WO1999036521A1 - A gene homologous to fox sperm acrosomal protein fsa-1 (cbcald05) - Google Patents

Abstract

CBCALD05 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CBCALD05 polypeptides and polynucleotides in the design of protocols for the treatment of infertility and diseases related to fertility, among others, and diagnostic assays for such conditions.

Description

A Gene Homologous to Fox Sperm Acrosomal Protein FSA-1 (CBCALD05)

FIELD OF INVENTION

This invention relates to newly identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to the gene family containing acrosomal protein, hereinafter referred to as CBCALD05. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

Te fox FSA-1 is a membrane protein, located on the acrosomal membrane, and it li^ several partners in many mammals, and it may involve in fsperm activity and/or fertil ation. The novel human candidate for this protein has little homology to the known human sperm acrosomal proteins. This indicates that the Gene family containing acrosomal protein has an established, proven history as therapeutic targets. Clearly there is a need for identification and characteri2a.tion of further members of the Gene family containing acrosomal protein which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, infertility and diseases related to fertility.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to CBCALD05 polypeptides and recombinant materials and methods for their production. Mother aspect of the invention relates to methods for using such CBGALD05 polypeptides and polynucleotides. Such uses include the treatment of infertility and diseases related to fertility, among others. In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with CBCALD05 imbalance with the identified compounds. Yet another aspect of the invention relates to diagnoΛic assays for detecting diseases associated with inappropriate CBCALD05 activity or levels.

DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided to facilitate understanding of certain terms used frequently herein. "CBCALD05" refers, among others, generally to a polypeptide having the ammo acid sequence set fo.rth in SEQ ID NO 2 or an allehc variant thereof

"CBCALD05 activity or CBG\LD05 polypeptide activity" or "biological activity of the

CBCALD05 or CBGALD05 polypeptide" refers to the metabolic or physiologic function of said CBCALD05 including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigemc and lmmunogemc activities of said CBCALD05

"CBCALD05 gene" refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 1 or allehc variants thereof and/or their complements

"Antibodies" as used herem mcludes polyclonal and monoclonal antibodies, chimenc, single cham, and humamzed antibodies, as well as Fab fragments, mcludmg the products of an Fab or other immunoglobulin expression library

"Isolated" means altered "by the hand of man" from the natural state If an "isolated" composition or substance occurs m nature, it has been changed or removed from its original environment, or both For example, a polynucleotide or a polypeptide naturally present in a living .animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting mateπals of its natural state is "isolated", as the teπn is employed herem

"Polynucleotide" generally refers to any polyπbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA 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, hybnd molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions In addition, "polynucleotide" refers to triple-stranded regions compnsing RNA or DNA or both RNA and DNA The term polynucleotide also mcludes 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 vanety of modifications has been made to DNA and RNA, thus, "polynucleotide" embraces chemically, enzymatically or metabo cally modified foπns of polynucleotides as typically found m nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells "Polynucleotide" also embraces relatively short polynucleotides, often referred to as ohgonucleotides

"Polypeptide" refers to any peptide or protem compnsing 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 chams, commonly refeιτed to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred 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 posttranslational processing, or by chemical modification techniques which are well known m the art Such modifications are well descnbed in basic texts and in more detailed monographs, as well as m a voluminous research literature Modifications can occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chains and the ammo or carboxyl termini It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites m a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods Modifications mclude acetylation, acylation,

amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deπvative, covalent attachment of a hpid or hpid denvative, covalent attachment of phosphotidyhnositol, cross-lmkmg, 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, mynstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to protems such as argrnylation, and ubiquitination See, for instance, PROTEINS - STRUCTURE , ND MOLEC UL.AR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York, 1993 and Wold, F , Posttranslational Protein Modifications Perspectives and Prospects, pgs 1-12 in

POSTTI NSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press. New York, 1983, Seifter et al ,

for protein modifications and nonprotem cofactors", Meth Enzymol (1990) 182 626-646 and Rattan et al , "Protein Synthesis Posttranslational Modifications and Aging", Ann NYAcadSci (1992) 663 48-62 "Vanant" as the teπn is used herem, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical vanant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the variant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions, .fusions and tnmcations in the polypeptide encoded by the reference sequence, as discussed below A typical vanant of a polypeptide differs in ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the vanant are closely similar overall and, in many regions, identical A variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions. deletions m any combination A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code A vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc vanant, or it may be a vanant that is not known to occur naturally Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis

"Identity" is a measure of the identity of nucleotide sequences or ammo acid sequences In general, the sequences are aligned so that the highest order match is obtained "Identity" per se has an art-recognized meaning and can be calculated using published techniques See, e g (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A M , ed , Oxford University Press, New York, 1988, BIOCOMPUTING INFORMATICS AND GENOME PROJECTS, Smith, D W , ed , Academic Press, New York, 1993, COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Gnffin, A M , and Gnffin, H G , eds , Humana Press, New Jersey, 1994, SEQUENCE .ANALYSIS IN MOLECULAR BIOLOGY, von Heiηje, G , Academic Press, 1987, and SEQUENCE ANALYSIS PRIMER Gnbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991) While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Canllo, H , and Lipton, D , SIAM J Applied Math (1988) 48 1073) Methods commonly employed to determine identity or similanty between two sequences mclude, but are not limited to, those disclosed m Guide to Huge Computers, Martin J Bishop, ed , Academic Press, San Diego, 1994, and Canllo, H , and Lipton, D , SLAM J Applied Math (1988) 48 1073 Methods to determine identity and similanty are codified in computer programs Preferred computer program methods to deteπrnne identity and similanty between two sequences include, but are not limited to, GCS program package (Devereux, J , et al , Nucleic Acids Research (1984) 12(1) 387), BLASTP, BLASTN, FASTA (Atschul, S F et al , JMolec Biol (1990) 215 403) As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO 1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO 1 In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence

