WO1999047665A1 - THE HUMAN CI-B14.5a HOMOLOG GENE: CBUACD09 - Google Patents

Abstract

CBUACD09 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CBUACD09 polypeptides and polynucleotides in the design of protocols for the treatment of cancer, AIDS, metabolic disorders, and IDDM, among others, and diagnostic assays for such conditions.

Description

The Human CI-B14.5a Homolog Gene: CBUACD09

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 NADH dehydrogenase complex family, hereinafter referred to as CBUACD09. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

The NADH dehydrogenase is a mitochondrial complex composed of many subunits, and is associated with metabolism. The CI-B14.5a protein is a subunit of NADH dehydrogenase. This indicates that the NADH dehydrogenase complex family has an established, proven history as therapeutic targets. Clearly there is a need for identification and characterization of further members of the NADH dehydrogenase complex family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, cancer, AIDS, metabolic disorders, and insulin dependent diabetes mellitus (IDDM).

SUMMARY OF THE INVENTION

In one aspect, the invention relates to CBUACD09 polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such CBUACD09 polypeptides and polynucleotides. Such uses include the treatment of cancer, AIDS, metabolic disorders, and IDDM, 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 CBUACD09 imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBUACD09 activity or levels.

DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided to facilitate understanding of certain terms used frequently herein. "CBUACD09" refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO 2 or an alle c variant thereof

"CBUACD09 activity or CBUACD09 polypeptide activity" or "biological activity of the CBUACD09 or CBUACD09 polypeptide" refers to the metabolic or physiologic function of said CBUACD09 including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigemc and lmmunogenic activities of said CBUACD09

"CBUACD09 gene" refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 1 or alle c variants thereof and/or their complements "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 immunoglobulin expression library

"Isolated" means altered "by the hand of man" from the natural state If an "isolated" composition or substance occurs in 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 materials of its natural state is "isolated", as the term is employed herein

"Polynucleotide" generally refers to any polyπbonucleotide or polydeoxπbonucleotide, 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, hybrid 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 tπple-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 variety of modifications has been 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 characteristic of viruses and cells "Polynucleotide" also embraces relatively short polynucleotides, often referred to as ohgonucleotides

"Polypeptide" refers to any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, l e , peptide isosteres "Polypeptide" refers to both short chains, commonly referred to as peptides, ohgopeptides or o gomers, and to

2 longer chains, generally referred to as proteins Polypeptides may contain amino acids other than the 20 gene-encoded ammo acids "Polypeptides" include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature Modifications can occur anywhere m a polypeptide, including the peptide backbone, the ammo acid side-chains and the amino 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 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 posttranslation natural processes or may be made by synthetic methods Modifications include acetylation, acylation, ADP-πbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a pid or hpid deπvative, covalent attachment of phosphotidylmositol, 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, lodmation, methylation, mynstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racermzation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitmation See, for instance, PROTEINS - STRUCTURE AND

MOLECULAR 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 POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York, 1983, Seifter et al , "Analysis for protein modifications and nonprotem cofactors", Meth Enzymol (1990) 182 626-646 and Rattan et al , "Protein Synthesis Posttranslational Modifications and Aging", Ann NYAcad Set (1992) 663 48-62

"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes in 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 ammo acid substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide Generally, differences are limited so that the

3 sequences of the reference polypeptide and the variant 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 in 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 variant, or it may be a vaπant 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," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (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, Griffin,

A M , and Griffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Heinje, G , Academic Press, 1987, and Sequence Analysis Primer, Gπbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Caπllo, H , and Lipman, D , SIAM J Applied Math , 48 1073 (1988) Preferred methods to determine identity are designed to give the largest match between the sequences tested Methods to determine identity and similarity are codified m publicly available computer programs Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S F et al . J Molec Biol 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NM Bethesda, MD 20894, Altschul, S , et al , J Mol Biol 215 403-410 (1990) The well known Smith Waterman algorithm may also be used to determine identity

Preferred parameters for polypeptide sequence comparison include the following 1) Algorithm Needleman and Wunsch. J Mol Biol 48 443-453 (1970)

Comparison matrix BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4

4 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps)

