WO1999021990A1 - Cbmaad07: a voltage dependent anion channel protein - Google Patents

Abstract

CBMAAD07 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CBMAAD07 polypeptides and polynucleotides in the design of protocols for the treatment of microsomal and neurological disorders, among others, and diagnostic assays for such conditions.

Description

CBMAAD07: A VOLTAGE DEPENDENT ANION CHANNEL PROTEIN

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 VDAC gene family, hereinafter referred to as CBMAAD07. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION

Voltage-dependent anion channel proteins are mitochondria! outer membrane proteins which function as pores for anion transfer. To date, two types of VDAC genes have been cloned in humans. Both the human type 1 and 2 VDAC share homology to proteins in organisms ranging from yeast to mouse. This indicates that the VDAC gene family has an established, proven history as therapeutic targets. Clearly there is a need for identification and characterization of further members of the VDAC gene family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, microsomal and neurological disorders.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to CBMAAD07 polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such CBMAAD07 polypeptides and polynucleotides. Such uses include the treatment of microsomal and neurological disorders, 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 CBMAAD07 imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBMAAD07 activity or levels.

DESCRIPTION OF THE INVENTION Definitions

The following definitions are provided to facilitate understanding of certain terms used frequently herein. "CBMAAD07" refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or an allelic variant thereof.

"CBMAAD07 activity or CBMAAD07 polypeptide activity" or "biological activity of the CBMAAD07 or CBMAAD07 polypeptide" refers to the metabolic or physiologic function of said CBMAAD07 including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and immunogenic activities of said CBMAAD07.

"CBMAAD07 gene" refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:l or allelic variants thereof and/or their complements. "Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, 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 polyribonucleotide or polydeoxribonucleotide, which may be unmodified R A 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 triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically 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 oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins Polypeptides may contain ammo acids other than the 20 gene-encoded amino acids "Polypeptides" include ammo 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 descπbed m basic texts and in more detailed monographs, as well as in a voluminous research literature Modifications can occur anywhere in a polypeptide, including 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 m 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 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 hpid or lipid derivative, covalent attachment of phosphotidylmositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystme, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodmation, methylation, myπstoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as argmylation, and ubiquitmation See, for instance, PROTEINS - STRUCTURE AND

MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York, 1993 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 Sci (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 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 ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical variant of a polypeptide differs m ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, m many regions, identical A variant and reference polypeptide may differ m 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 variant of a polynucleotide or polypeptide may be a naturally occurπng such as an allelic variant, or it may be a variant 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, Griffin, A M , and Griffin, H G , eds , Humana Press, New Jersey, 1994, SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Hemje, G , Academic Press, 1987, and

SEQUENCE ANALYSIS PRIMER, Gπbskov, 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 (Caπllo, H , and Lipton, D , SIAMJ Applied Math (1988) 48 1073) Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed m Guide to Huge Computers, Martin J Bishop, ed , Academic Press, San Diego, 1994, and Caπllo, H , and Lipton, D , SIAM J Applied Math (1988) 48 1073 Methods to determine identity and similarity are codified m computer programs Preferred computer program methods to determine identity and similarity 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 m 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 m the reference sequence or m 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 amino 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 include up to five ammo acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO 2 In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference ammo acid sequence, up to 5% of the ammo acid residues m the reference sequence may be deleted or substituted with another ammo acid, or a number of ammo acids up to 5% of the total amino acid residues m the reference sequence may be inserted into the reference sequence These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence

Polypeptides of the Invention

In one aspect, the present invention relates to CBMAAD07 polypeptides (or CBMAAD07 proteins) The CBMAAD07 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 preferred Also included within CBMAAD07 polypeptides are polypeptides having the amino acid sequence which have at least 80% identity to the polypeptide having 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 CBMAAD07 polypeptide exhibit at least one biological activity of CBMAAD07 The CBMAAD07 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 ammo acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid m purification such as multiple histidme residues, or an additional sequence for stability duπng recombinant production Fragments of the CBMAAD07 polypeptides are also included in the invention. A fragment is a polypeptide having an amino acid sequence that entirely is the same as part, but not all, of the ar ino acid sequence of the aforementioned CBMAAD07 polypeptides. As with CBMAAD07 polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61- 80, 81-100, and 101 to the end of CBMAAD07 polypeptide. In this context "about" includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes. Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of CBMAAD07 polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl teπninus. Also preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet- forming regions, turn and tam-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface- fon ing regions, substrate binding region, and high antigenic index regions. Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate CBMAAD07 activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human. Preferably, all of these polypeptide fragments retain the biological activity of the CBMAAD07, including antigenic activity. Variants of the defined sequence and fragments also form part of the present invention. Preferred variants are those that vary from the referents by conservative amino acid substitutions — i.e., those that substitute a residue with another of like characteristics. 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 variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination. The CBMAAD07 polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include 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 in the art. Polynucleotides of the Invention

Another aspect of the invention relates to CBMAAD07 polynucleotides. CBMAAD07 polynucleotides include isolated polynucleotides which encode the CBMAAD07 polypeptides and fragments, and polynucleotides closely related thereto. More specifically, CBMAAD07 polynucleotide of the invention include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding a CBMAAD07 polypeptide of SEQ ID NO: 2, and polynucleotide having the particular sequence of SEQ ID NO: 1. CBMAAD07 polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 82% identity over its entire length to a nucleotide sequence encoding the CBMAAD07 polypeptide of SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence that is at least 82% 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% being the most preferred. Also included under CBMAAD07 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 CBMAAD07 polynucleotides.

CBMAAD07 of the invention is structurally related to other proteins of the VDAC gene family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO: 1) encoding human CBMAAD07. The cDNA sequence of SEQ ID NO: 1 contains an open reading frame (nucleotide number 100 to 948) encoding a polypeptide of 283 amino acids of SEQ ID NO:2. The amino acid sequence of Table 2 (SEQ ID NO:2) has about 98% identity (using FASTA) in 283 amino acid residues with mouse VDAC3 (Sampson MJ, et al. Genomics, 1996;36:192-196). The nucleotide sequence of Table 1 (SEQ ID NO:l) has about 81.7% identity (using FASTA) in 1414 nucleotide residues with mouse VDAC3 (Sampson MJ, et al. Genomics, 1996;36:192-196). Thus, CBMAAD07 polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art.

Table 1"

GGGTTTGAAGACCTTCAGCGTTGCCCTGGCGGACAGAGACAGGCCCTCGGGGTGGAGGTCTTTGGTTTC ATAAGAGCCTGAGAGAGATTTTTCTAAGATATGTGTAACACACCAACGTACTGTGACCTAGGAAAGGCT GCTAAGGATGTCTTCAACAAAGGATATGGCTTTGGCATGGTCAAGATAGACCTGAAAACCAAGTCTTGT AGTGGAGTGGAATTTTCTACTTCTGGTCATGCTTACACTGATACAGGGAAAGCATCAGGCAACCTAGAA ACCAAATATAAGGTCTGTAACTATGGACTTACCTTCACCCAGAAATGGAACACAGACAATACTCTAGGG ACAGAAATCTCTTGGGAGAATAAGTTGGCTGAAGGGTTGAAACTGACTCTTGATACCATATTTGTACCG AACACAGGAAAGAAGAGTGGGAAATTGAAGGCCTCCTATAAACGGGATTGTTTTAGTGTTGGCAGTAAT GTTGATATAGATTTTTCTGGACCAACCATCTATGGCTGGGCTGTGTTGGCCTTCGAAGGGTGGCTTGCT GGCTATCAGATGAGTTTTGACACAGCCAAATCCAAACTGTCACAGAATAATTTCGCCCTGGGTTACAAG GCTGCGGACTTCCAGCTGCACACACATGTGAACGATGGCACTGAATTTGGAGGTTCTATCTACCAGAAG GTGAATGAGAAGATTGAAACATCCATAAACCTTGCTTGGACAGCTGGGAGTAACAACACCCGTTTTGGC ATTGCTGCTAAGTACATGCTGGATTGTAGAACTTCTCTCTCTGCTAAAGTAAATAATGCCAGCCTGATT GGACTGGGTTATACTCAGACCCTTCGACCAGGAGTCAAATTGACTTTATCAGCTTTAATCGATGGGAAG AACTTCAGTGCAGGAGGTCACAAGGTTGGCTTGGGATTTGAACTGGAAGCTTAATGTGGTTTGAGGAAA GCATCAGATTTGTCCCTGGAAGTGAAGAGAAATGAACCCACTATGTTTTGGCCTTAAAATTCTTCTGTG AAATTTCAAAAGTGTGAACTTTTTATTCTTCCAAAGAATTGTAATCCTCCCCACACTGAAGTCTAGGGG TTGCGAATCCCTCCTGAGGGAGATGCTTGAAGGCATGCCTGGAAGTTGTCATGTTTGTGCCACGTTTCA GTTCAGTTCTGAAGTGTTATTAAATGTGTTCCTCAGCGACAGTGTAGCGTCATGTTAGAGGAGACGATC TGACCCACCAGTTTGTACATCACGTCCTGCATGTCCCACACCATTTTTTCATGACCTTGTAATATACTG GTCTCTGTGCTATAGTGGAATCTTTGGTTTTGCATCATAGTAAAATAAAATAAACCCATCACATTTGGA ACATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