Similarly, by a polypeptide having an ammo acid sequence having at least, for example, 95% "identity" to a reference ammo acid sequence of SEQ ID NO 2 is intended that the ammo acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may mclude up to five ammo acid alterations per each 100 ammo acids of the reference ammo acid of SEQ ID NO 2 In other words, to obtain a polypeptide havmg an ammo acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the ammo acid residues in the reference sequence may be deleted or substituted with another ammo acid, or a number of ammo acids up to 5% of the total ammo acid residues in the reference sequence may be inserted into the reference sequence These alterations of the reference sequence may occur at the ammo or carboxy terminal positions of the reference ammo acid sequence or anywhere between those terminal positions, interspersed either individually among residues m the reference sequence or m one or more contiguous groups within the reference sequence

Polypeptides of the Invention

In one aspect, the present invention relates to CBCALD05 polypeptides (or CBGALD05 proteins) The CBCALD05 polypeptides include the polypeptide of SEQ ID NO 2, as well as polypeptides comprising the ammo acid sequence of SEQ ID NO 2, and polypeptides comprising the amino acid sequence which have at least 80% identity to that of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% are highly preferred Also included within CBGALD05 polypeptides are polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide havmg the ammo acid sequence of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and still more preferably at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% are highly preferred Preferably CBCALD05 polypeptide exhibit at least one biological activity of CBCALD05

The CBCALD05 polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein It is often advantageous to mclude an additional ammo acid sequence which contains secretory or leader sequences, pro-seqύences, sequences which aid in purification such as multiple histidme residues, or an additional sequence for stability dunng recombinant production

Fragments of the CBCALD05 polypeptides are also included in the invention A fragment is a polypeptide havmg an ammo acid sequence that entirely is the same as part, but not all, of the ammo acid sequence of the aforementioned CBCALD05 polypeptides As with CBGALD05 polypeptides. fragments may be "free-standing," or composed within a larger polypeptide of which they form a part or region, most preferably as a single continuous region Representative examples of polypeptide frasjments of the invention, mclude, for example, fragments from about ammo acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of CBCALD05 polypeptide In this context "about" mcludes the paiticularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 ammo acid at either extreme or at both extremes

Preferred fragments mclude, for example, truncation polypeptides havmg the ammo acid sequence of CBC LD05 polypeptides, except for deletion of a continuous senes of residues that mcludes the ammo termmus, or a continuous senes of residues that mcludes the carboxyl termmus or deletion of two continuous senes of residues, one mcludmg the ammo termmus and one mcludmg the carboxyl teπmnus Also prefeired are fragments charactenzed by structural or functional attnbutes such as fragments that compπse alp.ha-he.hx and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and ton-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alplia amphipatluc regions, beta amphipathic regions, flexible regions, sur&ce-formmg regions, substrate binding region, and high antigenic mdex regions Other prefeired fragments are biologically active fragments Biologically active fragments are those that mediate CBCALD05 activity, mcludmg those with a similar activity or an improved activity, or with a decreased undesirable activity Also mcluded are those that are antigenic or lmmunogenic m an animal, especially m a human

Preferably, all of these polypeptide fragments retain the biological activity of the CBCALD05, mcludmg antigenic activity Vanants of the defined sequence and fr.agments also form part of the present mvention Preferred vanants are those that vary from the referents by conservative ammo acid substitutions - l e , those that substitute a residue with another of like charactenstics Typical such substitutions are among Ala, Val, Leu and lie, among Ser and Thr, among the acidic residues .Asp and Glu. among Asn and Gin, and among the basic residues Lys and Aig, or aromatic residues Phe and Tyr Particularly preferred are vanants m which several, 5-10, 1-5, or 1-2 ammo acids are substituted, deleted, or added m any combination The CBCALD05 polypeptides of the mvention can be prepared in any suitable manner Such polypeptides mclude isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods Means for prepanng such polypeptides are well understood m the art

Polynucleotides of the Invention other aspect of the mvention relates to CBCALD05 polynucleotides CBCALD05 polynucleotides mclude isolated polynucleotides which encode the CBCALD05 polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBC^LD05 polynucleotide of the mvention mclude a polynucleotide compnsing the nucleotide sequence contamed m SEQ ID NO 1 encoding a CBCALD05 polypeptide of SEQ ID NO 2, and polynucleotide havmg the particular sequence of SEQ ID NO 1 CBCALD05 polynucleotides fu.rther mclude a polynucleotide compnsing a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBG^D05 polypeptide of SEQ ID NO 2, and a polynucleotide compnsing a nucleotide sequence that is at least 80% identical to of SEQ ID NO 1 over its entire length In this regard, polynucleotides at least 90% identical are particularly preferred, and those with at least 95% are especially preferred Furthermore, those with at least 97% are highly preferred and those with at least 98-99% are most highly prefeπed, with at least 99% bemg the most prefeired Also mcluded under CBCALD05 polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contamed m SEQ ID NO 1 to hybndize under conditions useable for amplification or for use as a probe or marker The mvention also provides polynucleotides which are complementary to such