Preferred parameters for polynucleotide comparison include the following

1) Algorithm Needleman and unsch, J Mol Biol 48 443-453 (1970) Compaπson matrix matches = +10, mismatch = 0 Gap Penalty 50 Gap Length Penalty 3 A program useful with these parameters is publicly available as the "gap" program from

Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for polynucleotide comparisons

Preferred polynucleotide embodiments further include an isolated polynucleotide compπsing a polynucleotide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polynucleotide reference sequence of SEQ ID NO 1 , wherem said reference sequence may be identical to the sequence of SEQ ID NO 1 or may mclude up to a certain mteger number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or msertion, and wherem said alterations may occur at the 5 ' or 3' termmal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides m the reference sequence or m one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO 1 by the numerical percent of the respective percent identity and subtracting that product from said total number of nucleotides m SEQ ID NO 1, or

nn £ xn - (xn ^• y),

wherem nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO 1, and y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and wherein any non-integer product of xn and y is rounded down to the nearest mteger prior to subtracting it from xn Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations

Preferred polypeptide embodiments further mclude an isolated polypeptide comprising a polypeptide havmg at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said reference sequence may be identical to the sequence of SEQ ID NO 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids m SEQ ID NO 2 by the numeπcal percent of the respective percent identity and subtracting that product from said total number of amino acids m SEQ ID NO 2, or

na £ xa - (xa y),

wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID NO 2, and y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and wherem any non-mteger product of xa and y is rounded down to the nearest integer pπor to subtracting it from xa

Polypeptides of the Invention

In one aspect, the present invention relates to CBUACD09 polypeptides (or CBUACD09 proteins) The CBUACD09 polypeptides include the polypeptide of SEQ ID NO 2, as well as polypeptides comprising the amino 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 prefeπed Also included within CBUACD09 polypeptides are polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide having the amino 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

6 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% are highly preferred Preferably CBUACD09 polypeptide exhibit at least one biological activity of CBUACD09

The CBUACD09 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 include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production

Fragments of the CBUACD09 polypeptides are also included in the invention A fragment is a polypeptide having an ammo acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned CBUACD09 polypeptides As with CBUACD09 polypeptides, fragments may be "free-standing," or compnsed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region Representative examples of polypeptide fragments of the invention, mclude, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61- 80, 81-100, and 101 to the end of CBUACD09 polypeptide In this context "about" mcludes the particularly 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 having the ammo acid sequence of CBUACD09 polypeptides, except for deletion of a continuous seπes of residues that mcludes the ammo terminus, or a continuous senes of residues that mcludes the carboxyl terminus or deletion of two continuous senes of residues, one mcludmg the ammo terminus and one mcludmg the carboxyl terminus Also preferred are fragments characterized by structural or functional attributes such as fragments that compπse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet- forming regions, turn and turn-forming regions, coil and coιl-foιτrung regions, hydrophύic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface- forming regions, substrate binding region, and high antigenic mdex regions Other preferred fragments are biologically active fragments Biologically active fragments are those that mediate CBUACD09 activity, including 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 CBUACD09, mcludmg antigenic activity Variants of the defined sequence and fragments also form part of the present invention Preferred vaπants are those that vary from the referents by conservative ammo acid substitutions — I e , those that substitute a residue with another of like characteπstics Typical such substitutions are among Ala, Val, Leu and He, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, 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 CBUACD09 polypeptides of the invention can be prepared m 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 preparing such polypeptides are well understood m the art

Polynucleotides of the Invention

Another aspect of the invention relates to CBUACD09 polynucleotides CBUACD09 polynucleotides mclude isolated polynucleotides which encode the CBUACD09 polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBUACD09 polynucleotide of the invention mclude a polynucleotide compnsing the nucleotide sequence contained m SEQ ED NO 1 encodmg a CBUACD09 polypeptide of SEQ ID NO 2, and polynucleotide having the particular sequence of SEQ ID NO 1 CBUACD09 polynucleotides further mclude a polynucleotide compnsing a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encodmg the CBUACD09 polypeptide of SEQ ID NO 2, and a polynucleotide comprising 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 preferred, with at least 99% bemg the most preferred Also included under

