Other methods include biophysical techniques such as surface plasma resonance and spectroscopy. These screening methods are also «0 00 • 0 · · • <00 • 00 • 0 0 • 000 00 • 0 00 0 · ·» · ··

0 0 · ·· · 0 0 • 0 · 0

0000 00 000 may use to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its possible recipes. Standard methods for carrying out such assays are well known in the art.

Examples of antagonists of the polypeptides of the invention are antibodies or, in some cases, oligonucleotides or proteins that are closely related, for example, to ligands, substrates, receptors and polypeptide enzymes, for example, a ligand, substrate, receptor and enzyme fragment, or a small molecule that binds to the polypeptide of the invention, but does not elicit a response, thus preventing the activity of the polypeptide.

Screening methods may also include transgenic technology and the ANIC-BP gene. The method of construction of transgenic animals is well established. For example, the ANIC-BP gene can be introduced by microinjection into the male pronucleus of fertilized germ cells, by retroviral transfer to embryos before or after embryo implantation, or by injection of genetically modified, for example, electroporation, embryonic stem cells into host blastocytes. Particularly suitable transgenic animals are so-called knock-in animals, in which the animal gene is replaced by a human equivalent within the genome of the animal. Knock-in animals are useful in drug discovery processes, for targeted recovery, where the compound is specific to the human target. Other suitable animals are so-called knock-out animals, wherein the expression of the animal ortholog of the polypeptide of the invention and encoded by the endogenous DNA sequence in the cell is partially or totally annulled. The gene knockout may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a result of technology constraints, or may occur in all or substantially all cells of an animal. Transgenic animal technology also offers a complete animal expression cloning system in which the introduced genes are expressed to provide a large number of polypeptides of the invention.

··························

fcfc fc fc fc · · · · · · · · fc ·· fc

The invention also relates to screening kits for use in the methods described above. Such a kit comprises:

(a) a polypeptide of the invention, (b) a recombinant cell expressing a polypeptide of the invention, (c) a cell membrane expressing a polypeptide of the invention, (d) an agent against a polypeptide of the invention, ¹ wherein the polypeptide is preferably SEQ ID No: 2 It is understood that any such kit (a), (b), (c), or (d) may contain a substantial component.

Dictionary of terms

The following definitions are used to help you understand some of the terms that often occur here.

Antibodies include polyclonal and monoclonal antibodies, single chain chimeric and humanized antibodies, as well as fragments of Eabs, including Fab products or other immunoglobulin new expression libraries.

Isolated means changed by the human hand from its natural state, ie if it occurs in nature, it has been altered and / or removed from its original environment. For example, a polynucleotide or polypeptide naturally contained in a living organism is not isolated. but the same polynucleotide or polypeptide separated from coexisting materials of its natural state is isolated in the sense as used herein. In addition, a polynucleotide or polypeptide that is introduced into an organism by transformation, gene manipulation, or any other recombinant method is isolated, although it is still present in said living or inanimate organism.

Generally, a polynucleotide is any polyribonucleotide (RNA) or polydeoxyribonucleotide (DNA), which may be unmodified

or modified RNA or DNA. As a polynucleotide, for example, without limitation, single and double stranded DNA, DNA which is a mixture of single-stranded or double-stranded regions, single-stranded or double-stranded RNA, which is a mixture of single-stranded or double-stranded regions, hybrid molecules containing DNA and RNA, which may be single-stranded or more usually double-stranded or mixtures of single-stranded or double-stranded regions. In addition, the term polynucleotide means a triple strand region comprising RNA and / or DNA. The term polynucleotide also includes DNA or RNA comprising one or more modified bases and DNA or RNA with frameworks modified for stability or for other reasons. Modified bases include, for example, tritylated bases and unusual bases such as inosine. Many modifications can be made to DNA and RNA, so that the term polynucleotide includes chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells. The term polynucleotide includes relatively short polynucleotides, often referred to as oligonucleotides.