polynucleotides

CBCALD05 of the mvention is structurally related to other proteins of the Gene family containing acrosomal protein, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO 1) encoding human CBCALD05 The cDNA sequence of SEQ ID NO 1 contains an open readmg frame (nucleotide number 11 to 879) encoding a polypeptide of 293 ammo acids of SEQ ID NO 2 The ammo acid sequence of Table 2 (SEQ ED NO 2) .has about 822% identity (using FASTA) 236 ammo acid residues with fox acrosomal protem FS A- 1 (S Beaton et al ,Reprod Fertil Dev 6 761-770J994) The nucleotide sequence of Table 1 (SEQ ID NO 1) lias about 88 5% identity (us g FASTA) m 574 nucleotide residues with fox spem acrosoaml protem FSA-1 (S Beaton et al ,Reprod Fertil Dev 6 761-770,1994) Thus, CBCALD05 polypeptides and polynucleotides of the present mvention are expected to have, mter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled m the art

Table T

GACTCCCAAG ATGGCGGACC TACTGGGCTC CATCCTGAGC TCCATGGAGA

51 AGCCACCCAG CCTCGGTGAC CAGGAGACTC GGCGCAAGGC CCGAGAACAG

101 GCCGCCCGCC TGAAGAAACT ACAAGAGCAA GAGAAACAAC AGAAAGTGGA

151 GTTTCGTAAA AGGATGGAGA AGGAGGTGTC AGATTTCATT CAAGACAGTG

201 GGCAGATCAA GAAAAAGTTT CAGCCAATGA ACAAGATCGA GAGGAGCATA

251 CTACATGATG TGGTGGAAGT GGCTGGCCTG ACATCCTTCT CCTTTGGGGA 301 AGATGATGAC TGTCGCTATG TCATGATCTT CAAAAAGGAG TTTGCACCCT

351 CAGATGAAGA GCTAGACTCT TACCGTCGTG GAGAGGAATG GGACCCCCAG

401 AAGGCTGAGG AGAAGCGGAA GCTGAAGGAG CTGGCCCAGA GGCAAGAGGA

451 GGAGGCAGCC CAGCAGGGGC CTGTGGTGGT GAGCCCTGCC AGCGACTACA

501 AGGACAAGTA CAGCCACCTC ATCGGCAAGG GAGCAGCCAA AGACGCAGCC

551 CACATGCTAC AGGCCAATAA GACCTACGGC TGTGTGCCCG TGGCCAATAA

601 GAGGGACACA CGCTCCATTG AAGAGGCTAT GAATGAGATC AGAGCCAAGA

651 AGCGTCTGCG GCAGAGTGGG GAAGAGTTGC CGCCAACCTC TAGGCGCCCC

701 GCCCAGCTCC CTTTGACCCC TGGGGCAGGG CAGGGGGCAG GGAGAGACAA

751 GGCTGCTGCT ATTAGAGCCC ATCCTGGAGC CCCACCTCTG AACCACCTCC

801 TACCAGCTGT CCCTCAGGCT GGGGGAAAAC AGGTGTTTGA TTTGTCACCG

851 TTGGAGCTTG GATATGTGCG TGGCATGTGT GTGTGTGTGT GAGAGTGTGA

901 ATGCACAGGT GGGTATTTAA TCTGTATTAT TCCCCGTTCT TGGAATTTTC

951 TTCCCCATGG GGCTGGGGTA CTTTACATTC AATAAATACT GTTTAACCCA

1001 AAAAAAAAAA AAAAAAAAAA AAAAAAA

A nucleotide sequence of a human CBCALD05 (SEQ ID NO: 1).

Table 2^b

1 MADL GSILΞ SMEKPPSLGD QETRRKAREQ aZVARLKKLQEQ EKQQKVEFRK

51 RMEKEVSDFI QDSGQI.KKKF QPMNKIERSI LHDWEVAGL TSFSFGEDDD

101 CRYVMIFKKE FAPSDEELDS YRRGEEWDPQ KAEE.KRKLKE LAQRQEEEiA

151 QQGPVWSPA SDYKDKYSHL IGKGAAKDAA HMLQ.ANKTYG CVPV.ANKRDT 201 RSIEEAI rEI .RAKKRLRQSG EELPPTSRRP AQLPLTPGAG QGAGRDIAAA

251 IR.AHPGAPPL HLLPAVPQA GGKQVFDLSP LELGYVRGMC VCV

An ammo acid sequence of a human CBCALD05 (SEQ ID NO 2)

One polynucleotide of the present mvention encoding CBCALD05 may be obtained usmg standard cloning and screening, from a cDNA library denved from mRNA m cells of human cord blood using the expressed sequence tag (EST) analysis (Adams, M D , et al Science (1991) 252 1651-1656, Adams, M D et al , Nature, (1992) 355 632-634, Adams, M D , et al , Nature (1995) 377 Supp 3-174) Polynucleotides of the mvention can also be obtained from natural sources such as genoπuc DNA branes or can be synthesized usmg well known and commercially available techniques The nucleotide sequence encoding CBCALD05 polypeptide of SEQ ID NO 2 may be identical to the polypeptide encodmg sequence contamed in Table 1 (nucleotide number 11 to 879 of SEQ ID NO 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO 2

When the polynucleotides of the invention are used for the recombinant production of CBCALD05 polypeptide, the polynucleotide may mclude the codmg sequence for the mature polypeptide or a fragment thereof, by itself, the codmg sequence for the mature polypeptide or fragment m readmg frame with other codmg sequences, such as those encodmg a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide poitions For example, a marker sequence which facilitates punfication of the fused polypeptide can be encoded In certain preferred embodiments of t is aspect of the mvention, the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed m 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 transcnbed, non-translated sequences, splicing and polyadenylation signals, nbosome binding sites and sequences that stabilize rriRNA

