WO2001009166A1 - Octoray, a g-protein coupled receptor - Google Patents

Abstract

Octoray polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing Octoray polypeptides and polynucleotides in diagnostic.

Description

OCTORAY, A G-PROTEIN COUPLED RECEPTOR

Field of the Invention

This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use m diagnosis and identifying compounds that may be agonists, antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.

Background of the Invention

The drug discovery process is currently undergoing a fundamental revolution as it embraces "functional genomics", that is, high throughput genome- or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly superseding earlier approaches based on "positional cloning" A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position

Functional genomics relies heavily on high-throughput DNA sequencing technologies and the vaπous tools of biomformatics to identfy gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and charactense further genes and their related polypeptides/protems, as targets for drug discovery.

It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 1991 , 351:353-354). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins. Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamme (Kobilka, B.K., et al., Proc Natl Acad. Sci., USA, 1987, 84:46-50; Kobilka, B.K., et al, Science, 1987, 238:650-656; Bunzow, J.R , et al , Nature, 1988, 336-783-787), G-protems themselves, effector proteins, e.g., phosphohpase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kmase A and protein kmase C (Simon, M I., et al., Science, 1991, 252:802-8) For example, m one form of signal transduction, the effect of hormone binding is activation of the enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide, GTP. GTP also influences hormone binding. A G-protem connects the hormone receptor to adenylate cyclase G- protein was shown to exchange GTP for bound GDP when activated by a hormone receptor The GTP- carrymg form then binds to activated adenylate cyclase Hydrolysis of GTP to GDP, catalyzed by the G-protem itself, returns the G-protem to its basal, inactive form. Thus, the G-protem serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal

The membrane protein gene superfamily of G-protem coupled receptors has been characteπzed as having seven putative transmembrane domains. The domains are believed to represent transmembrane -hehces connected by extracellular or cytoplasrmc loops. G-protem coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.

G-protem coupled receptors (otherwise known as 7TM receptors) have been charactenzed as including these seven conserved hydrophobic stretches of about 20 to 30 ammo acids, connecting at least eight divergent hydrophilic loops. The G-protem family of coupled receptors includes dopamine receptors which bmd to neuroleptic drugs used for treating psychotic and neurological disorders Other examples of members of this family include, but are not limited to, calcitonm, adrenergic, endothelm, cAMP, adenosme, muscannic, acetylchohne, serotonin, histamine, thromb , kmin, follicle stimulating hormone, opsins, endothehal differentiation gene-1, rhodopsms, odorant, and cytomegalovirus receptors

Most G-protem coupled receptors have single conserved cysteme residues m each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated m signal transduction. Phosphorylation and hpidation (palmitylation or farnesylation) of cysteme residues can influence signal transduction of some G-protem coupled receptors. Most G-protem coupled receptors contain potential phosphorylation sites within the third cytoplasrmc loop and/or the carboxy terminus. For several G-protem coupled receptors, such as the β- adrenoreceptor, phosphorylation by protein kmase A and/or specific receptor kinases mediates receptor desensitization. For some receptors, the ligand binding sites of G-protem coupled receptors are believed to compnse hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains, said sockets being surrounded by hydrophobic residues of the G-protem coupled receptors The hydrophilic side of each G-protem coupled receptor transmembrane helix is postulated to face inward and form a polar ligand binding site. TM3 has been implicated in several G-protem coupled receptors as having a ligand bmdmg site, such as the TM3 aspartate residue TM5 sennes, a TM6 asparagine and

TM6 or TM7 phenylalanmes or tyrosmes are also implicated in ligand binding

G-protem coupled receptors can be lntracellularly coupled by heterotπmenc G-protems to vaπous mtracellular enzymes, ion channels and transporters (see, Johnson et al , Endoc Rev , 1989, 10:317-331). Different G-protem α-subumts preferentially stimulate particular effectors to modulate vaπous biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protem coupled receptors has been identified as an important mechanism for the regulation of G-protem couplmg of some G-protem coupled receptors. G-protem coupled receptors are found m numerous sites within a mammalian host.

Over the past 15 years, nearly 350 therapeutic agents targeting 7 transmembrane (7 TM) receptors have been successfully introduced onto the market.