The term polypeptide refers to any polypeptide comprising two or more amino acids linked together by peptide bonds or modified peptide bonds, for example, peptide isosteres. A polypeptide refers to both short chains, generally called peptides, oligopeptides or oligomers, and longer chains, generally called proteins. Polypeptides may comprise amino acids other than the 20 genes of the encoded amino acid. Polypeptides include amino acid sequences modified by natural processes, such as post-translational processing or chemical modification techniques, which are well known in the art. Such modifications are described in basic texts and in more detailed monographs, as well as in extensive research literature. Modifications can occur anywhere in the polypeptide, including the peptide backbone, in amino acid side chains

.. • ί · · ·

and at the amino or carboxy termini. It will be appreciated that the same type of modification may be at the same or different degrees at multiple sites within a given polypeptide. A given polypeptide may also contain modifications of various types. The polypeptides may be branched ubiquitinations or may be cyclic with or without branching. Cyclic, branched and branched cyclic polypeptides may be the result of post-translation of natural processes or may be produced by synthetic methods. As a modification states acetylation, acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a proportion of heme, covalent attachment of a nucleotide or its derivative, covalent attachment of a lipid or a derivative, covalent attachment fosfotidy1 iNOS aqueous polymerization medium, crosslinking, ^cyclization: disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, cheap form, gamma-carboxylation, glycosylation, CPI anchorage, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulface, a transferred RNA-mediated addition of amino acids to proteins such as arginylation and ubiquitination (e.g., Proteins Structure and Molecular Properties 2nd edition, TECreighton, WH Freeman and Comp., New York 1993; Wold F. Post-translational Protein Modifications, Perspectives and Prospects, 1 to 12 in Post-translational Covalent Modification of Proteins, BC Johnson, ed., Academic Press, New York 1983; Seifter et al. Analysis for protein modifications and nonprotein cofactors Meth.Enzymol. 182: 626-646 (1990); and Rattan et al. Protein Synthesis; Post Translational Modifications and Aging Ann. NY Acad Sci. 663: 48-62 (1992).

A fragment of a polypeptide sequence is a sequence of polypeptides that is shorter than the reference sequence but that retains substantially the same biological function or activity as the reference polypeptide.

- ..

A fragment of a polynucleotide sequence is a polynucleotide sequence that is shorter than the reference sequence of SEQ ID NO = 1.

By variant is meant a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains substantially its properties. A typical variant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the polypeptide sequence encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and splicing of the polypeptide encoded by the reference sequence, as set forth below. A typical variant of a polypeptide differs in amino acid sequence from the reference polypeptide. In general, the changes are limited so that the sequences of the reference polypeptide and the variants are generally closely similar and are identical in many regions. The variant and the reference polypeptide may differ in amino acid sequence by one or more substitutions, incorporation or deletion in any combination. A substituted or incorporated amino acid residue may or may not be encoded by the genetic code. Typical conservative substitutions are Gly, Ala, Val,

Glu, Asn, Gln, Ser, Thr, Lys, Arg, and Phe, and Tyr.

the nucleotide or polypeptide may be naturally occurring as an allele, or may be a variant in nature not occurring. Non-naturally occurring variants of polynucleotides and polypeptides can be obtained by mutagenesis techniques or by direct synthesis. Also disclosed are polypeptides having one or more post-translational modifications, such as glycosylation, phosphorylation, methylation, ADP ribosylation. Inclusion includes methylation of the N-terminal amino acid, phosphorylation of a number of threonines, and modification of the C-terminal glycines.

Ile, Leu, Asp, polyAlela variants mean one or more alternative forms of a gene occurring at a given site in the genome.

Polymorphism refers to a change in the nucleotide sequence of the Ca encoded polypeptide sequence (if it is severe) at a given position in the genome within the population.

A single nucleotide polymorphism (SNP) refers to the occurrence of nucleotide variability at a single nucleotide position in a genome within a population. SNPs can occur within the gene or within the intergenic regions of the genome. SNP cases can be examined using the allele specific amplification (ASA). At least 3 primers are required for the process. A common primer in the reverse complement to the polymorphism under investigation is used. The common primer may be between 50 to 1500 base pairs from the polymorphic base. The other at least two primers are identical except that the 3 bases fluctuate to one of both or several alleles that produce the polymorphism. At least two PCR reactions are then performed on the DNA sample, each using a common primer and one of the allele specific primers.

As used herein, the term cleavage variant (CSplice Variant) refers to cDNA molecules formed from RNA molecules originally transcribed from the same genomic DNA sequence but which have undergone alternative RNA cleavage. Alternative RNA cleavage occurs when the primary RNA transcript is subjected to cleavage generally to remove introns, resulting in the production of more than one mRNA molecule, each of which can encode different amino acid sequences. The term cleavage variant also extends to proteins encoded by the above cDNA molecules.

Identity (Identity) means the relationship between two or more polypeptide sequences or between two or more polynucleotide sequences determined by sequence comparison. Generally, identity means that the nucleotide exactly matches the nucleotide, or the amino acid exactly matches the amino acid, the sequence of the polynucleotide exactly matches the sequence of the polynucleotide, and the sequence of the polypeptides corresponds exactly to the sequence of the polypeptides along the length of the sequences being compared.