Further prefened embodiments are polynucleotides encodmg CBCALD05 vanants compose the ammo acid sequence CBCALD05 polypeptide of Table 2 (SEQ ID NO 2) m wl ich several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acid residues are substituted, deleted or added, m any combination

The present mvention further relates to polynucleotides that hybndize to the herem above-descπbed sequences In this regard, the present mvention especially relates to polynucleotides which hybndize under stringent conditions to the herem above-descnbed polynucleotides As herem used, the term ".stringent conditions" means hybndι.zatιon will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences

Polynucleotides of the mvention. which are identical or sufficiently identical to a nucleotide sequence contamed m SEQ .ID NO 1 or a fragment thereof, may be used as hybndization probes for cDNA and genonuc DN.A, to isolate full-length cDNAs and genomic clones encodmg CBCALD05 polypeptide and to isolate cDNA and genomic clones of other genes (mcludmg genes encodmg homologs and orthologs from species other than human) that have a high sequence similanty to the CBC^D05 gene Such hybndization techniques are .known to those of skill in the art Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent The probes generally will compnse at least 15 nucleotides Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides Particularly preferred probes will range between 30 and 50 nucleotides

In one embodiment, to obtain a polynucleotide encodmg CBCALD05 polypeptide, mcludmg homologs and oithologs from species other man human, compnses the steps of screening an appropnate library under stmgent hybndization conditions with a labeled probe havmg the SEQ ID NO 1 or a fragment thereof, and isolating fiill-length cDNA and genomic clones contammg said polynucleotide sequence Thus m another aspect, CBCALD05 polynucleotides of the present mvention further mclude a nucleotide sequence compnsing a nucleotide sequence that hybndize under stnngent condition to a nucleotide sequence havmg SEQ ID NO 1 or a fragment thereof Also mcluded with CBCALD05 polypeptides are polypeptide composing ammo acid sequence encoded by nucleotide sequence obtained by the above hybndization condition Such hybndization techniques are well known to those of skill m the art Stnngent hybndrzauon conditions are as defined above or, alternatively, conditions under overnight incubation at 42°C m a solution compnsmg 50% formamide, 5xSSC (150mM NaCl, 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10 % dextxan sulfate. and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by waslung the filters m 0 lx SSC at about 65°C The polynucleotides and polypeptides of the present mvention may be employed as research reagents and matenals for discovery of treatments and diagnostics to animal and human disease

Vectors, Host Cells, Expression

The present mvention also relates to vectors which compnse a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engmeered with vectors of the mvention and to the production of polypeptides of the mvention by recombinant techmques Cell-free translation systems can also be employed to produce such proteins usmg RN^\s deπved from the DNA con&ructs of the present mvention

For recombinant production, host cells can be genetically engmeered to incorporate expression systems or portions thereof for polynucleotides of the present mvention Introduction of polynucleotides mto host cells can be effected by methods descnbed m many standard laboratory manuals, such as Davis et al, BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring .Harbor Laboratory Press, Cold Spring Harbor, N Y (1989) such as calcium phosphate transfection,

mediated txansfection, transvection, microinjection. catiomc lipid-mediated transfection, electroporation, traisduction, scrape loadmg, ballistic mtroduction or infection

Representative examples of appropnate ho.sts mclude bactenal cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtihs cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophύa 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 vanety of expression systems can be used Such systems mclude, among others, chromosomal, episomal and virus-denved systems, e g , vectors denved from bactenal plasmids, from bactenopliage, from txansposons. from yeaΛ episomes. from insertion elements, from yeast chromosomal elements, from viruses such as baculovinises, papova viruses, such as SN40, vaccinia vrruses, adenoviruses, fowl pox vmises, pseudorabies viruses and retrovrruses, and vectors denved from combinations thereof, such as those denved from plasmid and bactenophage 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 suitable to maintain, propagate or express polynucleotides to produce a polypeptide m a host may be used The appropnate nucleotide sequence may be inserted mto an expression system by any of a vanety of well-lαiown and routine techmques, such as, for example, those set foith m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL (supra)

For secretion of the translated protem mto the lumen of the endoplasmic reticulum. mto the penplasmic space or mto the extracellular environment, appropnate secretion sign^s may be incorporated mto the desired polypeptide These sigirals may be endogenous to the polypeptide or they may be heterologous sign s

If the CBC/ .D05 polypeptide is to be expressed for use m screening assays, generally, it is preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested pnor to use m the screening assay If CBCALD05 polypeptide is secreted mto the medium, the medium can be recovered m order to recover and punfy the polypeptide, if produced intracellularly, the cells must first be lysed before the polypeptide is recovered

CBCALD05 polypeptides can be recovered and punfied from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin diromatography Most preferably, high performance liquid chromatography is employed for purification Well .known techmques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification

Diagnostic Assays

This mvention also relates to the use of CBCALD05 polynucleotides for use as diagnostic reagents Detection of a mutated form of CBGALD05 gene 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 expression of CBG/^LD05 Individuals caιτymg mutations m the CBCALD05 gene may be detected at the DNA level by a vanety of techmques

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 may be amplified enzymatically by usmg PCR or other amplification techmques pnor to

