WO1999063069A1 - Human mitochondrial processing protease beta subunit (mppb) homolog gene - Google Patents

Abstract

The CBDAOH03 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilising CBDAOH03 polypeptides and polynucleotides in therapy, and diagnostic assays for such.

Description

Human Mitochondria! Processing Protease Beta Subunit (MPPB) Homolog Gene

Field of the Invention

This mvention relates to newly identified polypeptides and polynucleotides encodmg such polypeptides, to their use m therapy and m identifying compounds which may be agomsts, antagonists and /or mhibitors which are potentially useful in therapy, and to production of such polypeptides and polynucleotides

Background of the Invention The drug discovery process is currently undergoing a fundamental revolution as it embraces

'functional genomics', that is, high throughput genome- or gene-based biology This approach is rapidly superceding earlier approaches based on 'positional cloning' A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available There is a continuing need to identify and characterise further genes and their related polypepudes/protens, as targets for drug discovery

Summary of the Invention

The present invention relates to CBDAOH03, m particular CBDAOH03 polypeptides and CBDAOH03 polynucleotides, recombinant materials and methods for their production In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of cancer, ADDS, metabolic disorders, and Diabetes Melhtus, hereinafter referred to as "the Diseases", amongst others In a further aspect, the invention relates to methods for identifymg agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with CBDAOH03 imbalance with the identified compounds In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropnate CBDAOH03 activity or levels

Description of the Invention

In a first aspect, the present mvention relates to CBDAOH03 polypeptides Such peptides include isolated polypetides comprising an ammo acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2 Such polypeptides mclude those compnsing the ammo acid of SEQ ID NO 2

Further peptides of the present invention include isolated polypeptides m which the ammo acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2 Such polypeptides mclude the polypeptide of SEQ ID NO 2

Further peptides of the present invention include isolated polypeptides encoded by a polynucleotide compπsmg the sequence contamed m SEQ ID NO 1

Polypeptides of the present invention are believed to be members of the metallopeptidase family of polypeptides They are therefore of interest because mitochondπal processing peptidase (MPP) removes the amino-terminal leading sequence from nuclearly-encoded mitochondπal protems, and MPPB is the beta-subunit of MPP These properties are hereinafter referred to as "CBDAOH03 activity" or "CBDAOH03 polypeptide activity" or "biological activity of CBDAOH03" Also included amongst these activities are antigenic and lmmunogenic activities of said CBDAOH03 polypeptides, in particular the antigenic and immunogenic activities of the polypeptide of SEQ ID NO 2 Preferably, a polypeptide of the present invention exhibits at least one biological activity of CBDAOH03 The polypeptides of the present invention may be m the form of the "mature" protem 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 puπfication such as multiple histidine residues, or an additional sequence for stability duπng recombmant production The present invention also mcludes mclude vaπants of the aforementioned polypetides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like characteπstics Typical such substitutions are among Ala, Val. Leu and lie, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin, and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly preferred are vaπants in which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acids are substituted, deleted, or added m any combmation Polypeptides of the present mvention 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 in the art In a further aspect, the present mvention relates to CBDAOH03 polynucleotides Such polynucleotides include isolated polynucleotides compπsmg a nucleotide sequence encodmg a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the ammo acid sequence of SEQ LD NO 2, over the entire length of SEQ ID NO 2 In this regard, polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred Such polynucleotides mclude a polynucleotide compπsmg the nucleotide sequence contamed m SEQ ID NO 1 encodmg the polypeptide of SEQ ID NO 2 Further polynucleotides of the present mvention mclude isolated polynucleotides compπsmg a nucleotide sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to a nucleotide sequence encodmg a polypeptide of SEQ ID NO 2, over the entire codmg region In this regard, polynucleotides which have at least 97% identity are highly prefeπed, whilst those with at least 98-99% identity are more highly prefeπed, and those with at least 99% identity are most highly prefeπed

Further polynucleotides of the present mvention mclude isolated polynucleotides compπsmg a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ID NO 1 over the entire length of SEQ ID NO 1 In this regard, polynucleotides which have at least 97% identity are highly prefeπed, whilst those with at least 98-99% identiy are more highly prefeπed, and those with at least 99% identity are most highly prefeπed Such polynucleotides mclude a polvnucleotide compπsmg the polynucleotide of SEQ ID NO 1 as well as the polynucleotide of SEQ ID NO 1