CBUACD09 polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO 1 to hybridize under conditions useable for amplification or for use as a probe or marker The invention also provides polynucleotides which are complementary to such CBUACD09 polynucleotides CBUACD09 of the invention is structurally related to other proteins of the NADH dehydrogenase complex family, as shown by the results of sequencmg the cDNA of Table 1 (SEQ ID NO 1) encodmg human CBUACD09 The cDNA sequence of SEQ ED NO 1 contains an open reading frame (nucleotide number 12 to 365) encodmg a polypeptide of 118 ammo acids of SEQ ID NO 2 The ammo acid sequence of Table 2 (SEQ ID NO 2) has about 79 7% identity (using FASTA) m 118 ammo acid residues with B taunts CI-B14 5a mRNA for NADH dehydrogenase (J M Anzmendi, et al FEBS Lett 313, 80-84, 1992) The nucleotide sequence of Table 1 (SEQ ID NO 1) has about 77 0% identity (usmg FASTA) in 449 nucleotide residues with B taunts CI-B 14 5a mRNA for NADH dehydrogenase X68585 (J M Aπzmendi, et al FEBS Lett 313, 80-84, 1992) Thus, CBUACD09 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 in the art

Table 1"

1 GGCCCTTCAG TATGCGCGGA CGGAAGATGG CGTCCGCCAC CCGTCTCATC

51 CAGCGGCTGC GGAACTGGGC GTCCGGGCAT GACCTGCAGG GGAAGCTGCA

101 GCTACGCTAC CAGGAGATCT CCAAGCGAAC TCAGCCTCCT CCCAAGCTCC

151 CTGTGGGTCC TAGCCACAAG CTCTCCAACA ATTACTATTG CACTCGCGAT

201 GGCCGCCGGG AATCTGTGCC CCCTTCCATC ATCATGTCGT CGCAGAAGGC

251 GCTGGTGTCA GGCAAGCCAG CAGAGAGCTC TGCTGTAGCT GCCACTGAGA

301 AGAAGGCGGT GACTCCAGCT CCTCCCATAA AGAGGTGGGA GCTGTCCTCG

351 GACCAGCCTT ACCTGTGACA CTGCACCCTC ACGGCCACCC GACTACTTTG

401 CCTCCTTGGA TTTCCTCCAG GGAGAATGTG ACCTAATTTA TGACAAATAC

451 GTAGAGCTCA GGTATCACTT CTAGTTTTAC TTTAAAAAAT AAAAAAATAG

501 AGACAGAGTC TCAAAAAAAA AAAAAAAAAA A

A nucleotide sequence of a human CBUACD09 (SEQ ID NO 1)

Table 2^b

1 MRGRKMASAT RLIQRLRNWA SGHDLQGKLQ LRYQEISKRT QPPPKLPVGP

51 ΞHKLSNNYYC TRDGRRESVP PSIIMSSQKA LVSGKPAESS AVAATEKKAV

101 TPAPPIKRWE LSSDQPYL

An ammo acid sequence of a human CBUACD09 (SEQ ID NO 2)

One polynucleotide of the present mvention encodmg CBUACD09 may be obtamed usmg standard clonmg and screemng, from a cDNA library deπved from mRNA m cells of human cord blood using the expressed sequence tag (EST) analysis (Adams, M O , 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 genomic DNA libraries or can be synthesized using well known and commercially available techniques

The nucleotide sequence encoding CBUACD09 polypeptide of SEQ ID NO 2 may be identical to the polypeptide encodmg sequence contained m Table 1 (nucleotide number 12 to 365 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

CBUACD09 polypeptide, the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself, the coding sequence for the mature polypeptide or fragment m reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions For example, a marker sequence which facilitates puπfication of the fused polypeptide can be encoded In certam prefeπed embodiments of this aspect of the mvention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc ) and descπbed m Gentz et al , Proc Natl Acad Set 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 binding sites and sequences that stabilize mRNA

Further prefeπed embodiments are polynucleotides encodmg CBUACD09 vaπants compnse the ammo acid sequence CBUACD09 polypeptide of Table 2 (SEQ ID NO 2) m which 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 hybπdize to the herem above- descπbed sequences In this regard, the present mvention especially relates to polynucleotides which hybπdize under stπngent conditions to the herem above-desenbed polynucleotides As herem used, the term "stπngent conditions" means hybndization 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 contained m SEQ ID NO 1 or a fragment thereof, may be used as hybndization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encodmg CBUACD09 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 CBUACD09