A nucleotide sequence of a human CBMAAD07 (SEQ ID NO: 1).

Table 2^b

MCNTPTYCDLGKAAKDVFNKGYGFGMVKIDLKTKSCSGVEFSTSGHAYTDTGKASG LETKYKVCNYGL TFTQKWNTDNT GTEIS ENK AEGLKLTLDTIFVPNTGKKSGKLKASYI RDCFSVGSNVDIDFSGPTI YG AVI-AFEG AGYQMSFDTAKSKLSQ NFALGYKAADFQLHTHVNDGTEFGGS IYQKVNEKIETSIN LAWTAGSNNTRFGIAAKYMLDCRTSLSAKWTNAS IGLGYTQT RPGVK TLSALIDGKNFSAGGHKVG LGFELEA

An amino acid sequence of a human CBMAAD07 (SEQ ID NO: 2).

One polynucleotide of the present invention encoding CBMAAD07 may be obtained using standard cloning and screening, from a cDNA library derived from mRNA in 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 l, Nature (1995) 377 Supp:3-174). Polynucleotides of the invention 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 CBMAAD07 polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 100 to 948 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 CBMAAD07 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 in 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 purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al. , Proc Natl Acad Sci USA (1989) 86:821- 824, or is an HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

Further preferred embodiments are polynucleotides encoding CBMAAD07 variants comprise the amino acid sequence CBMAAD07 polypeptide of Table 2 (SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 arnino acid residues are substituted, deleted or added, in any combination.

The present invention further relates to polynucleotides that hybridize to the herein above- described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the term "stringent conditions" means hybridization 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 invention, which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding CBMAAD07 polypeptide and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to the CBMAAD07 gene. Such hybridization 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 comprise 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 encoding CBMAAD07 polypeptide, including homologs and orthologs from species other than human, comprises the steps of screening an appropriate library under stingent hybridization 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 in another aspect, CBMAAD07 polynucleotides of the present invention further include a nucleotide sequence compnsing a nucleotide sequence that hybridize under stringent condition to a nucleotide sequence having SEQ ID NO 1 or a fragment thereof Also mcluded with CBMAAD07 polypeptides are polypeptide compnsing amino acid sequence encoded by nucleotide sequence obtained by the above hybndization condition Such hybndization techniques are well known to those of skill in the art Stringent 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 in 0 lx SSC at about 65°C

The polynucleotides and polypeptides of the present invention may be employed as research reagents and matenals for discover}' of treatments and diagnostics to animal and human disease

Vectors, Host Cells, Expression

The present invention also relates to vectors which compnse a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs denved from the DNA constructs of the present invention

For recombinant production, host cells can be genetically engmeered to incorporate expression systems or portions thereof for polynucleotides of the present invention Introduction of polynucleotides into 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 Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd E , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, canonic hpid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E coli, Streptomyces and Bacillus subtihs cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as D osophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells, and plant cells A great variety of expression systems can be used. Such systems include, among others, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.