RNA or cDNA may also be used m similar fashion Deletions and insertions can be detected by a change m size of the amplified product in companson to the noπrøl genotype Pomt mutations can be identified by hybndizmg amplified DNA to labeled CBCALD05 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments m gels, with or without denaturing agents, or by direct DNA sequencmg See, e g , Myers et αl , Science (1985) 230 1242 Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method See Cotton et αl , Proc Nαtl Acαd Sci USA (1985) 85 4397-4401 In .another embodiment, an anay of oligonucleotides probes compnsing CBCALD05 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g , genetic mutations Aπay technology methods are well known and have general applicability and can be used to address a vanety of questions in molecular genetics mcludmg gene expression, genetic lnώ-age, and genetic vanability (See for example M Chee et al , Science, Vol 274, pp 610-613 (1996))

The diagnostic assays offer a process for diagnosing or determining a susceptibility to lnfeitility and diseases related to fertility through detection of mutation m the CBCALD05 gene by the methods descnbed In addition, infertility and diseases related to fertility, can be diagnosed by methods compnsing determining from a sample denved from a subject an abnormally decreased or increased level of

CBCALD05 polypeptide or CBCALD05 mRNA Decreased or increased expression can be measured at the .RNA level using any of the methods well known m the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods .Assay techniques that can be used to determine levels of a protem, such as an CBC LD05 polypeptide, in a sample denved from a host are well-lαiown to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-bmdmg assays, Western Blot airalysis and ELISA assays

Thus in another aspect, the present invention relates to a diagonostic .kit for a disease or suspectabi ty to a disease, particularly infertility and diseases related to fertility, which compnses (a) a CBCALD05 polynucleotide, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof ,

(b) a nucleotide sequence complementary to that of (a),

(c) a CBGALD05 polypeptide, preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or

(d) an antibody to a CBCALD05 polypeptide, preferably to the polypeptide of SEQ ID NO 2 It will be appreciated that in any such kit, (a), (b), (c) or (d) may compnse a substantial component

Chromosome Assays

The nucleotide sequences of the present mvention are also valuable for chromosome identification The sequence is specifically targeted to and can hybndize with a particular location on an individual human chromosome The mappmg of relevant sequences to chromosomes accordmg to the present mvention is an important first step m coιτelating 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, for example, m V McKusick, Mendehan Inhentance m Man (available on lme through Johns Hoplαns 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 (coinhentance of physically adjacent genes)

The differences m the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease

Antibodies

The polypeptides of the mvention or their fragments or analogs thereof, or cells expressmg them can also be used as immunogens to produce antibodies lmmunospecific for the CBCALD05 polypeptides The term "lmmunospecific" means that the antibodies have substantiall greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides m the pnor art

.Antibodies generated against the CBCALD05 polypeptides can be obtained by administering the polypeptides or epitope-beanng fragments, an ogs or cells to an animal, preferably a nonhuman, usmg routme protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell lme cultures can be used Examples mclude the hybndoma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the tnoma technique, the human B-cell hybπdoma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hybndoma technique (Cole et al , MONOCLON.AL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985)

TeCahmques for the production of smgle chain antibodies (U S Patent No 4,946,778) can also be adapted to produce smgle cham antibodies to polypeptides of this mvention .Also, transgeiuc mice, or other organisms mcludmg other mammals, may be used to express humanized antibodies

The above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to purify the polypeptides by affinity chromatography

Antibodies againΛ CBCALD05 polypeptides may also be employed to treat infertility and diseases related to fertility, among others

Vaccines

.Another aspect of the invention relates to a method for mducmg an lmmunological response m a mammal which compnses inoculating the mammal with

polypeptide, or a fragment thereof. adequate to produce antibody and/or T cell immune response to protect said ammal from infertility and diseases related to fertility, among others Yet another aspect of the mvention relates to a method of mducmg lmmunological response m a mammal which compnses, delivering CBCALD05 polypeptide via a vector directmg expression of CBCALD05 polynucleotide in vivo m order to mduce such an lmmunological response to produce antibody to protect said animal from diseases Further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an lmmunological response in that mammal to a CBCALD05 polypeptide wherein the composition compnses a CBCALD05 polypeptide or CBCALD05 gene The vaccine formulation may further compnse a suitable earner Since

polypeptide may be broken down in the stomach, it is preferably admrmstered parenterally (mcludmg subcutaneous, intramuscular, intravenous, lntradermal etc injection) Formulations suitable for parenteral administration include aqueous and non-aqueous stenle injection solutions which may contam anti-oxidants, buffers, bactenostats and solutes which render the foπnulation mstonic with the blood of the recipient, and aqueous and non-aqueous stenle suspensions which may mclude suspending agents or thickening agents The foπnulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dned condition requmng only the addition of the stenle liquid earner immediately pnor to use The vaccme formulation may also include adjuvant systems for enh.ancιng the lmmunogenicity of the foπnulation, 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 experimentation Screening Assays

The CBCALD05 polypeptide of the present mvention may be employed m a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called lnlubitors) the CBCALD05 polypeptide of the present mvention Thus, polypeptides of the mvention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical hbranes, and natural product mixtures These agonists or antagonists may be natural or modified substrates, hgands, receptors, enzymes, etc , as the case may be, of the polypeptide of the present mvention, or may be structural or functional mimetics of the polypeptide of the present mvention See Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)

CBCALD05 polypeptides are responsible for many biological functions, mcludmg many pathologies Accordingly, it is desirous to find compounds and drags which .stimulate CBCALD05 polypeptide on the one hand and which can inhibit the function of CBCALD05 polypeptide on the other hand In general, agomsts are employed for therapeutic and prophylactic purposes for such conditions as infertility and diseases related to fertility .Antagonists may be employed for a vanety of therapeutic and prophylactic purposes for such conditions as infertility and diseases related to fertility