10 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 prefeπed probes will range between 30 and 50 nucleotides

In one embodiment, to obtain a polynucleotide encodmg CBUACD09 polypeptide, mcludmg homologs and orthologs from species other than human, compπses the steps of screemng an appropnate library under stingent hybndization conditions with a labeled probe having the SEQ ID NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence Thus m another aspect, CBUACD09 polynucleotides of the present mvention further mclude a nucleotide sequence compnsing a nucleotide sequence that hybndize under stπngent condition to a nucleotide sequence having SEQ ID NO 1 or a fragment thereof Also mcluded with CBUACD09 polypeptides are polypeptide compnsing ammo acid sequence encoded by nucleotide sequence obtamed by the above hybndization condition Such hybndization techniques are well known to those of skill in the art Stπngent hybndization conditions are as defined above or, alternatively, conditions under overnight incubation at 42°C in a solution compnsing 50% formamide, 5xSSC (150mM NaCl, 15mM tnsodium citrate), 50 mM sodium phosphate (pH7 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 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 techniques Cell-free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs 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 descπbed m many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sa brook 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, DEAE-dextran mediated transfection,

11 transvection, microinjection, cationic pid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection

Representative examples of appropπate hosts mclude bacteπal cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtths cells, fiingal cells, such as yeast cells and Aspergillus cells, insect cells such as DrosopMa 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 deπved from bactenal plasmids, from bactenophage, 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 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 in a host may be used The appropπate nucleotide sequence may be inserted mto an expression system by any of a vanety of well-known and routine techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL {supra)

For secretion of the translated protein mto the lumen of the endoplasmic reticulum, mto the penplasmic space or mto the extracellular environment, appropπate secretion signals may be incorporated mto the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals

If the CBUACD09 polypeptide is to be expressed for use m screening assays, generally, it is prefeπed that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested pnor to use in the screemng assay If CBUACD09 polypeptide is secreted mto 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 CBUACD09 polypeptides can be recovered and purified from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, aruon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affimty chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance liquid chromatography is employed for punfication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or punfication

12 Diagnostic Assays

This mvention also relates to the use of CBUACD09 polynucleotides for use as diagnostic reagents Detection of a mutated form of CBUACD09 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 CBUACD09 Individuals carrying mutations m the CBUACD09 gene may be detected at the DNA level by a vanety of techniques

Nucleic acids for diagnosis may be obtamed from a subject's cells, such as from blood, urine, sahva, tissue biopsy or autopsy mateπal The genomic DNA may be used directly for detection or may be amplified enzymatica-ly by usmg PCR or other amplification techniques pπor to analysis 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 m companson to the normal genotype Pomt mutations can be identified by hybndizing amplified DNA to labeled CBUACD09 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in meltmg temperatures DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing See, e g , 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 NatlAcadSci USA (1985) 85 4397-4401 In another embodiment, an array of oligonucleotides probes compnsing CBUACD09 nucleotide sequence or fragments thereof can be constructed to conduct efficient screemng of e g , genetic mutations Array technology methods are well known and have general applicability and can be used to address a vanety of questions m molecular genetics mcludmg gene expression, genetic linkage, and genetic vanabihty (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 cancer, AIDS, metabolic disorders, and IDDM through detection of mutation m the CBUACD09 gene by the methods descπbed

In addition, cancer, AIDS, metabolic disorders, and IDDM, can be diagnosed by methods comprising determining from a sample denved from a subject an abnormally decreased or increased level of CBUACD09 polypeptide or CBUACD09 mRNA 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, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods Assay techniques that can be used to determine

13 levels of a protein, such as an CBUACD09 polypeptide, m a sample deπved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays

Thus in another aspect, the present invention relates to a diagonostic kit for a disease or suspectability to a disease, particularly cancer, AIDS, metabolic disorders, and IDDM, which comprises

(a) a CBUACD09 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 CBUACD09 polypeptide, preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or

(d) an antibody to a CBUACD09 polypeptide, preferably to the polypeptide of SEQ ID NO 2 It will be appreciated that m any such kit, (a), (b), (c) or (d) may comprise 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 mapping of relevant sequences to chromosomes according to the present mvention is an important first step m coπelatmg 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 coπelated with genetic map data Such data are found, for example, m V McKusick, Mendehan Inhentance m 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 (coinhentance of physically adjacent genes)