The invention also provides polynucleotides which are complementary to all the above descπbed polynucleotides The nucleotide sequence of SEQ ID NO 1 shows homology with rat mitochondπal processing peptidase beta-subunit (MPPB) LI 2965 (N Paces,et al Proc Νatl Acad Sci U S A 1993,90 5355- 5358 ) The nucleotide sequence of SEQ ID NO 1 is a cDNA sequence and compnses a polypeptide encodmg sequence (nucleotide 14 to 1480) encodmg a polypeptide of 489 ammo acids, the polypeptide of SEQ ID NO 2 The nucleotide sequence encoding the polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in SEQ ID NO 1 or it may be a sequence other than the one contained in SEQ ID NO 1, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO 2 The polypeptide of the SEQ ID NO 2 is structurally related to other protems of the metallopeptidase family, havmg homology and/or structural similaπty with Rat mitochondπal processing protease beta subunit precursor Q03346 (V Paces,et al Proc Natl Acad Sci U S A 1993,90 5355-5358 )

Prefeπed polypeptides and polynucleotides of the present mvention are expected to have, inter aha, similar biological functions/properties to their homologous polypeptides and polynucleotides Furthermore, prefeπed polypeptides and polynucleotides of the present mvention have at least one CBDAOH03 activity

Polynucleotides of the present mvention may be obtamed, usmg standard cloning and screemng techniques, from a cDNA hbrary deπved from mRNA m cells of human umbilical 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 O , et al , Nature (1995) 377

Supp 3-174) Polynucleotides of the mvention can also be obtamed from natural sources such as genomic DNA hbranes or can be synthesized usmg well known and commercially available techniques

When polynucleotides of the present mvention are used for the recombmant production of polypeptides of the present mvention, the polynucleotide may mclude the codmg sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide m reading frame with other codmg 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 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 Sci USA (1989) 86 821-824. or is an HA tag The polynucleotide may also contain non-coding 5 ' and 3 sequences, such as transcnbed, non-translated sequences, splicing and polyadenylation signals, πbosome binding sites and sequences that stabilize mRNA Further embodiments of the present mvention mclude polynucleotides encodmg polypeptide vaπants which compπse the ammo acid sequence of SEQ ID NO 2 and m which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, ammo acid residues are substituted, deleted or added, m am combination

Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contamed in SEQ ID NO 1, may be used as hybπdization probes for cDNA and genomic DNA or as pπmers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encodmg polypeptides of the present mvention and to isolate cDNA and genomic clones of other genes (mcludmg genes encoding homologs and orthologs from species other than human) that have a high sequence similaπty to SEQ ID NO 1 Typically these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95 % identical to that of the referent The probes or pπmers will generally compπse at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides Particularly prefeπed probes will have between 30 and 50 nucleotides A polynucleotide encodmg a polypeptide of the present mvention, mcludmg homologs and orthologs from species other than human, may be obtamed by a process which compnses the steps of screemng an appropπate hbrary under stringent hybπdization conditions with a labeled probe havmg the sequence of SEQ ID NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containmg said polynucleotide sequence Such hybπdization techniques are well known to the skilled artisan Prefeπed stringent hybπdization conditions mclude overnight incubation at 42°C m a solution compπsmg 50% forrnamide, 5xSSC (150mM NaCl, 15mM tπsodium 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 Thus the present mvention also mcludes polynucleotides obtamable by screemng an appropπate library under stingent hybπdization conditions with a labeled probe havmg the sequence of SEQ LD NO 1 or a fragment thereof

The skilled artisan will appreciate that, m many cases, an isolated cDNA sequence will be incomplete, m that the region codmg for the polypeptide is cut short at the 5' end of the cDNA This is a consequence of reverse transcπptase, an enzyme with inherently low 'processivrty' (a measure of the ability of the enzyme to remam attached to the template durmg the polymeπsation reaction), failing to complete a DNA copy of the mRNA template during 1st strand cDNA synthesis

There are several methods available and well known to those skilled in the art to obtain full-length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman et al , PNAS USA 85, 8998- 9002, 1988) Recent modifications of the technique, exemplified by the Marathon™' technology (Clontech Laboratories Inc ) for example, have significantly simplified the search for longer cDNAs In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence hgated onto each end Nucleic acid amplification (PCR) is then earned out to amplify the 'missing' 5' end of the cDNA using a combination of gene specific and adaptor specific oligonucleotide primers The PCR reaction is then repeated usmg 'nested' primers, that is, pπmers designed to anneal within the amplified product (typically an adaptor specific pπmer that anneals further 3' m the adaptor sequence and a gene specific pnmer that anneals further 5' in the known gene sequence) The products of this reaction can then be analysed by DNA sequencmg and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR usmg the new sequence information for the design of the 5' primer