Summary of the Invention

The present invention relates to Octoray, in particular Octoray polypeptides and Octoray polynucleotides, recombmant mateπals and methods for their production. Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to infections such as bacteπal, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pam; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoπs, myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, dehπum, dementia, and severe mental retardation; and dyskmesias, such as Huntmgton's disease or Gilles dela Tourett's syndrome hereinafter referred to as "diseases of the invention." In a further aspect, the invention relates to methods for identifying agonists and antagonists (e.g , inhibitors) using the mateπals provided by the invention, and treating conditions associated with

Octoray imbalance with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with mappropπate Octoray activity or levels.

Description of the Invention

In a first aspect, the present invention relates to Octoray polypeptides. Such polypeptides include:

(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%>, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2,

(c) an isolated polypeptide comprising the polypeptide sequence of SEQ ID NO:2; (d) an isolated polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID N0 2,

(e) the polypeptide sequence of SEQ ID NO:2; and (f) an isolated polypeptide having or compπsmg a polypeptide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ ID NO:2;

(g) fragments and variants of such polypeptides m (a) to (f).

Polypeptides of the present invention are believed to be members of the 7-TM (G-Protem Coupled) family of polypeptides. They are therefore of interest because 7-TM receptors are proven to be an important therapeutic target for drug discovery.

The biological properties of the Octoray are hereinafter referred to as "biological activity of Octoray" or "Octoray activity." Preferably, a polypeptide of the present invention exhibits at least one biological activity of Octoray. Polypeptides of the present invention also mcludes vaπants of the aforementioned polypeptides, including all allehc forms and splice vaπants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative, or any combination thereof. Particularly preferred vanants are those in which several, for mstance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acids are inserted, substituted, or deleted, m any combination.

Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids from the ammo acid sequence of SEQ ID NO: 2, or an isolated polypeptide compπsmg an ammo acid sequence having at least 30, 50 or 100 contiguous ammo acids truncated or deleted from the ammo acid sequence of SEQ ID NO: 2. Preferred fragments are biologically active fragments that mediate the biological activity of Octoray, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also preferred are those fragments that are antigenic or lmmunogenic in an animal, especially in a human.

Fragments of the polypeptides of the invention may be employed for producing the coπespondmg full-length polypeptide by peptide synthesis; therefore, these vaπants may be employed as intermediates for producing the full-length polypeptides of the invention. The polypeptides of the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional ammo acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance multiple histidine residues, or an additional sequence for stability during recombinant production.

Polypeptides of the present invention can be prepared in any suitable manner, for mstance by isolation form naturally occurring sources, from genetically engineered host cells compπsmg expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesizers, or a combination of such methods. Means for preparing such polypeptides are well understood in the art.

In a further aspect, the present invention relates to Octoray polynucleotides. Such polynucleotides include- (a) an isolated polynucleotide comprising a polynucleotide sequence having at least 95%, 96%, 97%,

98%, or 99% identity to the polynucleotide sequence of SEQ ID NO: 1 ;

(b) an isolated polynucleotide compπsmg the polynucleotide of SEQ ID NO: 1 ,

(c) an isolated polynucleotide having at least 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide of SEQ ID NO.1 , (d) the isolated polynucleotide of SEQ ID NO: 1 ;

(e) an isolated polynucleotide compπsmg 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;

(f) an isolated polynucleotide compπsmg a polynucleotide sequence encodmg the polypeptide of SEQ ID NO:2, (g) 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;

(h) an isolated polynucleotide encoding the polypeptide of SEQ ID NO:2;

(I) an isolated polynucleotide having or comprising a polynucleotide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0 99 compared to the polynucleotide sequence of SEQ ID NO. l, 0) an isolated polynucleotide having or comprising a polynucleotide sequence encoding a polypeptide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ ID NO:2; and

(k) polynucleotides that are fragments and vaπants of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides, over the entire length thereof. Preferred fragments of polynucleotides of the present mvention include an isolated polynucleotide comprising an nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID NO 1 , or an isolated polynucleotide comprising an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ED NO. 1 Preferred vaπants of polynucleotides of the present invention include splice vaπants, allehc variants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).