If the CBMAAD07 polypeptide is to be expressed for use in 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 prior to use in the screening assay. If CBMAAD07 polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered. CBMAAD07 polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.

Diagnostic Assays

This invention also relates to the use of CBMAAD07 polynucleotides for use as diagnostic reagents. Detection of a mutated form of CBMAAD07 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 CBMAAD07. Individuals carrying mutations in the CBMAAD07 gene may be detected at the DNA level by a vanety of techniques

Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy matenal The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques pnor to analysis RNA or cDNA may also be used in similar fashion Deletions and insertions can be detected by a change m size of the amplified product in comparison to the normal genotype Pomt mutations can be identified by hybndizing amplified DNA to labeled CBMAAD07 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting 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 etal , Proc Natl Acad Sci USA (1985) 85 4397-4401 In another embodiment, an array of oligonucleotides probes compnsing CBMAAD07 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening 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 in molecular genetics including gene expression, genetic linkage, and genetic variability (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 microsomal and neurological disorders through detection of mutation in the CBMAAD07 gene by the methods descπbed

In addition, microsomal and neurological disorders, can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of CBMAAD07 polypeptide or CBMAAD07 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 protein, such as an CBMAAD07 polypeptide, in a sample denved from a host are well-known to those of skill in the art Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays

Thus m another aspect, the present invention relates to a diagonostic kit for a disease or suspectability to a disease, particularly microsomal and neurological disorders, which compπses (a) a CBMAAD07 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 CBMAAD07 polypeptide, preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or

(d) an antibody to a CBMAAD07 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 comprise a substantial component

Chromosome Assays

The nucleotide sequences of the present invention 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 according to the present invention is an important first step in correlating those sequences with gene associated disease Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data Such data are found, for example, in V McKusick, Mende an Inheritance in Man (available on line through Johns Hopkins Umversity 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 m any normal individuals, then the mutation is likely to be the causative agent of the disease

Antibodies

The polypeptides of the invention or their fragments or analogs thereof, or cells expressmg them can also be used as rmmunogens to produce antibodies immunospecific for the CBMAAD07 polypeptides The term "immunospecific" means that the antibodies have substantiall greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the pnor art

Antibodies generated against the CBMAAD07 polypeptides can be obtained by admmisteπng the polypeptides or epitope-beanng fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al. , Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole etal, 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 single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms including other mammals, may be used to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.

Antibodies against CBMAAD07 polypeptides may also be employed to treat microsomal and neurological disorders, among others.

Vaccines Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with CBMAAD07 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from microsomal and neurological disorders, among others. Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering CBMAAD07 polypeptide via a vector directing expression of CBMAAD07 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 CBMAAD07 polypeptide wherein the composition comprises a CBMAAD07 polypeptide or CBMAAD07 gene. The vaccine formulation may further comprise a suitable carrier. Since CBMAAD07 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 include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use The vaccine formulation may also include adjuvant systems for enhancing the lmmunogemcity of the formulation, such as oil-m 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 CBMAAD07 polypeptide of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBMAAD07 polypeptide of the present mvention Thus, polypeptides of the invention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures These agonists or antagomsts may be natural or modified substrates, ligands, receptors, enzymes, as the case may be, of the polypeptide of the present invention, or may be structural or functional mimetics of the polypeptide of the present invention See Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991) CBMAAD07 polypeptides are responsible for many biological functions, mcludmg many pathologies Accordingly, it is desirous to find compounds and drugs which stimulate CBMAAD07 polypeptide on the one hand and which can inhibit the function of CBMAAD07 polypeptide on the other hand In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as microsomal and neurological disorders Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as microsomal and neurological disorders

In general, such screening procedures may involve using appropπate cells which express the CBMAAD07 polypeptide or respond to CBMAAD07 polypeptide of the present mvention Such cells mclude cells from mammals, yeast, Drosophύa or E coh Cells which express the CBMAAD07 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBMAAD07 polypeptide are then contacted with a test compound to observe bmdmg, 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 CBMAAD07 activity