The differences in 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

14 Antibodies

The polypeptides of the mvention or their fragments or analogs thereof, or cells expressmg them can also be used as -mmunogens to produce antibodies immunospecific for the CBUACD09 polypeptides The term "immunospecific" means that the antibodies have substantiall greater affimty for the polypeptides of the mvention than their affimty for other related polypeptides m the pπor art

Antibodies generated against the CBUACD09 polypeptides can be obtamed by administering the polypeptides or epitope-beaπng fragments, analogs 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 include the hybπdoma 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 , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985) Techniques for the production of single chain antibodies (U S Patent No 4,946,778) can also be adapted to produce smgle chain antibodies to polypeptides of this mvention Also, transgenic 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 punfy the polypeptides by affimty chromatography Antibodies against CBUACD09 polypeptides may also be employed to treat cancer, AIDS, metabolic disorders, and IDDM, among others

Vaccines

Another aspect of the invention relates to a method for inducmg an immunological response in a mammal which comprises inoculating the mammal with CBUACD09 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from cancer, AIDS, metabolic disorders, and IDDM, among others Yet another aspect of the invention relates to a method of inducing immunological response m a mammal which compnses, delivering CBUACD09 polypeptide via a vector directing expression of CBUACD09 polynucleotide in vivo in order to induce such an immunological 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 immunological response in that mammal to a CBUACD09 polypeptide wherein the composition comprises a CBUACD09

15 polypeptide or CBUACD09 gene The vaccine formulation may further compnse a suitable carrier Since CBUACD09 polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc injection) Formulations suitable for parenteral administration mclude aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bactenostats and solutes which render the formulation lnstonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may mclude suspending agents or thickening agents The formulations may be presented m unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dπed condition requiring only the addition of the sterile liquid earner immediately pnor to use The vaccme formulation may also mclude 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 experimentation

Screening Assays

The CBUACD09 polypeptide of the present mvention may be employed m a screemng process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBUACD09 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, gands, 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 Cohgan et al , Current Protocols m Immunology 1(2) Chapter 5 (1991) CBUACD09 polypeptides are responsible for many biological functions, mcludmg many pathologies Accordingly, it is desirous to find compounds and drugs which stimulate CBUACD09 polypeptide on the one hand and which can inhibit the function of CBUACD09 polypeptide on the other hand In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as cancer, AIDS, metabolic disorders, and IDDM Antagonists may be employed for a vanety of therapeutic and prophylactic purposes for such conditions as cancer, AIDS, metabolic disorders, and IDDM In general, such screemng procedures may mvolve usmg appropπate cells which express the

CBUACD09 polypeptide or respond to CBUACD09 polypeptide of the present mvention Such cells mclude cells from mammals, yeast, DrosopMa or E colt Cells which express the CBUACD09 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBUACD09 polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a

16 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 CBUACD09 activity

The assays may simply test binding of a candidate compound wherem adherence to the cells beanng the CBUACD09 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 CBUACD09 polypeptide, using detection systems appropπate to the cells bearmg the CBUACD09 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 assays may simply comprise the steps of mixmg a candidate compound with a solution containing a CBUACD09 polypeptide to form a mixture, measuring CBUACD09 activity in the mixture, and comparing the CBUACD09 activity of the mixture to a standard

The CBUACD09 cDNA, protein and antibodies to the protem may also be used to configure assays for detecting the effect of added compounds on the production of CBUACD09 mRNA and protein in cells For example, an ELISA may be constructed for measuring secreted or cell associated levels of CBUACD09 protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of CBUACD09 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues

The CBUACD09 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 binding and crosslinking assays in which the CBUACD09 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 include biophysical techniques such as surface plasmon resonance and spectroscopy In addition to being used for purification and cloning of the receptor, these binding assays can be used to identify agonists and antagonists of CBUACD09 which compete with the binding of CBUACD09 to its receptors, if any Standard methods for conductmg screening assays are well understood in the art Examples of potential CBUACD09 polypeptide antagonists mclude antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc , as the case may be, of the CBUACD09 polypeptide, e g , a fragment of the ligands,