Polynucleotides of the present mvention also include polynucleotides encodmg polypeptide vaπants that compπse the ammo acid sequence of SEQ ID NO:2 and m which several, for mstance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 ammo acid residues are substituted, deleted or added, in any combination.

In a further aspect, the present mvention provides polynucleotides that are RNA transcπpts of the DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that:

(a) comprises an RNA transcπpt of the DNA sequence encoding the polypeptide of SEQ ID NO:2;

(b) is the RNA transcπpt of the DNA sequence encoding the polypeptide of SEQ ID NO:2;

(c) comprises an RNA transcπpt of the DNA sequence of SEQ ID NO: 1 ; or (d) is the RNA transcπpt of the DNA sequence of SEQ ID NO: 1 ; and RNA polynucleotides that are complementary thereto.

The polynucleotide sequence of SEQ ID NO:l shows homology with KIAAOOOl (Nomura N., Miyajima N., Sazuka T, Tanaka A, Kawarabayashi Y, Sato S, Nagase T, Seki N, Ishikawa K, Tabata S 1994. DNA Res. 1, 27-35). The polynucleotide sequence of SEQ ID NO:l is a cDNA sequence that encodes the polypeptide of SEQ ED NO:2. The polynucleotide sequence encoding the polypeptide of

SEQ ID NO:2 may be identical to the polypeptide encodmg sequence of SEQ ID NO: 1 or it may be a sequence other than SEQ ED NO: 1, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2. The polypeptide of the SEQ ID NO:2 is related to other protems of the 7-TM family, having homology and/or structural sirmlaπty with KIAAOOOl (Normura N, Miyajima N, Sazuka T, Tanaka A, Kawarabayashi Y, Sato S, Nagase T, Seki

N, Ishikawa K, Tabata S 1994. DNA Res. 1, 47-56).

Preferred polypeptides and polynucleotides of the present invention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one Octoray activity

Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library deπved from mRNA in cells of human placenta and testis, (see for mstance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spπng Harbor, N.Y. (1989)). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbraπes or can be synthesized using well known and commercially available techniques. When polynucleotides of the present invention are used for the recombinant production of polypeptides of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide m reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- prote sequence, or other fusion peptide portions. For example, a marker sequence that facilitates puπfication of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and descπbed in Gentz et al, Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcπbed, non-translated sequences, splicing and polyadenylation signals, πbosome bmdmg sites and sequences that stabilize mRNA.

Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ID NO: 1 , may be used as hybπdization probes for cDNA and genomic DNA or as pπmers for a nucleic acid amplification reaction (for instance, PCR). Such probes and pπmers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present mvention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similaπty to SEQ ID NO.1 , typically at least 95% identity. Preferred probes and pπmers will generally compπse at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred pπmers will have between 20 and 25 nucleotides.

A polynucleotide encoding a polypeptide of the present invention, mcludmg homologs from species other than human, may be obtained by a process compπsmg the steps of screening a library under stπngent hybπdization conditions with a labeled probe having the sequence of SEQ ID NO" 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybπdization techniques are well known to the skilled artisan. Preferred stπngent hybπdization conditions include overnight incubation at 42°C in a solution compπsmg: 50% formamide, 5xSSC (150mM NaCl, 15mM tπsodium citrate), 50 mM sodium phosphate (pH 7 6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters m 0 lx SSC at about 65°C. Thus the present invention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screenmg a library under stπngent hybπdization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides.

The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, m that the region coding for the polypeptide does not extend all the way through to the

5' terminus This is a consequence of reverse transcπptase, an enzyme with inherently low "processivity" (a measure of the ability of the enzyme to remain attached to the template duπng the polymerisation reaction), failing to complete a DNA copy of the mRNA template duπng first strand cDNA synthesis. There are several methods available and well known to those skilled m the art to obtain full- length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman et al., Proc Nat Acad Sci USA 85, 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon (trade mark) technology (Clontech Laboratoπes Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon (trade mark) technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the cDNA using a combination of gene specific and adaptor specific ohgonucleotide pπmers. The PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal withm the amplified product (typically an adaptor specific pπmer that anneals further 3' m the adaptor sequence and a gene specific pπmer that anneals further 5' in the known gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full- length PCR using the new sequence information for the design of the 5' pπmer. Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells compπsmg expression systems. Accordingly, m a further aspect, the present invention relates to expression systems compπsmg a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems and to the production of polypeptides of the invention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs deπved from the DNA constructs of the present mvention

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Polynucleotides may be introduced into host cells by methods descπbed in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al.(ibid). Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection. Representative examples of appropriate hosts include bacterial cells, such as Streptococci,

Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

A great variety of expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g. , vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression.