The assays may simply test bmdmg of a candidate compound wherein adherence to the cells bearing the CBMAAD07 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 CBMAAD07 polypeptide, using detection systems appropπate to the cells bearing the CBMAAD07 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 mixing a candidate compound with a solution containing a CBMAAD07 polypeptide to form a mixture, measuπng CBMAAD07 activity in the mixture, and comparing the CBMAAD07 activity of the mixture to a standard

The CBMAAD07 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 CBMAAD07 mRNA and protein m cells For example, an ELISA may be constructed for measuring secreted or cell associated levels of CBMAAD07 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 CBMAAD07 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues

The CBMAAD07 protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known the art These include, but are not limited to, ligand binding and crosshnkmg assays m which the CBMAAD07 is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotmylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy In addition to being used for purification and cloning of the receptor, these binding assays can be used to identify agonists and antagonists of CBMAAD07 which compete with the binding of CBMAAD07 to its receptors, if any Standard methods for conducting screening assays are well understood m the art

Examples of potential CBMAAD07 polypeptide antagonists mclude antibodies or, in some cases, ohgonucleotides or proteins which are closely related to the hgands, substrates, receptors, enzymes, as the case may be, of the CBMAAD07 polypeptide, e g , a fragment of the hgands, substrates, receptors, enzymes, or small molecules which bind 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 agonists, antagonists, hgands, receptors, substrates, enzymes for CBMAAD07 polypeptides, or compounds which decrease or enhance the production of CBMAAD07 polypeptides, which comprises

(a) a CBMAAD07 polypeptide, preferably that of SEQ ID NO 2,

(b) a recombinant cell expressing a CBMAAD07 polypeptide, preferably that of SEQ ID NO 2,

(c) a cell membrane expressing a CBMAAD07 polypeptide, preferably that of SEQ ID NO 2, or (d) antibody to a CBMAAD07 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 invention provides methods of treating abnormal conditions such as, microsomal and neurological disorders, related to both an excess of and insufficient amounts of CBMAAD07 polypeptide activity.

If the activity of CBMAAD07 polypeptide is in excess, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the CBMAAD07 polypeptide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of CBMAAD07 polypeptides still capable of binding the ligand, substrate, enzymes, receptors in competition with endogenous CBMAAD07 polypeptide may be administered. Typical embodiments of such competitors comprise fragments of the CBMAAD07 polypeptide.

In still another approach, expression of the gene encoding endogenous CBMAAD07 polypeptide can be inhibited using 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 in 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 oligomers can be administered er se or the relevant oligomers can be expressed in vivo. For treating abnormal conditions related to an under-expression of CBMAAD07 and its activity, several approaches are also available. One approach comprises aαlministering to a subject a therapeutically effective amount of a compound which activates CBMAAD07 polypeptide, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of CBMAAD07 by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding 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 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 CBMAAD07 polypeptides m combination with a suitable pharmaceutical earner

Formulation and Administration Peptides, such as the soluble form of CBMAAD07 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated m combination with a suitable pharmaceutical earner Such formulations compπse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient Such earners include 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 in conjunction with other compounds, such as therapeutic compounds Preferred forms of systemic admimstration of the pharmaceutical compositions include injection, typically by mtravenous injection Other injection routes, such as subcutaneous, intramuscular, or mtrapentoneal, can be used Alternative means for systemic admimstration mclude transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents In addition, if properly formulated m entenc or encapsulated formulations, oral admimstration may also be 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 in the range of 0 1-100 μg/kg of subject Wide vanations in the needed dosage, however, are to be expected in view of the vanety of compounds available and the differing efficiencies of vanous routes of admimstration For example, oral administration would be expected to require higher dosages than admimstration 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 in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above. Thus, for example, cells from a subject may be engineered 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 into the subject

All publications, including but not limited to patents and patent applications, cited in this specification are herein 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: ZHANG, QING-HUA WANG, YA-XIN