In general, such screening procedures may mvolve usmg appropnate cells which express the CBCALD05 polypeptide or respond to CBCALD05 polypeptide of the present mvention Such cells mclude cells from mamnrals, yea , Drosophila or E coli Cells which express the CBG LD05 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBCALD05 polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response The ability of the cells which were contacted with the candidate compounds is compared with the same cells which were not contacted for CBCALD05 activity

The assays may simply test binding of a candidate compound wherein adherence to the cells bearmg the CBCALD05 polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor Further, these assays may test whether the candidate compound results in a signal generated by activation of the CBGALD05 polypeptide, using detection systems appropnate to the cells bearmg the CBCALD05 polypeptide Inhibitors of activation are generally assayed in the presence of a known agomst and the effect on activation by the agonist by the presence of the candidate compound is observed

Further, the assays may simply comprise the steps of mixmg a candidate compound with a solution containing a CBCALD05 polypeptide to form a mixture, measuring CBCALD05 activity m the mixture, and companng the CBCALD05 activity of the mixture to a standard The CBGALD05 cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of CBCALD05 mRNA and protein m cells For example, an ELISA may be constructed for measuring secreted or cell associated levels of CBCALD05 protein using monoclonal and polyclonal antibodies by standard methods known m the art, and this can be used to discover agents which may inhibit or enhance the production of

CBGALD05 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues The CBCALD05 protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques .known in the art These mclude, but are not limited to, hgand bindmg and crosshnking assays m which the CBCALD05 is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), 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 mclude biophysical techmques such as surface plasmon resonance and spectroscopy In addition to being used for punfication and cloning of the receptor, these bmdmg assays can be used to identify agomsts and antagonists of CBC/\LD05 which compete with the bmdmg of CBCALD05 to its receptors, if any Standard methods for conductmg screening assays are well understood m the art

Examples of potential CBCALD05 polypeptide antagonists mclude antibodies or, m some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be, of the CBCALD05 polypeptide, e g , a fragment of the ligands, substrates, receptors, enzymes, etc , or small molecules which bmd to the polypetide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented

Thus in another aspect, the present invention relates to a screening kit for identifying agomsts, antagonists, ligands, receptors, substrates, enzymes, etc for CBGALD05 polypeptides, or compounds which decrease or enhance the production of CBCALD05 polypeptides, which compnses (a) a CBCALD05 polypeptide. preferably that of SEQ ID NO 2,

(b) a recombinant cell expressing a CBCALD05 polypeptide, preferably that of SEQ ID NO 2,

(c) a cell membrane expressing a CBCALD05 polypeptide, preferably that of SEQ ID NO 2, or

(d) antibody to a CBCALD05 polypeptide, preferably that of SEQ ID NO 2

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

Prophylactic and Therapeutic Methods

This mvention provides methods of treating abnormal conditions such as, infertility and diseases related to fertility, related to both an excess of and insufficient amounts of CBCALD05 polypeptide activity If the activity of CBG LD05 polypeptide is m excess, seveial approaches are available One approach compnses administering to a subject an inhibitor compound (antagonist) as hereinabove descnbed along with a pharmaceutically acceptable earner in an amount effective to inhibit the function of the CBCALD05 polypeptide, such as, for example, by blocking the bmdmg of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnorm condition In another approach, soluble foπns of CBCALD05 polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc m competition with endogenous CBCALD05 polypeptide may be administered Typical embodiments of such competitors comprise fragments of the CBCALD05 polypeptide

In still another approach, expression of the gene encoding endogenous CBCALD05 polypeptide can be inhibited using expression blocking techniques Known such techmques involve the use of antisense sequences, either internally generated or separately administered See, for example, O'Connor, J Neurochem (1991) 56 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988) Alternatively, o gonucleotides which form tnple helices with the gene can be supplied See, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, De.rvan et -?/ , Science (1991) 251 1360 These oligomers can be administered per se or the relevant oligomers can be expressed in vivo

For treating abnormal conditions related to an under-expression of CBGALD05 and its activity, several approaches are also available One approach compnses admimstenng to a subject a therapeutically effective amount of a compound which activates CBCALD05 polypeptide, 1 e , an agonist as descnbed above, in combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition Alternatively, gene therapy may be employed to effect the endogenous production of CBCALD05 by the relevant cells in the subject For example, a polynucleotide of the mvention may be engmeered for expression m a replication defective retroviral vector, as discussed above The retroviral expression construct may then be isolated and introduced mto a packaging cell transduced with a retroviral plasmid vector contammg .RNA encodmg a polypeptide of the present mvention such that the packaging cell now produces infectious viral particles containing the gene of interest These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in v vo For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) m Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996) Another approach is to admmister a therapeutic amount of CBCALD05 polypeptides m combination with a suitable pharmaceutical earner Foπnulation and Administration

Peptides, such as the soluble form of CBGALD05 polypeptides, and agomsts and antagonist peptides or small molecules, may be formulated m combination with a suitable pharmaceutical earner Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a phaπnaceutically acceptable earner or excipient Such earners mclude but are not hmited to, salme, buffered salme, dextrose, water, glycerol, etlianol, and combinations thereof Formulation should suit the mode of admimstration, and is well within the skill of the art The mvention further relates to pharmaceutical packs and kits compnsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention Polypeptides and other compounds of the present mvention may be employed alone or in conjunction with other compounds, such as therapeutic compounds