Recombinant polypeptides of the present mvention may be prepared by processes well known in the art from genetically engmeered host cells compπsmg expression systems Accordingly, m a further aspect, the present mvention relates to expression systems which compπse a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engmeered with such expression sytems 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 deπved from the DNA constructs of the present mvention For recombmant 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 m Molecular Biology (1986) and Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989) Prefeπed such methods mclude, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micromjection, cationic hpid-medtated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection

Representative examples of appropπate hosts mclude bacteπal cells, such as streptococci, staphylococci, E cob, Strepto yces and Bacillus subtihs cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosophtla 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 vaπety of expression systems can be used, for instance, chromosomal, episomal and virus-deπved systems, e g vectors denved from bacteπal plasmids, from bacteπophage, 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 retrovruses, and vectors deπved from combinations thereof, such as those deπved from plasmid and bacteπophage genetic elements, such as cosmids and phagenuds The expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide m a host may be used The appropπate nucleotide sequence may be inserted mto an expression system by any of a vaπety of well-known and routme techniques, such as, for example, those set forth m Sambrook et al , MOLECULAR CLONING, A LABORATORY MANUAL (supra) Appropπate secretion signals may be mcorporated mto the desired polypeptide to allow secretion of the translated protem mto the lumen of the endoplasmic reticulum, the peπplasrmc space or the extracellular environment These signals may be endogenous to the polypeptide or they may be heterologous signals

If a polypeptide of the present mvention is to be expressed for use m screemng assays, it is generally prefeπed that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested pnor to use m the screemng assay If the polypeptide is secreted mto the medium, the medium can be recovered m order to recover and puπfy the polypeptide If produced lntracellularly, the cells must first be lysed before the polypeptide is recovered

Polypeptides of the present mvention can be recovered and purified from recombmant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Most preferably, high performance hquid chromatography is employed for puπfication Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or puπfication This mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of the gene characteπsed by the polynucleotide of SEQ LD NO 1 which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over- expression or altered expression of the gene Individuals carrying mutations m the gene may be detected at the DNA level by a vaπety of techniques

Nucleic acids for diagnosis may be obtamed 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 usmg PCR or other amplification techniques pnor 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 amphfied product m companson to the normal genotype Pomt mutations can be identified by hybndizmg amphfied DNA to labeled CBDAOH03 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 m gels, with or without denaturing agents, or by direct DNA sequencmg (ee, e g , Myers et al , Science (1985) 230 1242) Sequence changes at specific locations may also be revealed b} nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cotton et al , Proc Natl Acad Sci USA (1985) 85 4397-4401) In another embodiment, an array of oligonucleotides probes compπsmg CBDAOH03 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g , genetic mutations Aπay technology methods are \\ ell known and have general applicability and can be used to address a vaπety 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 the Diseases through detection of mutation m the CBDAOH03 gene by the methods descπbed In addition, such diseases may be diagnosed by methods compnsmg determining from a sample deπved from a subject an abnormally decreased or increased level of polypeptide or mRNA Decreased or mcreased expression can be measured at the RNA level usmg any of the methods well known m the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for mstance PCR, RT-PCR, RNase protection, Northern blottmg and other hybπdization methods Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present mvention, m a sample denved 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 m another aspect, the present mvention relates to a diagonostic kit which comprises

(a) a polynucleotide of the present mvention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof ,

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

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

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

It will be appreciated that in any such kit, (a), (b), (c) or (d) may compnse a substantial component Such a kit will be of use in diagnosing a disease or suspectabi ty to a disease, particularly cancer, AIDS, metabolic disorders, and Diabetes Melhtus. amongst others

The nucleotide sequences of the present mvention are also valuable for chromosome identification The sequence is specifically targeted to. and can hybπdize with, a particular location on an mdividual human chromosome The mapping of relevant sequences to chromosomes according to the present mvention is an important first step m correlating those sequences with gene associated disease Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be coπelated with genetic map data Such data are found m, for example, V McKusick, Mendehan Inheπtance m Man (available on-lme 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 (coinheπtance of physically adjacent genes)

The differences m the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed m 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

The polypeptides of the mvention or their fragments or analogs thereof, or cells expressmg them, can also be used as rmmunogens to produce antibodies lrnmunospecific for polypeptides of the present mvention The term "lrnmunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides m the pπor art

Antibodies generated against polypeptides of the present mvention may be obtained by administering the polypeptides or epitope-beaπng fragments, analogs or cells to an animal, preferably a non-human animal, usmg routine protocols For preparation of monoclonal antibodies, any techmque which provides antibodies produced by continuous cell line cultures can be used Examples mclude the hybπdoma techmque (Kohler, G and Milstem, C , Nature (1975) 256 495-497), the tπoma techmque, the human B-cell hybndoma techmque (Kozbor et al , Immunology Today (1983) 4 72) and the EBV- hybndoma techmque (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985)