Generally, any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropriate polynucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, (ibid). Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.

If a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before the polypeptide is recovered.

Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and/or purification. Polynucleotides of the present mvention may be used as diagnostic reagents, through detecting mutations in the associated gene. Detection of a mutated form of the gene characteπzed by the polynucleotide of SEQ ID NO.1 in the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations m the gene may be detected at the DNA level by a vaπety of techniques well known in the art.

Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy mateπal. The genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques pnor to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in compaπson to the normal genotype. Pomt mutations can be identified by hybπdizmg amplified DNA to labeled Octoray nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence difference may also be detected by alterations m the electrophoretic mobility of DNA fragments in gels, with or without denatuπng agents, or by direct DNA sequencing (see, for instance, Myers et al , Science (1985) 230: 1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cotton et al , Proc Natl Acad Sci USA (1985) 85: 4397-4401). An array of oligonucleotides probes compπsmg Octoray polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g , genetic mutations. Such arrays are preferably high density arrays or gπds Array technology methods are well known and have general applicability and can be used to address a vaπety of questions in molecular genetics including gene expression, genetic linkage, and genetic vaπabihty, see, for example, M.Chee et al., Science, 274, 610- 613 (1996) and other references cited therein.

Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for mstance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample deπved from a host are well-known to those of skill m the art Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Thus in another aspect, the present invention relates to a diagnostic kit comprising:

(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment or an RNA transcript thereof;

(b) a nucleotide sequence complementary to that of (a), (c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or

(d) an antibody to a polypeptide of the present mvention, preferably to the polypeptide of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others

The polynucleotide sequences of the present mvention are valuable for chromosome localisation studies. The sequence is specifically targeted to, and can hybπdize with, a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first step m correlating those sequences with gene associated disease Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendehan Inheπtance in Man (available on-lme through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (co- mheπtance of physically adjacent genes). Precise human chromosomal localisations for a genomic sequence (gene fragment etc.) can be determined using Radiation Hybrid (RH) Mapping (Walter, M. Spillett, D., Thomas, P., Weissenbach, J., and Goodfellow, P., (1994) A method for constructing radiation hybπd maps of whole genomes, Nature Genetics 7, 22-28). A number of RH panels are available from Research Genetics (Huntsville, AL, USA) e g. the GeneBπdge4 RH panel (Hum Mol

Genet 1996 Mar;5(3):339-46 A radiation hybπd map of the human genome. Gyapay G, Schmitt K, Fizames C, Jones H, Vega-Czarny N, Spillett D, Muselet D, Prud'Homme JF, Dib C, Auffray C, Moπssette J, Weissenbach J, Goodfellow PN). To determine the chromosomal location of a gene using this panel, 93 PCRs are performed using primers designed from the gene of interest on RH DNAs Each of these DNAs contains random human genomic fragments maintained in a hamster background (human / hamster hybrid cell lines) These PCRs result in 93 scores indicating the presence or absence of the PCR product of the gene of interest These scores are compared with scores created using PCR products from genomic sequences of known location. This compaπson is conducted at http://www.genome.wi.mit.edu/.

The polynucleotide sequences of the present mvention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides m tissues, by detecting the mRNAs that encode them. The techniques used are well known in the art and include m situ hydπdisation techniques to clones arrayed on a gπd, such as cDNA microarray hybπdization (Schena et al, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques such as PCR. A preferred method uses the TAQMAN (Trade mark) technology available from Perk Elmer. Results from these studies can provide an indication of the normal function of the polypeptide in the organism. In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene (for example, one having an alteration m polypeptide coding potential or a regulatory mutation) can provide valuable insights into the role of the polypeptides of the present invention, or that of inappropnate expression thereof in disease. Such mappropπate expression may be of a temporal, spatial or simply quantitative nature.