Prefened forms of systemic admimstration of the pharmaceutical compositions mclude injection, typically by mtravenous injection Other injection routes, such as subcutaneous, lntiamuscular, or mtrapentoneal, can be used Alternative means for systemic admimstration mclude transmucosal and transdermal admimstration usmg penetiants such as bile salts or fusidic acids or other detergents In addition, if properly formulated m entenc or encapsulated foimulations, oral admimstration may also be possible Admimstration of these compounds may also be topical and/or localized, m the form of salves, pastes, gels

The dosage range required depends on the choice of peptide, the route of ----ministration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attendmg practitioner Suitable dosages, however, are m the range of 0 1 - 100 μg/kg of subject Wide vanations in the needed dosage, however, are to be expected m view of the vanety of compounds available and the differing efficiencies of vanous routes of admimstration For example, oral admιm.stratιon would be expected to require higher dosages than admιnι.stration by mtravenous injection Vanations m these dosage levels can be adjusted usmg standard empincal routines for optimization, as is well understood in the art

Polypeptides used m treatment can also be generated endogenously in the subject, m treatment modalities often refened to as "gene therapy" as descnbed above Thus, for example, cells from a subject may be engmeered with a polynucleotide, such as a DNA or RN.A, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector The cells are then introduced mto the subject

All publications, mcludmg but not limited to patents and patent applications, cited in this specification are herem incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: SHANGHAI SECOND MEDICAL UNIVERSITY

(ii) TITLE OF THE INVENTION: A GENE HOMOLOGOUS TO FOX SPERM ACROSOMAL PROTEIN FSA-1 (CBCALD05)

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE .ADDRESS:

(A) ADDRESSEE: RATNER &. PRESTIA (B) STREET: P.O. BOX 980

(C) CITY: VALLEY FORGE

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19482

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS (D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: TO BE ASSIGNED

(B) FILING DATE: (C) CLASSIFICATION: UNKNOWN

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: PRESTIA, PAUL F (B) REGISTRATION NUMBER: 23,031

(C) REFERENCE/DOCKET NUMBER: GP-70355

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 610-407-0700

(B) TELEFAX: 610-407-0701

(C) TELEX: 846169

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 1027 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

GACTCCCAAG ATGGCGGACC TACTGGGCTC CATCCTGAGC TCCATGGAGA AGCCACCCAG 60

CCTCGGTGAC CAGGAGACTC GGCGCAAGGC CCGAGAACAG GCCGCCCGCC TGAAGAAACT 120 ACAAGAGCAA GAGAAACAAC AGAAAGTGGA GTTTCGTAAA AGGATGGAGA AGGAGGTGTC 180

AGATTTCATT CAAGACAGTG GGCAGATCAA GAAAAAGTTT CAGCCAATGA ACAAGATCGA 240

GAGGAGCATA CTACATGATG TGGTGGAAGT GGCTGGCCTG ACATCCTTCT CCTTTGGGGA 300

AGATGATGAC TGTCGCTATG TCATGATCTT CAAAAAGGAG TTTGCACCCT CAGATGAAGA 360

GCTAGACTCT TACCGTCGTG GAGAGGAATG GGACCCCCAG AAGGCTGAGG AGAAGCGGAA 420 GCTGAAGGAG CTGGCCCAGA GGCAAGAGGA GGAGGCAGCC CAGCAGGGGC CTGTGGTGGT 480

GAGCCCTGCC AGCGACTACA AGGACAAGTA CAGCCACCTC ATCGGCAAGG GAGCAGCCAA 540

AGACGCAGCC CACATGCTAC AGGCCAATAA GACCTACGGC TGTGTGCCCG TGGCCAATAA 600

GAGGGACACA CGCTCCATTG AAGAGGCTAT GAATGAGATC AGAGCCAAGA AGCGTCTGCG 660

GCAGAGTGGG GAAGAGTTGC CGCCAACCTC TAGGCGCCCC GCCCAGCTCC CTTTGACCCC 720 TGGGGCAGGG CAGGGGGCAG GGAGAGACAA GGCTGCTGCT ATTAGAGCCC ATCCTGGAGC 780

CCCACCTCTG AACCACCTCC TACCAGCTGT CCCTCAGGCT GGGGGAAAAC AGGTGTTTGA 840

TTTGTCACCG TTGGAGCTTG GATATGTGCG TGGCATGTGT GTGTGTGTGT GAGAGTGTGA 900

ATGCACAGGT GGGTATTTAA TCTGTATTAT TCCCCGTTCT TGGAATTTTC TTCCCCATGG 960

GGCTGGGGTA CTTTACATTC AATAAATACT GTTTAACCCA AAAAAAAAAA AAAAAAAAAA 1020 AAAAAAA 1027

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 293 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear ( ii ) MOLECULE TYPE : protein

(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :

Met Ala Asp Leu Leu Gly Ser lie Leu Ser Ser Met Glu Lys Pro Pro

1 5 10 15

Ser Leu Gly Asp Gin Glu Thr Arg Arg Lys Ala Arg Glu Gin Ala Ala

20 25 30 Arg Leu Lys Lys Leu Gin Glu Gin Glu Lys Gin Gin Lys Val Glu Phe

35 40 45

Arg Lys Arg Met Glu Lys Glu Val Ser Asp Phe lie Gin Asp Ser Gly

50 55 60

Gin lie Lys Lys Lys Phe Gin Pro Met Asn Lys lie Glu Arg Ser lie 65 70 75 80

Leu His Asp Val Val Glu Val Ala Gly Leu Thr Ser Phe Ser Phe Gly

85 90 95

Glu Asp Asp Asp Cys Arg Tyr Val Met lie Phe Lys Lys Glu Phe Ala

100 105 110 Pro Ser Asp Glu Glu Leu Asp Ser Tyr Arg Arg Gly Glu Glu Trp Asp

115 120 125.