Techniques for the production of smgle cham antibodies, such as those descπbed m U S Patent No 4,946,778, can also be adapted to produce smgle cham antibodies to polypeptides of this mvention Also, transgenic mice, or other organisms, mcludmg other mammals, may be used to express humanized antibodies

The above-descπbed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to purify the polypeptides by affinity chromatography Antibodies against polypeptides of the present mvention may also be employed to treat the

Diseases, amongst others

In a further aspect, the present invention relates to genetically engineered soluble fusion protems compπsmg a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of vaπous subclasses (IgG, IgM, IgA, IgE) Prefeπed as an immunoglobulm is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hmge region In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa Furthermore, this invention relates to processes for the preparation of these fusion protems by genetic engineering, and to the use thereof for drug screemng, diagnosis and therapy A further aspect of the mvention also relates to polynucleotides encodmg such fusion protems Examples of fusion protem technology can be found in International Patent Application Nos W094/29458 and W094/22914

Another aspect of the mvention relates to a method for mducmg an immunological response m a mammal which compnses moculatmg the mammal with a polypeptide of the present mvention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases herembefore mentioned, amongst others Yet another aspect of the invention relates to a method of mducmg immunological response m a mammal which compnses, delivering a polypeptide of the present mvention via a vector directing expression of the polynucleotide and codmg for the polypeptide in vivo m order to mduce such an immunological response to produce antibody to protect said animal from diseases

A further aspect of the mvention relates to an lmmunological/vaccme formulation (composition) which, when mtroduced mto a mammalian host, mduces an immunological response in that mammal to a polypeptide of the present mvention wherem the composition compnses a polypeptide or polynucleotide of the present mvention The vaccme formulation may further compnse a suitable earner Smce a polypeptide may be broken down m the stomach, it is preferably administered parenterally (for mstance, subcutaneous, intramuscular, mtravenous, or intradermal injection) Formulations suitable for parenteral admimstration mclude aqueous and non-aqueous stenle injection solutions which may contain anti-oxidants, buffers, bactenostats and solutes which render the formulation mstomc with the blood of the recipient, and aqueous and non- aqueous sterile suspensions which may mclude suspendmg agents or thickenmg agents The formulations may be presented m umt-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored m a ffeeze-dπed condition requiring only the addition of the sterile liquid earner immediately pnor to use The vaccine formulation may also include adjuvant systems for enhancing the lmmunogemcity of the formulation, such as oil-in water systems and other systems known m the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routme experimentation

Polypeptides of the present mvention are responsible for many biological functions, mcludmg many disease states, m particular the Diseases herembefore mentioned It is therefore desirous to devise screemng methods to identify compounds which stimulate or which inhibit the function of the polypeptide Accordingly, m a further aspect the present mvention provides for a method of screemng compounds to identify those which stimulate or which inhibit the function of the polypeptide In general, agomsts or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned Compounds may be identified from a vaπety of sources, for example, cells, cell-free preparations, chemical hbraπes, and natural product mixtures Such agomsts, antagonists or inhibitors so-identified may be natural or modified substrates, hgands, receptors, enzymes, etc , as the case may be, of the polypeptide, or may be structural or functional mimetics thereof (see Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)) The screemng method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearmg the polypeptide, or a fusion protem thereof by means of a label directly or indirectly associated with the candidate compound Alternatively, the screemng method may mvolve competition with a labeled competitor Further, these screemng methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, usmg detection systems appropπate to the cells bearing the polypeptide Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agomst by the presence of the candidate compound is observed Constitutively active polpypeptides may be employed m screenmg methods for inverse agonists or inhibitors, m the absence of an agomst or inhibitor, by testmg whether the candidate compound results m inhibition of activation of the polypeptide Further, the screemng methods may simply compnse the steps of mixing a candidate compound with a solution containing a polypeptide of the present mvention, to form a mixture, measuring CBDAOH03 activity in the mixture, and comparing the CBDAOH03 activity of the mixture to a standard Fusion protems, such as those made from Fc portion and CBDAOH03 polypeptide, as hereinbefore descnbed, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D Bennett et al , J Mol Recognition, 8 52-58 (1995), and K Johanson et al , 1 Biol Chem, 270(16) 9459-9471 (1995))

The polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide m cells For example, an ELISA assay may be constructed for measunng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagomst or agonist, respectively) from suitably manipulated cells or tissues

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

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

Thus, m another aspect, the present mvention relates to a screemng kit for identifymg agomsts, antagonists, hgands, receptors, substrates, enzymes, etc for polypeptides of the present mvention, or compounds which decrease or enhance the production of such polypeptides, which compnses