A further aspect of the present mvention relates to antibodies. The polypeptides of the invention or their fragments, or cells expressmg them, can be used as lmmunogens to produce antibodies that are lmmunospecific for polypeptides of the present invention. The term "lmmunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides the pnor art.

Antibodies generated against polypeptides of the present mvention may be obtained by administering the polypeptides or epitope-beaπng fragments, or cells to an animal, preferably a non- human animal, using routine protocols. For preparation of monoclonal antibodies, any techmque which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybπdoma technique (Kohler, G. and Milstem, C, Nature (1975) 256:495-497), the tπoma technique, the human B-cell hybπdoma technique (Kozbor et al , Immunology Today (1983) 4:72) and the EBV- hybπdoma technique (Cole et al , Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985). Techniques for the production of single chain antibodies, such as those descπbed m U.S. Patent

No 4,946,778, can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgemc mice, or other organisms, including other mammals, may be used to express humanized antibodies The above-descπbed antibodies may be employed to isolate or to identify clones expressing the polypeptide or to punfy the polypeptides by affinity chromatography. Antibodies against polypeptides of the present invention may also be employed to treat diseases of the invention, amongst others.

Polypeptides and polynucleotides of the present invention may also be used as vaccines. Accordingly, in a further aspect, the present invention relates to a method for inducing an lmmunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established withm the individual or not. An lmmunological response m a mammal may also be induced by a method compπses dehveπng a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an lmmunological response to produce antibody to protect said animal from diseases of the invention. One way of admmisteπng the vector is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may compπse DNA, RNA, a modified nucleic acid, or a DNA/RNA hybπd. For use a vaccine, a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition). The formulation may further comprise a suitable carrier. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or mtradermal injection). Formulations suitable for parenteral administration include aqueous and non- aqueous steπle injection solutions that may contain anti-oxidants, buffers, bacteπostats and solutes that render the formulation mstonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dπed condition requiπng only the addition of the steπle liquid earner immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the lmmunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine expeπmentation.

Polypeptides of the present invention have one or more biological functions that are of relevance in one or more disease states, m particular the diseases of the invention herembefore mentioned. It is therefore useful to identify compounds that stimulate or inhibit the function or level of the polypeptide. Accordingly, m a further aspect, the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function or level of the polypeptide Such methods identify agonists or antagonists that may be employed for therapeutic and prophylactic purposes for such diseases of the mvention as hereinbefore mentioned. Compounds may be identified from a vaπety of sources, for example, cells, cell-free preparations, chemical hbraπes, collections of chemical compounds, and natural product mixtures. Such agonists or antagonists so-identified may be natural or modified substrates, gands, receptors, enzymes, etc., as the case may be, of the polypeptide, a structural or functional mimetic thereof (see Cohgan et al , Current Protocols m Immunology l(2):Chapter 5 (1991)) or a small molecule. Such small molecules preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules.

The screening method may simply measure the bmdmg of a candidate compound to the polypeptide, or to cells or membranes beaπng the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve measuring or detecting (qualitatively or quantitatively) the competitive binding of a candidate compound to the polypeptide against a labeled competitor (e.g agonist or antagonist). Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropπate to the cells beaπng the polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Further, the screening methods may simply compπse the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring a Octoray activity in the mixture, and compaπng the Octoray activity of the mixture to a control mixture which contains no candidate compound.

Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats. Such HTS formats include not only the well-established use of 96- and, more recently, 384- well micotiter plates but also emerging methods such as the nanowell method described by Schullek et al, Anal Biochem., 246,

20-29, (1997)

Fusion proteins, such as those made from Fc portion and Octoray polypeptide, as hereinbefore descπbed, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995), and K Johanson et al , J Biol Chem, 270(16) 9459-9471 (1995))

An EST (W79920) was identified from the public database as a possible 7TM receptor Pπmers were designed against the 5' and the 3' region of this EST and were used to screen the PAPA pacenta and testis cDNA braπes Positive clones were identified and sequenced. Two clones which contain the full length gene were further analyzed The polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells. For example, an ELISA assay may be constructed for measuπng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known m the art. This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.

A polypeptide of the present invention may be used to identify membrane bound or soluble receptors, if any, through standard receptor bmdmg techniques known the art. These include, but are not limited to, ligand binding and crosslmkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, ^^1), chemically modified (for instance, biotmylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art.