Percentage of identity (¾ identity). For sequences that do not match exactly, the percent identity is determined. Generally, the two sequences to be compared are aligned so that there is a maximum correlation between the two sequences. This may involve inclusion in the gap in one or both sequences to facilitate the degree of alignment. Percent identity can be determined for the entire length of each of the aligned sequences (the so-called global alignement), which is particularly advantageous for sequences of the same or very similar length, or a shorter defined length (the so-called local alignement), which is better suited for unequal length sequences.

Similarity is another, more sophisticated measure of the relationship between two polypeptide sequences. Generally, the word similarity means a comparison between amino acids of two polypeptide chains based on residue to residue, respecting not only the exact correspondence between the residue pairs, comparing one of each sequence being compared (as for identity), but also where there is no exact correspondence on the developmental basis. the rest is a likely substitute for another. This probability has a common score from which the% similarity (¾ similarity) of both sequences can be determined

Methods for comparing the identity and similarity of two or more sequences are well known in the art. Thus, for example, the programs available in the Visconsin Sequence Analysis Package, version 9.1 (Devereux J. et al., Nucleic Acid Res. 12, pp. 387-395, 1984, a commercial product of Genetics Computer Group, Madison, Visconsin, USA). ), for example, the BESTFIT and GAP programs can be used to determine the% identity of two polynucleotides and the% identity and% similarity of two polypeptide sequences. BESTFIT uses the local homo31 algorithm

logie (local homology), developed by Smith and Waterman (J. Mol.Biol. 147: 195-197, 1981; Advances in Applied Mathematics 2, 1981; 482-489), and found no better separate area of similarity. between two sequences. The BESTFIT program is better suited to compare two non-length polypeptide-polypeptide or polypeptide sequences, and the program assumes that the shorter sequence is part of the longer sequence. When comparing GAP, it aligns the two sequences and finds the maximum similarity according to the algorithm of Neddleman and Vunch (J. Mol. Biol. 48, 443-453, 1970). GAP is better suited for comparing sequences of approximately the same length and alignment is expected along the entire length. Preferably, the Gap Veight and Lenght Veight parameters used in each program are 50 and 3 for polynucleotide sequences and 12 and 4 for polypeptide sequences. Preferably,% identity and% similarity are determined when the two sequences to be compared are optimally aligned.

Other programs to define identity and / or similarity between sequences are also known in the art, for example, the BLAST family of programs (Altschul SF et al., J. Mol. Biol. 215, 403-410, 1990; Altschul SF et al. Nucleic Acids Res., 25: 389-340 (1997), a commercial product of the National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA) and can be obtained from the NCBI website www.ncbi.nlm.nih. gov) and FASTA (Pearson VR, Methods in Enzymology 183, pp. 63-99, 1990; Pearson VR and Lipman DJ, Proc. Nat. Acad. Sci USA 85, pp. 2444-2448, 1988, the commercial product of Visconsin Sequence Analysis). Package).

Preferably, the amino acid substitution matrix (BLSSUM62) is used to compare polypeptide sequences (Henikoff S. and Henikoff JG, Proc. Nat. Acad. Sci. USA 89: 10915-10919, 1992) including when nucleotide sequences are translated into sequences before comparison. amino acids.

Preferably, the BESTFIT program is used to determine the percent identity of the polynucleotide or polypeptide sequence of interest relative to a reference polynucleotide or polypeptide sequence, wherein the α-reference sequence of interest is optimally aligned and the program parameters are set to a scarcity value as described above.

The identity index is a measure of sequence relatedness that can be used to compare the candidate sequence (polynucleotide or polypeptide) and the reference sequence. For example, a polynucleotide sequence having, for example, an identity index of I / 95 compared to a reference sequence is identical to a reference sequence, with the candidate polynucleotide sequence having, on average, up to 5 differences per second. every 100 nucleotides of the reference sequence. Such differences are selected from the group consisting of at least one deletion, substitution, including transfer and t-ransversion or nucleotide incorporation. These differences can occur at the 5 and 3 end positions of the reference polynucleotide sequence, or anywhere between these end positions, whether individually between nucleotides in the reference sequence or in one or more adjacent groups within the reference sequence. Thus, to obtain a polynucleotide sequence with an identity index of 0.95 as compared to a reference polynucleotide sequence, on average up to 5 of each 100 nucleotides in the reference sequence may be deleted, replaced or incorporated, or any combination thereof, as mentioned above. The same applies mutatis mutandis to other identity index values, for example 0.96, 0.97, 0.98 and 0.99.