(a) a polypeptide of the present mvention,

(b) a recombmant cell expressmg a polypeptide of the present mvention,

(c) a cell membrane expressmg a polypeptide of the present mvention, or

(d) antibody to a polypeptide of the present mvention, which polypeptide is preferably that of SEQ ID NO 2

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

It will be readily appreciated by the skilled artisan that a polypeptide of the pres nt mvention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by

(a) determining m the first mstance the three-dimensional structure of the polypeptide,

(b) deducing the three-dimensional structure for the likely reactive or bmdmg sιte(s) of an agonist, antagomst or inhibitor,

(c) synthesmg candidate compounds that are predicted to bind to or react with the deduced binding or reactive site, and

(d) testmg whether the candidate compounds are mdeed agonists, antagonists or inhibitors It will be further appreciated that this will normally be an mterative process In a further aspect, the present mvention provides methods of treating abnormal conditions such as, for instance, cancer, AIDS, metabolic disorders, and Diabetes Melhtus, related to either an excess of, or an under-expression of, CBDAOH03 polypeptide activity

If the activity of the polypeptide is in excess, several approaches are available One approach compnses administering to a subject m need thereof an inhibitor compound (antagomst) as hereinabove descnbed, optionally m combination with a pharmaceutically acceptable earner, m an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the bmdmg of hgands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of bmdmg the ligand, substrate, enzymes, receptors, etc m competition with endogenous polypeptide may be administered Typical examples of such competitors mclude fragments of the CBDAOH03 polypeptide

In still another approach, expression of the gene encodmg endogenous CBDAOH03 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 m Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, oligonucleotides which form tπple 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 l , Science (1991) 251 1360) These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo

For treating abnormal conditions related to an under-expression of CBDAOH03 and its activity several approaches are also available One approach compnses administering to a subject a therapeutically effective amount of a compound which activates a polypeptide of the present mvention. l e . an agomst as descπbed above, m combination with a pharmaceutically acceptable earner, to thereb\ alleviate the abnormal condition Alternatively, gene therapy may be employed to effect the endogenous production of CBDAOH03 by the relevant cells in the subject For example, a polynucleotide of the mvention may be engmeered for expression m a replication defective retroviral vector, as discussed above The retroviral expression construct may then be isolated and introduced mto a packaging cell transduced with a retroviral plasmid vector containmg RNA encodmg a polypeptide of the present mvention such that the packaging cell now produces infectious viral particles containmg 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 an 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 a polypeptide of the present mvention in combination with a suitable pharmaceutical earner

In a further aspect, the present mvention provides for pharmaceutical compositions compπsmg a therapeutically effective amount of a polypeptide, such as the soluble form of a polypeptide of the present mvention, agonist/antagonist peptide or small molecule compound, m combination with a pharmaceutically acceptable earner or excipient Such earners mclude, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof The mvention further relates to pharmaceutical packs and kits compnsmg 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

The composition will be adapted to the route of admimstration, for instance by a systemic or an oral route Prefeπed forms of systemic admimstration mclude injection, typically by intravenous injection Other injection routes, such as subcutaneous, intramuscular, or intrapentoneal, can be used Alternative means for systemic admimstration mclude transmucosal and transdermal adrrumstration usmg penetrants such as bile salts or fusidic acids or other detergents In addition, if a polypeptide or other compounds of the present mvention can be formulated m an entenc or an encapsulated formulation, oral admimstration may also be possible Admimstration 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 or other compounds of the present mvention, the route of admimstration, 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 vaπations m the needed dosage, however, are to be expected in view of the vaπety of compounds available and the differing efficiencies of vaπous routes of admimstration For example, oral admimstration would be expected to require higher dosages than admimstration by intravenous injection Vaπations m these dosage levels can be adjusted usmg standard empmcal routines for optimization, as is well understood m the art

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

Polynucleotide and polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology This is most easily facilitated by storing the sequence m a computer readable medium and then usmg the stored data to search a sequence database usmg well known searchmg tools, such as GCC Accordmgly, m a further aspect, the present mvention provides for a computer readable medium having stored thereon a polynucleotide compπsmg the sequence of SEQ ID NO 1 and/or a polypeptide sequence encoded thereby

The following definitions are provided to facilitate understanding of certam terms used frequently herembefore

"Antibodies" as used herem includes polyclonal and monoclonal antibodies, chimenc, smgle chain, and humanized antibodies, as well as Fab fragments, mcludmg the products of an Fab or other immunoglobulm 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 onginal environment, or both For example, a polynucleotide or a polypeptide naturally present m a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting mateπals of its natural state is "isolated", as the term is employed herem