(ii) TITLE OF THE INVENTION: CBMAAD07: A VOLTAGE DEPENDENT

ANION CHANNEL PROTEIN

(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-70304 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 610-407-0700

(B) TELEFAX: 610-407-0701

(C) TELEX: 846169

(2) INFORMATION FOR SEQ ID NO:l;

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1414 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single CD) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

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

GGGTTTGAAG ACCTTCAGCG TTGCCCTGGC GGACAGAGAC AGGCCCTCGG GGTGGAGGTC 60

TTTGGTTTCA TAAGAGCCTG AGAGAGATTT TTCTAAGATA TGTGTAACAC ACCAACGTAC 120 TGTGACCTAG GAAAGGCTGC TAAGGATGTC TTCAACAAAG GATATGGCTT TGGCATGGTC 180

AAGATAGACC TGAAAACCAA GTCTTGTAGT GGAGTGGAAT TTTCTACTTC TGGTCATGCT 240

TACACTGATA CAGGGAAAGC ATCAGGCAAC CTAGAAACCA AATATAAGGT CTGTAACTAT 300

GGACTTACCT TCACCCAGAA ATGGAACACA GACAATACTC TAGGGACAGA AATCTCTTGG 360

GAGAATAAGT TGGCTGAAGG GTTGAAACTG ACTCTTGATA CCATATTTGT ACCGAACACA 420 GGAAAGAAGA GTGGGAAATT GAAGGCCTCC TATAAACGGG ATTGTTTTAG TGTTGGCAGT 480

AATGTTGATA TAGATTTTTC TGGACCAACC ATCTATGGCT GGGCTGTGTT GGCCTTCGAA 540

GGGTGGCTTG CTGGCTATCA GATGAGTTTT GACACAGCCA AATCCAAACT GTCACAGAAT 600

AATTTCGCCC TGGGTTACAA GGCTGCGGAC TTCCAGCTGC ACACACATGT GAACGATGGC 660

ACTGAATTTG GAGGTTCTAT CTACCAGAAG GTGAATGAGA AGATTGAAAC ATCCATAAAC 720 CTTGCTTGGA CAGCTGGGAG TAACAACACC CGTTTTGGCA TTGCTGCTAA GTACATGCTG 780

GATTGTAGAA CTTCTCTCTC TGCTAAAGTA AATAATGCCA GCCTGATTGG ACTGGGTTAT 840

ACTCAGACCC TTCGACCAGG AGTCAAATTG ACTTTATCAG CTTTAATCGA TGGGAAGAAC 900

TTCAGTGCAG GAGGTCACAA GGTTGGCTTG GGATTTGAAC TGGAAGCTTA ATGTGGTTTG 960

AGGAAAGCAT CAGATTTGTC CCTGGAAGTG AAGAGAAATG AACCCACTAT GTTTTGGCCT 1020 TAAAATTCTT CTGTGAAATT TCAAAAGTGT GAACTTTTTA TTCTTCCAAA GAATTGTAAT 1080

CCTCCCCACA CTGAAGTCTA GGGGTTGCGA ATCCCTCCTG AGGGAGATGC TTGAAGGCAT 1140

GCCTGGAAGT TGTCATGTTT GTGCCACGTT TCAGTTCAGT TCTGAAGTGT TATTAAATGT 1200

GTTCCTCAGC GACAGTGTAG CGTCATGTTA GAGGAGACGA TCTGACCCAC CAGTTTGTAC 1260

ATCACGTCCT GCATGTCCCA CACCATTTTT TCATGACCTT GTAATATACT GGTCTCTGTG 1320 CTATAGTGGA ATCTTTGGTT TTGCATCATA GTAAAATAAA ATAAACCCAT CACATTTGGA 1380

ACATAAAAAA AAAAAAAAAA AAAAAAAAAA AAAA 1414

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 283 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Cys Asn Thr Pro Thr Tyr Cys Asp Leu Gly Lys Ala Ala Lys Asp 1 5 10 15