Examples of antagonists of polypeptides of the present invention include antibodies or, in some cases, ohgonucleotides or proteins that are closely related to the hgands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g , a fragment of the hgands, substrates, receptors, enzymes, etc.; or a small molecule that bind to the polypeptide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented.

Screening methods may also involve the use of transgenic technology and Octoray gene. The art of constructing transgenic animals is well established. For example, the Octoray gene may be introduced through microinjection into the male pronucleus of fertilized oocytes, retroviral transfer into pre- or post-implantation embryos, or injection of genetically modified, such as by electroporation, embryonic stem cells into host blastocysts. Particularly useful transgenic animals are so-called "knock- " animals in which an animal gene is replaced by the human equivalent within the genome of that animal. Knock-m transgenic animals are useful in the drug discovery process, for target validation, where the compound is specific for the human target. Other useful transgenic animals are so-called "knock-out" animals in which the expression of the animal ortholog of a polypeptide of the present invention and encoded by an endogenous DNA sequence m a cell is partially or completely annulled The gene knock-out may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all, cells m the animal. Transgenic animal technology also offers a whole animal expression-clonmg system in which introduced genes are expressed to give large amounts of polypeptides of the present invention

Screening kits for use m the above described methods form a further aspect of the present invention. Such screening kits comprise:

(a) a polypeptide of the present invention;

(b) a recombinant cell expressing a polypeptide of the present invention;

(c) a cell membrane expressing a polypeptide of the present invention; or

(d) an antibody to a polypeptide of the present invention; which polypeptide is preferably that of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may compπse a substantial component.

Glossary

The following definitions are provided to facilitate understanding of certain terms used frequently hereinbefore.

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeπc, single chain, and humanized antibodies, as well as Fab fragments, including the products of an

Fab or other lmmunoglobulm expression library.

"Isolated" means altered "by the hand of man" from its natural state, i e., if it occurs in nature, it has been changed or removed from its oπgmal environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present m said organism, which organism may be living or non-living.

"Polynucleotide" generally refers to any polyπbonucleotide (RNA) or polydeoxπbonucleotide (DNA), which may be unmodified or modified RNA or DNA. "Polynucleotides" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tπtylated bases and unusual bases such as inosme. A vaπety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteπstic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as ohgonucleotides. "Polypeptide" refers to any polypeptide compπsing two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally refeπed to as proteins. Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids "Polypeptides" include ammo acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well descπbed in basic texts and m more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the ammo acid side-chams and the ammo or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-πbosylation, amidation, biotmylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a hpid or hpid derivative, covalent attachment of phosphotidyhnositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as argmylation, and ubiquitmation (see, for instance, Proteins - Structure and Molecular Properties, 2nd Ed , T E Creighton, W H Freeman and Company, New York, 1993, Wold, F., Post-translational Protein Modifications Perspectives and Prospects, 1-12, m Post-translational Covalent Modification of Proteins, B C Johnson, Ed , Academic Press, New York, 1983; Seifter et al , "Analysis for protem modifications and nonprotem cofactors", Meth Enzymol, 182, 626-646, 1990, and Rattan et al , "Protein Synthesis: Post-translational Modifications and Agmg", Ann NY Acad Sci, 663, 48-62, 1992).

"Fragment" of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. "Fragment" of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ID NO: 1.

"Vaπant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof. A typical vaπant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of the vaπant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical vaπant of a polypeptide differs m ammo acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the vaπant are closely similar overall and, m many regions, identical. A variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, insertions, deletions m any combination. A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe and Tyr. A variant of a polynucleotide or polypeptide may be naturally occurring such as an allele, or it may be a vaπant that is not known to occur naturally Non-naturally occurπng vaπants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis Also included as vaπants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation, ADP πbosylation and the like. Embodiments include methylation of the N-termmal ammo acid, phosphorylations of seπnes and threonmes and modification of C-terminal glycmes

"Allele" refers to one of two or more alternative forms of a gene occurπng at a given locus in the genome

"Polymorphism" refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position m the genome withm a population