Pro Gin Lys Ala Glu Glu Lys Arg Lys Leu Lys Glu Leu Ala Gin Arg

130 135 140

Gin Glu Glu Glu Ala Ala Gin Gin Gly Pro Val Val Val Ser Pro Ala 145 150 155 160

Ser Asp Tyr Lys Asp Lys Tyr Ser His Leu lie Gly Lys Gly Ala Ala

165 170 175

Lys Asp Ala Ala His Met Leu Gin Ala Asn Lys Thr Tyr Gly Cys Val

180 185 190 Pro Val Ala Asn Lys Arg Asp Thr Arg Ser lie Glu Glu Ala Met Asn

195 200 205

Glu lie Arg Ala Lys Lys Arg Leu Arg Gin Ser Gly Glu Glu Leu Pro

210 215 220

Pro Thr Ser Arg Arg Pro Ala Gin Leu Pro Leu Thr Pro Gly Ala Gly 225 230 235 240

Gin Gly Ala Gly Arg Asp Lys Ala Ala Ala lie Arg Ala His Pro Gly

245 250 255

Ala Pro Pro Leu Asn His Leu Leu Pro Ala Val Pro Gin Ala Gly Gly

260 265 270 Lys Gin Val Phe Asp Leu Ser Pro Leu Glu Leu Gly Tyr Val Arg Gly

275 280 285

Met Cys Val Cys Val 290

Claims

What is claimed is:

1 An isolated polynucleotide compnsmg a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encodmg the CBG\LD05 polypeptide of SEQ ID NO 2, or a nucleotide sequence complementary to said isolated polynucleotide

2 The polynucleotide of claim 1 wherein said polynucleotide compnses the nucleotide sequence contained in SEQ ID NO 1 encodmg the CBCALD05 polypeptide of SEQ ID N02

3 The polynucleotide of claim 1 wherem said polynucleotide compnses a nucleotide sequence that is at least 80% identical to that of SEQ ID NO 1 over its entire length

4 The polynucleotide of claim 3 which is polynucleotide of SEQ ID NO 1

5 The polynucleotide of claun 1 which is DNA or RNA

6 A DNA or RNA molecule compnsing an expression system, wherein said expression system is capable of producmg a CBCALD05 polypeptide compnsmg an ammo acid sequence, which has at least 80% identity with the polypeptide of SEQ ID NO 2 when said expression system is present in a compatible host cell

7 A host cell compnsing the expression system of claim 6

8 A process for producing a CBCALD05 polypeptide compnsmg cultunng a host of claim 7 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture

9 A process for producing a cell which produces a CBCALD05 polypeptide thereof compnsing transforming or transfecting a host cell with the expression system of claim 6 such that the host cell, under appropnate culture conditions, produces a CBCALD05 polypeptide

10 A CBCALD05 polypeptide compnsmg an ammo acid sequence which is at least 80%) identical to the ammo acid sequence of SEQ ID NO 2 over its entire length

11 The polypeptide of claim 10 which compnses the amino acid sequence of SEQ ID O 2

12 .An antibody lmmunospecific for the

polypeptide of claim 10

13 A method fc the treatment of a subject in need of enhanced activity or expression of CBGALD05 polypeptide of claim 10 compnsing

(a) admmistenng to the subject a therapeutically effective amount of an agonist to said polypeptide. and/or

(b) providing to the subject an isolated polynucleotide compnsmg a nucleotide sequence that has at least 80% identity to a nucleotide sequence encodmg the CBGALD05 polypeptide of SEQ ID NO 2 over its entire length, or a nucleotide sequence complementaiy to said nucleotide sequence in a form so as to effect production of said polypeptide activity in vivo

14 A method for the treatment of a subject havmg need to inhibit activity or expression of CBGALD05 polypeptide of claun 10 compnsing (a) admmistenng to the subject a therapeutically effective amount of an antagonist to said polypeptide, and/or

(b) admmistenng to the subject a nucleic acid molecule that inhibits the expression of the nucleotide sequence encoding said polypeptide, and/or

(c) administering to the subject a therapeutically effective amount of a polypeptide that competes with said polypeptide for its hgand, substrate , or receptor

15 A process for diagnosing a disease or a susceptibility to a disease m a subject related to expression or activity of CBCALD05 polypeptide of claim 10 m a subject compnsmg

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said CBC^\LD05 polypeptide m the genome of said subject, and/or

(b) analyzing for the presence or amount of the CBCALD05 polypeptide expression m a sample denved from said subject

16. A method for identifying compounds which inhibit (antagonize) or agonize the CBGALD05 polypeptide of claim 10 which comprises:

(a) contacting a candidate compound with cells which express the CBCALD05 polypeptide (or cell membrane expressing CBCALD05 polypeptide) or respond to CBCALD05 polypeptide; and

(b) observing the binding, or stimulation or inhibition of a functional response; or comparing the ability of the cells (or cell membrane) which were contacted with the candidate compounds with the same cells which were not contacted for CBCALD05 polypeptide activity.

17. An agonist identified by the method of claim 16.

18. .An antagonist identified by the method of claim 16.

19. A recombinant host cell produced by a method of Claim 9 or a membrane thereof expressing a CBCALD05 polypeptide.