Val Phe Asn Lys Gly Tyr Gly Phe Gly Met Val Lys lie Asp Leu Lys

20 25 30

Thr Lys Ser Cys Ser Gly Val Glu Phe Ser Thr Ser Gly His Ala Tyr 35 40 45

Thr Asp Thr Gly Lys Ala Ser Gly Asn Leu Glu Thr Lys Tyr Lys Val

50 55 60

Cys Asn Tyr Gly Leu Thr Phe Thr Gin Lys Trp Asn Thr Asp Asn Thr 65 70 75 80 Leu Gly Thr Glu lie Ser Trp Glu Asn Lys Leu Ala Glu Gly Leu Lys

85 90 95

Leu Thr Leu Asp Thr lie Phe Val Pro Asn Thr Gly Lys Lys Ser Gly

100 105 110

Lys Leu Lys Ala Ser Tyr Lys Arg Asp Cys Phe Ser Val Gly Ser Asn 115 120 125

Val Asp lie Asp Phe Ser Gly Pro Thr lie Tyr Gly Trp Ala Val Leu

130 135 140

Ala Phe Glu Gly Trp Leu Ala Gly Tyr Gin Met Ser Phe Asp Thr Ala 145 150 155 160 Lys Ser Lys Leu Ser Gin Asn Asn Phe Ala Leu Gly Tyr Lys Ala Ala

165 170 175

Asp Phe Gin Leu His Thr His Val Asn Asp Gly Thr Glu Phe Gly Gly

180 185 190

Ser lie Tyr Gin Lys Val Asn Glu Lys lie Glu Thr Ser lie Asn Leu 195 200 205

Ala Trp Thr Ala Gly Ser Asn Asn Thr Arg Phe Gly lie Ala Ala Lys

210 215 220

Tyr Met Leu Asp Cys Arg Thr Ser Leu Ser Ala Lys Val Asn Asn Ala 225 230 235 240 Ser Leu lie Gly Leu Gly Tyr Thr Gin Thr Leu Arg Pro Gly Val Lys

245 250 255

Leu Thr Leu Ser Ala Leu lie Asp Gly Lys Asn Phe Ser Ala Gly Gly

260 265 270

His Lys Val Gly Leu Gly Phe Glu Leu Glu Ala 275 280

Claims

What is claimed is:

1. An isolated polynucleotide comprising a nucleotide sequence that has at least 82% identity over its entire length to a nucleotide sequence encoding the CBMAAD07 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:l encoding the CBMAAD07 polypeptide of SEQ ED N02.

3. The polynucleotide of claim 1 wherein said polynucleotide comprises a nucleotide sequence that is at least 82% 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.

. 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 CBMAAD07 polypeptide, which has at least 99% 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 producing a CBMAAD07 polypeptide comprising culturing 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 CBMAAD07 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 CBMAAD07 polypeptide.

10. A CBMAAD07 polypeptide comprising an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO:2 over its entire length.

11. The polypeptide of claim 10 which comprises the amino acid sequence of SEQ ID NO:2.

12. An antibody immunospecific for the CBMAAD07 polypeptide of claim 10.

13. A method for the treatment of a subject in need of enhanced activity or expression of CBMAAD07 polypeptide of claim 10 comprising:

(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 comprising a nucleotide sequence that has at least 82% identity to a nucleotide sequence encoding the CBMAAD07 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 CBMAAD07 polypeptide of claim 10 comprising: (a) administering to the subject a therapeutically effective amount of an antagonist to said polypeptide; and/or

(b) administering 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 ligand, substrate , or receptor.

15. A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of CBMAAD07 polypeptide of claim 10 in a subject comprising:

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said CBMAAD07 polypeptide in the genome of said subject; and/or

(b) analyzing for the presence or amount of the CBMAAD07 polypeptide expression in a sample derived from said subject.

16. A method for identifying compounds which inhibit (antagonize) or agonize the CBMAAD07 polypeptide of claim 10 which comprises:

(a) contacting a candidate compound with cells which express the CBMAAD07 polypeptide (or cell membrane expressing CBMAAD07 polypeptide) or respond to CBMAAD07 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 CBMAAD07 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 CBMAAD07 polypeptide.