"Single Nucleotide Polymorphism" (SNP) refers to the occurrence of nucleotide vaπabihty at a single nucleotide position in the genome, withm a population. An SNP may occur withm a gene or withm mtergemc regions of the genome SNPs can be assayed using Allele Specific Amplification (ASA) For the process at least 3 pπmers are required. A common primer is used m reverse complement to the polymorphism being assayed. This common primer can be between 50 and 1500 bps from the polymorphic base. The other two (or more) pπmers are identical to each other except that the final 3' base wobbles to match one of the two (or more) alleles that make up the polymorphism. Two (or more) PCR reactions are then conducted on sample DNA, each using the common primer and one of the Allele Specific Primers.

"Splice Vaπant" as used herein refers to cDNA molecules produced from RNA molecules initially transcπbed from the same genomic DNA sequence but which have undergone alternative RNA splicing. Alternative RNA splicing occurs when a primary RNA transcπpt undergoes splicing, generally for the removal of mtrons, which results in the production of more than one mRNA molecule each of that may encode different ammo acid sequences. The term splice variant also refers to the proteins encoded by the above cDNA molecules.

"Identity" reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by compaπng the sequences. In general, identity refers to an exact nucleotide to nucleotide or ammo acid to ammo acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared.

"% Identity" - For sequences where there is not an exact correspondence, a "% identity" may be determined In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.

"Similarity" is a further, more sophisticated measure of the relationship between two polypeptide sequences. In general, "similaπty" means a comparison between the ammo acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated "score" from which the "% similarity" of the two sequences can then be determined.

Methods for compaπng the identity and similarity of two or more sequences are well known in the art Thus for instance, programs available m the Wisconsin Sequence Analysis Package, version 9 1 (Devereux J et al, Nucleic Acids Res, 12, 387-395, 1984, available from Genetics Computer Group, Madison, Wisconsin, USA), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % similaπty between two polypeptide sequences BESTFIT uses the "local homology" algoπthm of Smith and Waterman (J Mol Biol, 147,195-197, 1981, Advances in Applied Mathematics, 2, 482- 489, 1981) and finds the best single region of similarity between two sequences. BESTFIT is more suited to compaπng two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer. In compaπson, GAP aligns two sequences, finding a "maximum similaπty", according to the algoπthm of Neddleman and Wunsch (J Mol Biol, 48, 443-453, 1970). GAP is more suited to compaπng sequences that are approximately the same length and an alignment is expected over the entire length. Preferably, the parameters "Gap Weight" and "Length Weight" used m each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively. Preferably, % identities and similaπties are determined when the two sequences being compared are optimally aligned

Other programs for determining identity and/or similaπty between sequences are also known in the art, for instance the BLAST family of programs (Altschul S F et al, J Mol Biol, 215,

403-410, 1990, Altschul S F et al, Nucleic Acids Res., 25:389-3402, 1997, available from the National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA and accessible through the home page of the NCBI at www.ncbi.nlm.mh.gov) and FASTA (Pearson W R, Methods in Enzymology, 183, 63-99, 1990; Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85, 2444-2448,1988, available as part of the Wisconsin Sequence Analysis Package).

Preferably, the BLOSUM62 ammo acid substitution matnx (Hemkoff S and Hemkoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into ammo acid sequences before comparison Preferably, the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a reference polynucleotide or a polypeptide sequence, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore descπbed

"Identity Index" is a measure of sequence relatedness which may be used to compare a candidate sequence (polynucleotide or polypeptide) and a reference sequence. Thus, for instance, a candidate polynucleotide sequence having, for example, an Identity Index of 0.95 compared to a reference polynucleotide sequence is identical to the reference sequence except that the candidate polynucleotide sequence may include on average up to five differences per each 100 nucleotides of the reference sequence Such differences are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion. These differences may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups withm the reference sequence. In other words, to obtain a polynucleotide sequence having an Identity Index of 0.95 compared to a reference polynucleotide sequence, an average of up to 5 in every 100 of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described. The same applies mutatis mutandis for other values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99. Similarly, for a polypeptide, a candidate polypeptide sequence having, for example, an

Identity Index of 0 95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 ammo acids of the reference sequence. Such differences are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion. These differences may occur at the ammo- or carboxy- terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the ammo acids m the reference sequence or in one or more contiguous groups withm the reference sequence. In other words, to obtain a polypeptide sequence having an Identity Index of 0.95 compared to a reference polypeptide sequence, an average of up to 5 m every 100 of the ammo acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore descπbed. The same applies mutatis mutandis for other values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99.