"Polynucleotide" generally refers to any polynbonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotides" include, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybrid molecules compnsing DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA The term "polynucleotide" also mcludes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases mclude, for example, tπtylated bases and unusual bases such as inosine A variety of modifications may be made to DNA and RNA, thus, "polynucleotide" embraces chemically, enzymatically or metabohcally modified forms of polynucleotides as typically found m nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides

"Polypeptide" refers to any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, I e , peptide isosteres "Polypeptide" refers to both short chains, commonly referred to as peptides, ohgopeptides or ohgomers, and to longer chains, generally referred to as proteins Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids "Polypeptides" mclude ammo acid sequences modified either by natural processes, such as post-translational processmg, or by chemical modification techniques which are well known m the art Such modifications are well descπbed m basic texts and m more detailed monographs, as well as m a voluminous research literature Modifications may occur anywhere m a polypeptide, mcludmg the peptide backbone, the ammo acid side-chams and the ammo or carboxyl termini It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites m a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched as a result of ubiquitrnation, and they may be cyclic, with or without branching Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods Modifications mclude acetylation, acylation, ADP-nbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide deπvative, covalent attachment of a hpid or hpid denvative, covalent attachment of phosphotidylmositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystme, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, mynstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to protems such as argrnylation, and ubiquitrnation (see, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York, 1993, Wold, F , Post-translational Protem 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 protem modifications and nonprotein cofactors", Meth Enzymol (1990) 182 626-646 and Rattan et al , "Protem Synthesis Post-translational Modifications and Aging", Ann NY Acad Sci (1992) 663 48-62) "Vanant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties A typical variant of a polynucleotide differs m nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the vanant may or ma} not alter the ammo acid sequence of a polypeptide encoded by the reference pohτmcleotιde Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below A typical vanant 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 vanant are closely similar overall and, in many regions, identical A vanant 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 vanant of a polynucleotide or polypeptide may be a naturally occurring such as an allehc vanant, or it may be a vanant that is not known to occur naturally Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis

"Identity," as known m the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determmed 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 determmed by the match between stnngs of such sequences "Identity" and "similanty" can be readily calculated by known methods, mcludmg but not limited to those descnbed m (Computational Molecular Biology, Lesk, A M , ed , Oxford Umversity 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, Gπffin, A M , and Gnffin, 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, and Canllo, H , and Lipman, D , SIAM J Applied Math , 48 1073 (1988) Prefened methods to determine identity are designed to give the largest match between the sequences tested Methods to determine identity and similanty are codified m publicly available computer programs Preferred computer program methods to determine identity and similarity between two sequences mclude, 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 ai , J Molec Bwl 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NIH Bethesda, MD 20894, Altschul, S , et al , J Mol Bwl 215 403-410 (1990) The well known Smith Waterman algoπthm may also be used to determine identity

Preferred parameters for polypeptide sequence comparison mclude the following 1) Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Comparison matnx BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 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 peptide comparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3 Available as: The "gap" program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid comparisons.

By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:l, that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence. Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The 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(divided by 100) and subtracting that product from said total number of nucleotides in SEQ ID NO: l, or: ⁿn ≤ ^xn " (^xn • y), wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO: l, and y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc, and wherein any non-integer product of x_n and y is rounded down to the nearest integer prior to subtracting it from x_n. 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.

Similarly, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%. Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and

\l wherem said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those termmal positions, mterspersed either mdividually among the ammo acids m the reference sequence or m one or more contiguous groups within the reference sequence The number of ammo acid alterations for a given % identity is determmed by multiplymg the total number of ammo acids m SEQ ID NO 2 by the numeπcal percent of the respective percent ιdentιty(dιvιded by 100) and then subtractmg that product from said total number of ammo acids m SEQ ID NO 2, or ⁿa^≤xa ^" (^xa * ^v)> wherem n_a is the number of ammo acid alterations, x_a is the total number of ammo acids m SEQ ID NO 2, and y is, for mstance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc , and wherem any non-mteger product of x_a and y is rounded down to the nearest mteger pπor to subtractmg it from ^xa

"Fusion protem" refers to a protem encoded by two, often unrelated, fused genes or fragments thereof In one example, EP-A-0 464 discloses fusion protems compnsmg vaπous portions of constant region of immunoglobulm molecules together with another human protem or part thereof In many cases, employing an immunoglobulin Fc region as a part of a fusion protem is advantageous for use m therapy and diagnosis resulting m, for example, improved pharmacokmetic properties [see, e g , EP-A 0232 262] On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and puπfied