Similarly, for a polypeptide, the candidate polypeptide sequence having, for example, an identity index of 0.95 as compared to the reference polypeptide sequence, is identical to the reference sequence, with the candidate polypeptide sequence having on average up to 5 differences for every 100 amino acids of the reference sequence. Such differences are selected from the group consisting of at least one deletion, substitution, including conservative and non-conservative amino acid substitution or incorporation. These differences may occur at the amino or carboxyl end position of the reference polypeptide sequence or anywhere between these end positions, regardless of whether individually between amino acids in the reference sequence or in one or more continuous groups within the reference sequence. Thus, to obtain a polypeptide sequence having an identity index of 0.95 as compared to a reference polypeptide sequence, on average up to 5 of each 100 amino acids in the reference sequence may be deleted, replaced or incorporated, or any combination thereof, as previously described. The same applies mutatis mutandis to other identity index values, for example 0.96, 0.97, 0.98 and 0.99.

The relationship between the number of nucleotides or amino acid differences and the identity index can be expressed by the following equation: our Xa - (x - I), where the number of nucleotides or different amino acids x and the total number of nucleotides in SEQ ID NQ: 1 or amino acids in SEQ ID NO: 2

Even the identity index to the closest number before subtraction from XaHomolog is a generic term used in the art to indicate that a polynucleotide or polypeptide sequence has a high degree of sequence relatedness to a reference sequence. Such affinity may be expressed by the degree of identity and / or similarity between the two sequences as defined above. This generic term also includes ortholog and paralog. Orthological is a polynucleotide or polypeptide that is functionally equivalent to a polynucleotide or polypeptide in other species. Paralogic is a polynucleotide or polypeptide that is functionally similar within the same species.

A fusion protein is a protein that is encoded by two unrelated fusion genes or fragments thereof. Examples are given in US 5541087, US 5726044, EP 574395, EP 493418 and EP 0232262. In the case of Fc-ANIC-BP using the Fc immunoglobulin region as part of a fusion protein, it is preferable to perform functional expression of Fc-ANIC-BP to improve pharmacokinetic properties Such a fusion protein, when used to treat and form a dimeric Fc-ANIC-BP. The Fc-ANIC-BP construct means direction 5 and 3, a secretion cassette, that is, a signal sequence that triggers export from mammalian cells, DNA encoding the immunoglobulin FC region fragment as a fusion partner, and DNA encoding Fc-ANIC-BP. In some uses, the ability to alter intrinsic functional properties (complement binding, Fc-receptor binding) would be desirable by mutating functional Fc sites while retaining the remainder of the fusion protein intact or deleting the Fc portion. All Publications and References, including the cited patents and patent applications, are incorporated herein by reference as if the individual publication or reference were specifically and individually indicated and incorporated by reference. Any application to which this application claims priority is also indicated.

List of figures in drawings

Fig. 1 Representative mRNA differential display gel with a differently regulated band in the cerebral hemisphere opposite the lesion side. The arrow indicates the differentially expressed band.

4R1: injured

4R2 = injured sham treated rats

4L1 = uninjured side-treated rats

Giant. 2 RT-PCR with a 270 bp Mo25 rat brain fragment in TB after TBI at cycles 5, 10, 15, 20, 25 and 30. There is strong expression in the L2 band for cycles 20, 25 and 30.

L2- · cDNA rat TBI intact side; R2-´cDNA TBI rats injured side.

Giant. 3 Analysis Dot. blot with radiolabel ³² P 270 bp of the Mo25 rat gene fragment with rat brain cerebral mRNA after TBI. Six individual rats, 3 TBI (1 to 3) and 3 apparently operated animals (4 to 6) were tested.

Giant. 4 Multiple tissue blots (Clontech Laboratories Inc, Palo Alto, CA, USA) A 270 bp ³² P-radiolabeled gene fragment in Mo25 rats hybridized to 8 rat tissues.

Giant. Hybridization of rat brain sections. Probes with sense and no sense specific to SICCBP and Mo25 are used. Non-sense probes are used to monitor strong signals that light up myelinated nerve pathways (Corpus Callos, Tractus opticus, Tractus aolfactorius Intermedius, cerebellum commissura anterior).