The relationship between the number of nucleotide or ammo acid differences and the Identity Index may be expressed in the following equation: n_a < x_a - (x_a • I),

in which. n_a is the number of nucleotide or ammo acid differences,

x_a is the total number of nucleotides or ammo acids in SEQ ID NO: 1 or SEQ ID NO.2, respectively,

I is the Identity Index , • is the symbol for the multiplication operator, and m which any non-mteger product of x_a and I is rounded down to the nearest integer pπor to subtracting it from x_a

"Homolog" is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence Such relatedness may be quantified by determining the degree of identity and/or similaπty between the two sequences as hereinbefore defined Falling withm this geneπc term are the terms "ortholog", and "paralog" "Ortholog" refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species "Paralog" refers to a polynucleotide or polypeptide that within the same species which is functionally similar "Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example, EP-A-0 464 533-A discloses fusion proteins compπsmg vaπous portions of constant region of immunoglobulin molecules together with another human protein or part thereof In many cases, employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokmetic properties [see, e g , EP-A 0232 262] On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and punned

All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth Any patent application to which this application claims pπoπty is also incorporated by reference herein in its entirety in the manner descπbed above for publications and references

Claims

What is claimed is:

1 An isolated polypeptide selected from the group consisting of

(a) an isolated polypeptide encoded by a polynucleotide compπsmg the sequence of SEQ ID NO: 1 ; (b) an isolated polypeptide compnsing a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO:2;

(c) an isolated polypeptide compπs g the polypeptide sequence of SEQ ID NO:2;

(d) an isolated polypeptide having at least 95% identity to the polypeptide sequence of SEQ ED NO:2;

(e) the polypeptide sequence of SEQ ID NO.2; and (f) fragments and vanants of such polypeptides m (a) to (e).

2. An isolated polynucleotide selected from the group consistmg of:

(a) an isolated polynucleotide compnsing a polynucleotide sequence having at least 95% identity to the polynucleotide sequence of SEQ ED NO: 1 ; (b) an isolated polynucleotide compπsmg the polynucleotide of SEQ ID NO: 1 ;

(c) an isolated polynucleotide having at least 95% identity to the polynucleotide of SEQ ID NO: 1 ;

(d) the isolated polynucleotide of SEQ ID NO: 1 ;

(e) an isolated polynucleotide compπsmg a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO:2; (f) an isolated polynucleotide compnsing a polynucleotide sequence encodmg the polypeptide of SEQ

JJ3 NO:2;

(g) an isolated polynucleotide having a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO:2,

(h) an isolated polynucleotide encodmg the polypeptide of SEQ ID NO:2; (I) an isolated polynucleotide with a nucleotide sequence of at least 100 nucleotides obtained by screenmg a library under stπngent hybπdization conditions with a labelled probe having the sequence of SEQ ID NO. 1 or a fragment thereof having at least 15 nucleotides,

(j) a polynucleotide which is the RNA equivalent of a polynucleotide of (a) to (I), and (k) a polynucleotide sequence complementary to said isolated polynucleotide and polynucleotides that are vaπants and fragments of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides, over the entire length thereof.

3. An antibody lmmunospecific for the polypeptide of claim 1.

4 An antibody as claimed in claim 3 which is a polyclonal antibody.

5. An expression vector compnsing a polynucleotide capable of producing the polypeptide of claim 1 when said expression vector is present in a compatible host cell.

6 A process for producing a recombinant host cell which compnses the step of introducing an expression vector compπsmg a polynucleotide capable of producing the polypeptide of claim 1 into a cell such that the host cell, under appropπate culture conditions, produces said polypeptide.

7 A recombinant host cell produced by the process of claim 6

A membrane of the recombinant host cell of claim 7 expressing said polypeptide.

9. A process for producing a polypeptide which compnses cultuπng the host cell of claim 8 under conditions sufficient for the production of said polypeptide and recoveπng the polypeptide from the culture