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

SEQUENCE INFORMATION SEQ ID NO:l

1 GTTCGTAGCA GAAATGGCGG CTGCGGCGGC TCGAGTGGTG TTGTCATCCG

51 CGGCGCGAGG CGGCCTCTGG GGTTTCAGCG AGAGTCTTCT AATCCGAGGC

101 GCTGCGGGAC GGTCATTATA TTTTGGAGAG AACAGATTAA GAAGTACACA

151 GGCTGCTACC CAAGTTGTTC TGAATGTTCC TGAAACAAGA GTAACATGTT

201 TAGAAAGTGG ACTCAGAGTA GCTTCGGAAG ACTCTGGGCT CTCAACATGC

251 ACAGTTGGAC TCTGGATTGA TGCTGGAAGT AGATACGAAA ATGAGAAGAA

301 CAATGGAACA GCACACTTTC TGGAGCATAT GGCTTTCAAG GGCACCAAGA

351 AGAGATCCCA GTTAGATCTG GAACTTGAGA TTGAAAATAT GGGTGCTCAT

401 CTCAATGCCT ATACCTCCAG AGAGCAGACT GTATACTATG CCAAAGCATT

451 CTCTAAAGAC TTGCCAAGAG CTGTAGAAAT TCTTGCTGAT ATAATACAAA

501 ACAGCACATT GGGAGAAGCA GAGATTGAAC GTGAGCGTGG AGTAATCCTT

551 AGAGAGATGC AGGAAGTTGA AACCAATTTA CAAGAAGTTG TTTTTGATTA

601 TCTTCATGCC ACAGCTTATC AAAATACTGC ACTTGGACGG ACAATTTTGG

651 GACCAACTGA AAATATCAAA TCTATAAGTC GTAAGGACTT AGTGGATTAT

701 ATAACCACAC ATTATAAGGG GCCAAGAATA GTGCTTGCTG CTGCTGGAGG

751 TGTTTCCCAT GATGAATTGC TTGACTTAGC AAAGTTTCAT TTCGGTGACT

801 CTTTATGCAC ACACAAAGGA GAAATACCAG CTCTGCCTCC CTGCAAATTC

851 ACAGGAAGTG AGATTCGTGT GAGGGATGAC AAGATGCCTT TGGCGCACCT

901 TGCAATAGCT GTTGAAGCTG TTGGTTGGGC ACATCCAGAT ACAATCTGTC 951 TCATGGTTGC AAACACGCTG ATTGGCAACT GGGATCGCTC TTTTGGGGGA

1001 GGAATGAATT TATCTAGCAA GCTGGCCCAG CTCACTTGTC ATGGCAATCT

1051 TTGCCATAGC TTTCAGTCTT TCAACACTTC CTACACAGAT ACAGGATTAT

1101 GGGGACTGTA TATGGTTTGT GAATCATCCA CTGTTGCAGA CATGCTACAT

1151 GTTGTTCAAA AAGAATGGAT GCGACTCTGT ACAAGTGTCA CAGAAAGTGA

1201 GGTTGCACGA GCCAGAAATC TTCTGAAAAC AAACATGTTG TTGCAGCTTG

1251 ATGGTTCAAC TCCAATTTGT GAAGATATTG GTAGGCAAAT GTTATGCTAT

1301 AATAGAAGGA TTCCCATCCC TGAGCTTGAA GCAAGAATTG ATGCTGTGAA

1351 TGCTGAGACA ATTCGAGAAG TATGTACCAA ATACATTTAT AATAGGAGTC

1401 CAGCTATTGC TGCTGTTGGT CCCATTAAGC AACTACCAGA TTTTAAACAG

1451 ATACGCAGTA ACATGTGTTG GCTTCGTGAT TAAAATGCTC CTAATCAAGA

1501 TTGTTTGAAC ACATGTATTT ATAAAACAGA GCTAGAGAAA AATAAAAATG

1551 AACATGTATA TACATTTGGA AATTTGAATT AAATACTGTA TCATACTTTC

1601 AAAGGATAAA AAGACTACCC CTCTGAAGGT TGTTTTGTAT TAATGGTCAG

1651 TCTTTGTTCT CTGAGAAATT ATGTTGGAAG CAGCATACTT TCAAATTATT

1701 ACCATGAGTA TAATTTTAAG AATGAAAATG TTTACAGTAT TTTCAGTTTT

1751 ATTATAAAAA TGCACACACA A

SEQ ID NO:2

1 MAAAAARWL SSAARGGLWG FSESLLIRGA AGRSLYFGEN RLRSTQAATQ

51 VVLNVPETRV TCLESGLRVA SEDSGLSTCT VGL IDAGSR YENEKNNGTA 101 HFLEHMAFKG TKKRSQLDLE LEIENMGAHL NAYTSREQTV YYAKAFSKDL

151 PRAVEILADI I NSTLGEAE IERERGVILR EMQEVETNLQ EWFDYLHAT