Giant. 6 Expression of TaqMan PCR ANIC-NP in rat at real time 7 days after traumatic brain injury. The uninjured cortical side (the first two columns), the injured cortical side (the middle columns) and the small brain (the last columns) in the seemingly operated rat brains and the brain tissue of rats sacrificed 7 days after the experiment are compared. Error lines indicate the standard error of the mean (S.E.M.). ANIC-BP expression after brain injury. The x-axis shows various parts of the brain, the y-axis shows ng ANIC-BP mRNA. An empty square means seemingly operated, a full square means 7 days after head injury.

The invention is illustrated, but not limited, by the following examples.

the benefits of practical implementation.

DETAILED DESCRIPTION OF THE INVENTION

AND

Example 1

Brain injury model

Identification of upstream and downstream regulated genes in response to traumatic brain injury (TBI) in rats using the lateral fluid method is studied. In male Sprague-Davley rats, mild brain damage (TBI) directed to the right parietal cortex is induced by liquid shock. Five days after the injury, the rats are sacrificed and the removed brain is analyzed in different ways. Protein re-regulation in the cerebellum of traumatized brains is confirmed by RT-PCR.

Control rats are anesthetized and the temporal muscle is removed, but no eraniotomy is performed. After 5 days of survival, all rats are anesthetized, sacrificed, and the brain removed and frozen in liquid nitrogen.

A second series of TBI rats was used for histochemical and immunohistochemical staining. One week after TBI, rats are anesthetized and perfused with brine followed by 3% paraformaldehyde. Brains were cut by freezing microtome into 30 µm coronal sections.

Example 2

Immunohistochemistry

Brain sections are labeled with monoclonal antibodies against a calcium binding protein. The immune complex is visualized by the avidin-biotin / DAB method as a positive control

Calbindin-D (28kD) as a Reliable Marker of Brain Purkifia Cells.

Example 3

In-situ hybridization

The oligodeoxynucleotides selected from SEQ ID NO: 1 (sense and non sense) are labeled according to standard methods known to those skilled in the art. These probes are used to in situ hybridization in rat brain sections by methods known to those skilled in the art, the flutoradiograms being visualized after 5 days of exposure to the naphilm Kodak BioMax.

Example 4

Isolation of RNA from TBI rats

A differential display of mRNA has been developed as a method to identify and analyze altered gene expression at the mRNA level in any eukariotic cell (Liang and Pardee, Science 257, 967, 1992). Herein, this method is used to study genes regulated up and down in response to traumatic brain injury, to gain a better insight into the molecular effects of CNS damage (Day Daas et al., Meeting of the American Neuroseience Society Washington DC USA, 1998). The animal model for traumatic brain injury is called the Lateral Liquid Percussion Model performed as follows ^: Mild traumatic brain damage directed to the right parietal cortex is performed by the Lateral Liquid Percussion method in male SpragueDawley rats. Control rats are anesthetized and the temporal muscle is removed, but no craniotomy is performed. After 5 days of survival, all rats are anesthetized, sacrificed, and the brain removed and frozen in liquid nitrogen. Whole brains are homogenized and whole RNA is isolated (Sambrook et al. 1989).

fc fc fc fc fc fcfc fcfc fcfc fcfc

Differential display

The RNA obtained is analyzed by differential RNA display. The reverse transcript of mRNA was performed using the oligo-dT primer with two additional nucleotides in all possible combinations (sense ^primer: 13 mere), thus anchored to the start of the reaction completion. poles (A). Enhancement of the cDNA was performed with the same primer 3 and the second decameric primer 5 (upstream primer). The amplified products are analyzed on a non-denaturing 10¾ polyacrylamide gel (Amersham Pharmacia Biotech, Germany). DNA is visualized with silver staining. After staining, the gels were dried at room temperature for 1 hour (Fig. 1). Differentially regulated bands are excised from the gel. DNA was eluted, re-amplified, and subcloned into pCR2.1 vector (Invitrogen, USA). Subcloned fragments are sequenced by the Sanger method (Sanger F. et al., PNAS, USA 74, pp. 5463-5467) and compared to genomic databases. Evaluation of the obtained gene fragment is performed by reverse transcription PCR (RT-PCR). To confirm differentially expressed rat Mo25, the sequence was analyzed by RT-PCR using the Titan one tube RT-PCR system (Boehringer Mannheim, Germany) with specific 19 mere and 21 mere primers. One µg of RNA from control and TBI animals were used for RT-PCR. Multiple genes are evaluated by dotblot technique. with probes from the cover Mo25 and Northern blot analysis performed with probes from

of human Mo25.