17 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 m another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc for CBUACD09 polypeptides, or compounds which decrease or enhance the production of CBUACD09 polypeptides, which compnses

(a) a CBUACD09 polypeptide, preferably that of SEQ ID NO 2,

(b) a recombinant cell expressing a CBUACD09 polypeptide, preferably that of SEQ ID NO 2,

(c) a cell membrane expressing a CBUACD09 polypeptide, preferably that of SEQ ID NO 2, or (d) antibody to a CBUACD09 polypeptide, preferably that of SEQ ID NO 2

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

Prophylactic and Therapeutic Methods This mvention provides methods of treating abnormal conditions such as, cancer, AIDS, metabolic disorders, and IDDM, related to both an excess of and insufficient amounts of CBUACD09 polypeptide activity

If the activity of CBUACD09 polypeptide is m excess, several approaches are available One approach compnses administering to a subject an inhibitor compound (antagonist) as hereinabove descπbed along with a pharmaceutically acceptable earner in an amount effective to inhibit the function of the CBUACD09 polypeptide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of CBUACD09 polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc in competition with endogenous CBUACD09 polypeptide may be admmistered Typical embodiments of such competitors comprise fragments of the CBUACD09 polypeptide

In still another approach, expression of the gene encoding endogenous CBUACD09 polypeptide can be inhibited usmg expression blocking techniques Known such techniques involve the use of antisense sequences, either internally generated or separately administered See, for example, O'Connor, J Neurochem (1991) 56 560 m Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988) Alternatively, oligonucleotides which form triple 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, Dervan et al , Science (1991) 251 1360 These o gomers can be administered per se or the relevant ohgomers can be expressed in vivo

18 For treating abnormal conditions related to an under-expression of CBUACD09 and its activity, several approaches are also avadable One approach compnses administering to a subject a therapeutically effective amount of a compound which activates CBUACD09 polypeptide, 1 e , an agonist as descnbed above, m combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition Alternatively, gene therapy may be employed to effect the endogenous production of CBUACD09 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 packagmg cell transduced with a retroviral plasmid vector containmg RNA encodmg a polypeptide of the present mvention such that the packagmg cell now produces infectious viral particles containmg the gene of interest These producer cells may be admmistered to a subject for engineering cells in vivo and expression of the polypeptide in vivo 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 administer a therapeutic amount of CBUACD09 polypeptides in combination with a suitable pharmaceutical earner

Formulation and Administration

Peptides, such as the soluble form of CBUACD09 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical earner Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient Such earners mclude but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof Formulation should suit the mode of administration, 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 m conjunction with other compounds, such as therapeutic compounds

Prefeπed forms of systemic administration of the pharmaceutical compositions mclude injection, typically by mtravenous injection Other injection routes, such as subcutaneous, intramuscular, or intrapentoneal, can be used Alternative means for systemic administration mclude transmucosal and transdermal admimstration usmg penetrants such as bile salts or fusidic acids or other detergents In addition, if properly formulated m enteπc or encapsulated formulations, oral administration may also be

19 possible Administration of these compounds may also be topical and/or localized, m the form of salves, pastes, gels and the like

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

Polypeptides used in treatment can also be generated endogenously m the subject, in treatment modalities often refeπed to as "gene therapy" as descπbed above Thus, for example, cells from a subject may be engmeered with a polynucleotide, such as a DNA or RNA 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, including but not limited to patents and patent applications, cited m 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

20 SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: WU, JI-SHENG ZHOU, JUAN XU, SHU-HUA

GU, BAI-WEI WANG, YA-XIN

(ii) TITLE OF THE INVENTION: The Human CI-B14.5a Homolog Gene: CBUACD09

(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:

21 ( viii ) ATTORNEY/AGENT INFORMATION :

(A) NAME: PRESTIA, PAUL F

(B) REGISTRATION NUMBER: 23,031

(C) REFERENCE/DOCKET NUMBER: GP-70407

(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: 531 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l;

GGCCCTTCAG TATGCGCGGA CGGAAGATGG CGTCCGCCAC CCGTCTCATC CAGCGGCTGC 60

GGAACTGGGC GTCCGGGCAT GACCTGCAGG GGAAGCTGCA GCTACGCTAC CAGGAGATCT 120 CCAAGCGAAC TCAGCCTCCT CCCAAGCTCC CTGTGGGTCC TAGCCACAAG CTCTCCAACA 180