Reverse PCR transcript (RT-PCR)

To confirm the differentially expressed, stroke-induced calcium-binding protein, it was analyzed by RT-PCR using Titan One Tube RT-PCR (Boehringer Mannheim, Germany) with specific 19 mere and 21 mere primers. One pg of total RNA from control and TBI animals was used for RT-PCR.

Real-time PCR ·· ·· • · ·

The distribution of ANIC-BP in rat brain after head trauma is examined by the Real Time TaqMan PCR technique in the prism of the ABI detection system with 7700 sequences (CPE, Applied Biosystems, Germany). In this way, absolute concentrations of mRNA with high sensitivity can be measured. Special primers of 25 and 29 bp in length and 32 mere TaqMan probe (reporter staining-FAM / quencher dye-TAMRA) were determined.

Binding of Ca ^2- * ·

Protein were separated by SDS-PAGE was blotted on nitrocellulose membranes and examined for binding of radiolabelled Ca ^₂₊ method described by Maruyama, K. et al. (J. Biochem., 1984, 95, 511-519). After incubation, the excess isotope is washed off and the membrane exposed to Kodak XOMAT-TM film for a suitable time. A band is formed at the binding protein site. The presence of the binding protein was confirmed using a binding protein specific antibody and the application of that antibody by Western blotting, well known in the art.

Industrial applicability

A protein for the manufacture of compositions for the treatment and prevention of disorders related to protein expression and for inhibiting or activating the effect of polynucleotides and polypeptides.

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Sequence list <110> Merck Patent GmbH <120> Acute neural calcium binding protein <130> ANIC3PJDDWS <140>

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<160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 1026 <212> DNA <213> Homo sapiens <220>

<221> CDS <222> (1) .. (1026) <400> 1

atg Met 1 ccg ttc Pro Phe ccg ttt ggg aag Lys tet Ser cac aaa tet approx. gca Pro Ala gac att Asp Ile 15 Dec gtg Wall 48 For Phe 5 Gly His Lys Ser aag aat ctg aag gag age atg get gtt ctg gaa aag caa gac att tet 96 Lys Asn Leu Lys Glu Ser Met Ala Wall Leu Glu Lys Gin Asp Ile Ser 20 May 25 30 gat aaa £ 53 gca gaa aag get aca gaa gaa gtt tcc aaa aat ctg gtt 144 Asp Lys Lys Ala Glu Lys Ala Thr Glu Glu Wall Ser Lys Asn Leu Wall 35 40 45 gcc atg aaa gaa att ctg melt ggc aca aat gaa aaa gag cct cag aca 192 Ala Met Lys Glu Ile Leu Tyr Gly Thr Asn Glu Lys Glu For Gin Thr 50 55 60 gaa gca gta get caa ctt get caa gaa ctc melt aat agt ggg ctc ctt 240 Glu Ala Wall Ala Gin Leu Ala Gin Glu Leu Tyr Asn Ser Gly Leu Leu 65 70 75 80 age acc ctg gta get gat tta cag ctc att gac ttt gag ggc aaa aaa 288 Ser Thr Leu Wall Ala Asp Leu Gin Leu Ile Asp Phe Glu Gly Lys Lys 85 90 95 gac gtg get caa att ttc aac aat att ctc aga aga caa att ggt acg 336 Asp Wall Ala Gin Ile Phe Asn Asn Ile Leu Arg Arg Gin Ile Gly Thr 100 ALIGN! 105 110 aga act cct act gtt gaa tray atc tgc acc caa cag aat att ttg ttc 384 Arg Thr For Thr Wall Glu Tyr Ile Cys Thr Gin Gin Asn Ile Leu Phe 115 120 125 atg tta ttg aaa ggg melt gaa tet approx gaa ata get cta aat tgt gga 432 Met Léu Leu Lys Gly Tyr Glu Ser For Glu Ile Ala Leu Asn Cys Gly 130 135 140