ATTACTATTG CACTCGCGAT GGCCGCCGGG AATCTGTGCC CCCTTCCATC ATCATGTCGT 240'

CGCAGAAGGC GCTGGTGTCA GGCAAGCCAG CAGAGAGCTC TGCTGTAGCT GCCACTGAGA 300

AGAAGGCGGT GACTCCAGCT CCTCCCATAA AGAGGTGGGA GCTGTCCTCG GACCAGCCTT 360

ACCTGTGACA CTGCACCCTC ACGGCCACCC GACTACTTTG CCTCCTTGGA TTTCCTCCAG 420 GGAGAATGTG ACCTAATTTA TGACAAATAC GTAGAGCTCA GGTATCACTT CTAGTTTTAC 480

TTTAAAAAAT AAAAAAATAG AGACAGAGTC TCAAAAAAAA AAAAAAAAAA A 531

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 118 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

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

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

22 Met Arg Gly Arg Lys Met Ala Ser Ala Thr Arg Leu lie Gin Arg Leu

1 5 10 15

Arg Asn Trp Ala Ser Gly His Asp Leu Gin Gly Lys Leu Gin Leu Arg 20 25 30 Tyr Gin Glu lie Ser Lys Arg Thr Gin Pro Pro Pro Lys Leu Pro Val 35 40 45

Gly Pro Ser His Lys Leu Ser Asn Asn Tyr Tyr Cys Thr Arg Asp Gly

50 55 60

Arg Arg Glu Ser Val Pro Pro Ser lie lie Met Ser Ser Gin Lys Ala 65 70 75 80

Leu Val Ser Gly Lys Pro Ala Glu Ser Ser Ala Val Ala Ala Thr Glu

85 90 95

Lys Lys Ala Val Thr Pro Ala Pro Pro lie Lys Arg Trp Glu Leu Ser 100 105 110 Ser Asp Gin Pro Tyr Leu 115

23

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 CBUACD09 polypeptide of SEQ ID NO 2, or a nucleotide sequence complementary to said isolated polynucleotide

2 The polynucleotide of claim 1 wherein said polynucleotide comprises the nucleotide sequence contained in SEQ ID NO 1 encodmg the CBUACD09 polypeptide of SEQ ID N02

3 The polynucleotide of claim 1 wherein said polynucleotide comprises 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 claim 1 which is DNA or RNA

6 A DNA or RNA molecule comprising an expression system, wherein said expression system is capable of producing a CBUACD09 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 comprising the expression system of claim 6

8 A process for producmg a CBUACD09 polypeptide comprising cultuπng 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 CBUACD09 polypeptide thereof comprising transforming or transfecting a host cell with the expression system of claim 6 such that the host cell, under appropriate culture conditions, produces a CBUACD09 polypeptide

24

10 A CBUACD09 polypeptide compnsmg an ammo acid sequence which is at least 0% identical to the amino acid sequence of SEQ ID NO 2 over its entire length

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

12 An antibody immunospecific for the CBUACD09 polypeptide of claim 10

13 A method for the treatment of a subject in need of enhanced activity or expression of CBUACD09 polypeptide of claim 10 compnsmg

(a) administering 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 CBUACD09 polypeptide of SEQ ID NO 2 over its entire length, or a nucleotide sequence complementary 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 having need to inhibit activity or expression of CBUACD09 polypeptide of claim 10 compnsmg (a) administenng to the subject a therapeutically effective amount of an antagonist to said polypeptide, and/or

(b) administenng to the subject a nucleic acid molecule that inhibits the expression of the nucleotide sequence encodmg 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 CBUACD09 polypeptide of claim 10 m a subject compnsmg

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said CBUACD09 polypeptide m the genome of said subject, and/or

(b) analyzing for the presence or amount of the CBUACD09 polypeptide expression in a sample derived from said subject

25

16. A method for identifying compounds which inhibit (antagonize) or agonize the CBUACD09 polypeptide of claim 10 which comprises:

(a) contacting a candidate compound with cells which express the CBUACD09 polypeptide (or cell membrane expressing CBUACD09 polypeptide) or respond to CBUACD09 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 CBUACD09 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 CBUACD09 polypeptide.

26