• ·

ata Ile 145 atg Met tta Leu aga Arg gaa Glu tgc Cys 150 atc aga cat gaa Glu approx For 155 ctt Leu gca Ala aaa atc att 480 Lys Ile Ile 160 ttg tgg tcg gaa cag ttt tat gat ttc ttc aga melt gtc gaa atg tca 528 -Leu Trp Ser- Glu- Gin- 165 Phe 'Tyr' Asp Pne • PKe ¹⁷⁰ Arg Tyr ’ Wall" Glu Met 175 Ser aca ttt gac ata act tca gat gca ttt gcc aca ttc aag gat tta ctt 576 Thr Phe Asp Ile 180 Ála Ser Asp Ala Phe Ala Thr Phe Lys Asp 190 Leu Leu aca aga cat 333 ttg ctc ag gca gaa ttt ttg gaa cag cat melt gat 624. Thr Arg His 195 Lys Leu Leu Ser Ala Glu Phe Leu Glu Gin 205 His Tyr Asp aga ttt ttc agt gaa melt gag aag tta ctt cat tca gaa aat melt gtg 672 . ^Ar 9. Phe 210 Phe _t Ser Glu Tyr Glu-Lys-Leu Leu His Ser 220 Glu Asn Tyr Wall aca aaa aga cag tca ctg aag ctt ctc ggt gaa cta cta cta aat aga 720 Thr 225 Lys Arg Gin Ser Leu 230 Lys Leu Leu Gly Glu 235 Leu Leu Leu Asp Arg 240 cac aac ttc aca 5’t atg aca aaa tac atc agt aaa cct gag aac ctc 768 His Asn Phe Thr Ile 245 Met Thr Lys Tyr Ile 250 Ser Lys For Glu Asn 255 Leu aaa tta atg atg aac ctg ctg ega gac aaa agt cgc aac atc cag ttt 816 Lys Leu Met Met 260 Asn Leu Leu Arg Asp 265 Lys Ser Arg Asn Ile 270 Gin Phe gag gcc ttt cac gtt ttt aag gtg ttt gta gcc aat cct aac aag acg 864 Glu Ala Phe 275 His Wall Phe Lys Val Phe 280 Wall Ala Asn For 285 Asn Lys Thr cag ccc atc cta gac atc ctc ctc aag aac cag gcc aaa ctc ata gag 912 Gin For 290 Ile Leu Asp Ile Leu Leu Lys Asn Gin Ala 300 Lys Leu Ile Glu ttc ctc age aag ttt cag aac gac agg acg gag gat gag cag ttt aac 960 Phe 305 Leu Ser Lys Phe Gin 310 Asn Asp Arg Thr Glu 315 Asp Glu Gin Phe Asn 320 gac gag aag acc melt tta gtt aaa cag atc agg gat ttg aag aga approx 1008 Asp Glu Lys Thr Tyr Leu Gin Ile Arg Asp Leu Lys Arg For

325 330 335 get a cag caa gaa get aa Ala Gin Gin Glu Ala

340

1026

<210 2 <211> 341 <212> PRT <213> Homo sapiens

Met 1 For Phe For Phe 5 Gly Lys Ser His Lys 10 Ser Pro Ala Asp Ile 15 Dec Wall Lys Asn Leu Lys Glu Ser Met Ala Wall Leu Glu Lys Gin Asp Ile Ser 20 May 25 30 Asp Lys Lys Ala Glu Lys Ala Thr Glu Glu Wall Ser Lys Asn , Leu Wall 35 40 45 Ala Met Lys Glu Ile Leu Tyr Gly Thr Asn Glu Lys Glu For Gin Thr 50 55 60 Glu Ala Wall Ala Gin Leu Ala Gin Glu Leu Tyr Asn Ser Gly Leu Leu 65 70 75 80 Ser Thr Leu Wall Ala Asp Leu Gin Leu Ile Asp Phe Glu Gly Lys Lys 85 90 95 Asp Wall Ala Gin Ile Phe Asn Asn Ile Leu Arg Arg Gin Ile Gly Thr 100 ALIGN! 105 110 Arg Thr For Thr Wall Glu Tyr Ile Cys Thr Gin Gin Asn Ile Leu Phe 115 120 125 Met Leu Leu Lys Gly Tyr Glu Ser For Glu Ile Ala Leu Asn Cys Gly 130 135 140 Ile Met Leu Arg Glu Cys Ile Arg His Glu For Leu Ala Lys Ile Ile 145 150 155 160 Leu Trp Ser Glu Gin Phe Tyr Asp Phe Phe Arg ' Tyr Wall Glu Met Ser 165 170 175 Thr Phe Asp Ile Ala Ser Asp Ala Phe Ala Thr Phe Lys Asp Leu Leu 180 185 190 Thr Arg His Lys Leu Leu Ser Ala Glu Phe Leu Glu Gin His Tyr Asp 195 200 205 Arg Phe Phe Ser Glu Tyr Glu Lys Leu Leu His Ser Glu Asn Tyr Wall 210 215 220 Thr Lys Arg Gin Ser Leu Lys Leu Leu Gly Glu Leu Leu Leu Asp Arg 225 230 235 240 His Asn Phe Thr Ile Met Thr Lys Tyr Ile Ser Lys For Glu Asn Leu 245 250 255 Lys Leu Met Met Asn Leu Leu Arg Asp Lys Ser Arg Asn Ile Gin Phe 260 265 270 Glu Ala Phe His Wall Phe Lys Wall Phe Wall Ala Asn For Asn Lys Thr 275 280 285 Gin For Ile Leu Asp Ile Leu Leu Lys Asn Gin Ala Lys Leu Ile Glu 290 295 300 Phe Leu Ser Lys Phe Gin Asn Asp Arg Thr Glu Asp Glu Gin Phe Asn 305 310 315 320 Asp Glu Lys Thr Tyr Leu Wall Lys Gin Ile Arg A.sp Leu Lys Arg For

(+420) 325 330 335

Ala Gin Gin Glu Ala 340? (/ W - VW »/ 1½

Claims (11)

PATENT CLAIMS 1- An isolated polypeptide selected from the group consisting of (a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO ^{: 1} , (b) an isolated polypeptide comprising a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID No = 2; Cc) An isolated polypeptide having at least 95% identity to the polypeptide sequence of SEQ ID No = 2, Cd) the polypeptide sequence of SEQ ID No = 2, Ce) fragments and variants of such polypeptides according to (a) to Cd). The isolated polypeptide of claim 1 comprising the sequence of the polypeptides of SEQ ID NO = 2 The isolated polypeptide of claim 1, which is a polypeptide of SEQ ID NO = 2. An isolated polynucleotide selected from the group consisting of Ca) an isolated polynucleotide comprising a polynucleotide sequence having at least 95% identity to the polynucleotide sequence of SEQ ID No ^: 1, Cb) an isolated polynucleotide having at least 95% identity to the polynucleotide of SEQ ID No = 1, Cc) an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to SEQ ID No = 2, Cd) an isolated polynucleotide having a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to SEQ ID No = 2, Ce) an isolated polynucleotide having a nucleotide sequence of at least 100 nucleotides obtained by screening the library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID No = 1, or a fragment thereof with at least 15 nucleotides, F) a polynucleotide whose RNA is equivalent to polynucleotide (a) to (e), or a polynucleotide sequence complementary to the isolated polynucleotide and polynucleotides that are variants and fragments of said polynucleotides or that are complementary to said polynucleotides over their entire length. The isolated polynucleotide of claim 4 selected from the group consisting of ¹ (a) an isolated polynucleotide comprising a polynucleotide of SEQ ID NQ: 1, (b) an isolated polynucleotide of SEQ ID NCh1, (c) an isolated polynucleotide comprising a polynucleotide sequence encoding the polypeptide of SEQ ID NO = 2 and Cd) an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 2. An expression system comprising a polynucleotide capable of producing the polypeptide of claim 1, contained in a compatible host cell. A recombinant host cell comprising the expression vector of claim 6 or a membrane expressing the polypeptide of claim 1. A method of producing a polypeptide according to claim 1, having the step of culturing the host cell as defined in claim 7 under conditions sufficient to produce the polypeptide of claim 1 and recovering the polypeptide from the culture medium. A fusion protein consisting of an immunoglobulin Fc region and any polypeptide of claim 1. The antibody immunospecific for the polypeptide of any one of claims 1 to 3. ♦ · ·· · 4 · '44 '4 4 4 44 44 4 < • ·· 4 4 * · • 444 4,444 4 4 4 4 4 4 4 4 ···· 44 4 4 * 4444 « 11. A screening method for identifying compounds that stimulate or inhibit the function or level of polypeptides of claim 1, wherein (a) measuring or determining, qualitatively or quantitatively, binding of the candidate compound to the polypeptides or cells or membranes expressing the polypeptide or its fusion protein by labeling directly or indirectly associated with the candidate compound, (Cb) measuring competition for binding of the candidate compound to the polypeptide or to cells or membranes expressing the polypeptide or its fusion protein in the presence of a labeled competitor; Cc) it is tested whether the candidate compound results in a signal generated by activation or inhibition of the polypeptides, * using a detection system appropriate to the cells or cell membranes expressing the polypeptide, Cd) mixing the candidate compound with a solution comprising the polypeptide of claim 1 to form a mixture, measuring the activity of the polypeptides in the mixture, and comparing the activity with a control mixture that does not contain a candidate compound, (Ce) determining the effect of the candidate compound on the production of mRNA encoding the polypeptide or polypeptide in cells, using, for example, an ELISA assay; and Cf) the compound is produced by standard biotechnological or chemical methods.