201 AYQNTALGRT ILGPTENIKS ISRKDLVDYI TTHYKGPRIV LAAAGGVSHD

251 ELLDLAKFHF GDSLCTHKGE IPALPPCKFT GSEIRVRDDK MPLAHLAIAV

301 EAVGWAHPDT ICLMVANTLI GNWDRSFGGG MNLSSKLAQL TCHGNLCHSF

351 QSFNTSYTDT GLWGLYMVCE SSTVADMLHV VQKEWMRLCT SVTESEVARA

401 RNLLKTNMLL QLDGSTPICE DIGRQMLCYN RRIPIPELEA RIDAVNAETI

451 REVCTKYIYN RSPAIAAVGP IKQLPDFKQI RSNMCWLRD

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: SHANGHAI SECOND MEDICAL UNIVERSITY

(ii) TITLE OF THE INVENTION: Processing Protease Beta Subunit (MPPB) Homolog Gene

(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- 70445 ( 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: 1771 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

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

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

GTTCGTAGCA GAAATGGCGG CTGCGGCGGC TCGAGTGGTG TTGTCATCCG CGGCGCGAGG 60

CGGCCTCTGG GGTTTCAGCG AGAGTCTTCT AATCCGAGGC GCTGCGGGAC GGTCATTATA 120 TTTTGGAGAG AACAGATTAA GAAGTACACA GGCTGCTACC CAAGTTGTTC TGAATGTTCC 180

TGAAACAAGA GTAACATGTT TAGAAAGTGG ACTCAGAGTA GCTTCGGAAG ACTCTGGGCT 240

CTCAACATGC ACAGTTGGAC TCTGGATTGA TGCTGGAAGT AGATACGAAA ATGAGAAGAA 300

CAATGGAACA GCACACTTTC TGGAGCATAT GGCTTTCAAG GGCACCAAGA AGAGATCCCA 360

GTTAGATCTG GAACTTGAGA TTGAAAATAT GGGTGCTCAT CTCAATGCCT ATACCTCCAG 420 AGAGCAGACT GTATACTATG CCAAAGCATT CTCTAAAGAC TTGCCAAGAG CTGTAGAAAT 480

TCTTGCTGAT ATAATACAAA ACAGCACATT GGGAGAAGCA GAGATTGAAC GTGAGCGTGG 540

AGTAATCCTT AGAGAGATGC AGGAAGTTGA AACCAATTTA CAAGAAGTTG TTTTTGATTA 600

TCTTCATGCC ACAGCTTATC AAAATACTGC ACTTGGACGG ACAATTTTGG GACCAACTGA 660

AAATATCAAA TCTATAAGTC GTAAGGACTT AGTGGATTAT ATAACCACAC ATTATAAGGG 720 GCCAAGAATA GTGCTTGCTG CTGCTGGAGG TGTTTCCCAT GATGAATTGC TTGACTTAGC 780

AAAGTTTCAT TTCGGTGACT CTTTATGCAC ACACAAAGGA GAAATACCAG CTCTGCCTCC 840

CTGCAAATTC ACAGGAAGTG AGATTCGTGT GAGGGATGAC AAGATGCCTT TGGCGCACCT 900

TGCAATAGCT GTTGAAGCTG TTGGTTGGGC ACATCCAGAT ACAATCTGTC TCATGGTTGC 960

AAACACGCTG ATTGGCAACT GGGATCGCTC TTTTGGGGGA GGAATGAATT TATCTAGCAA 1020 GCTGGCCCAG CTCACTTGTC ATGGCAATCT TTGCCATAGC TTTCAGTCTT TCAACACTTC 1080

CTACACAGAT ACAGGATTAT GGGGACTGTA TATGGTTTGT GAATCATCCA CTGTTGCAGA 1140

CATGCTACAT GTTGTTCAAA AAGAATGGAT GCGACTCTGT ACAAGTGTCA CAGAAAGTGA 1200

GGTTGCACGA GCCAGAAATC TTCTGAAAAC AAACATGTTG TTGCAGCTTG ATGGTTCAAC 1260

TCCAATTTGT GAAGATATTG GTAGGCAAAT GTTATGCTAT AATAGAAGGA TTCCCATCCC 1320 TGAGCTTGAA GCAAGAATTG ATGCTGTGAA TGCTGAGACA ATTCGAGAAG TATGTACCAA 1380

ATACATTTAT AATAGGAGTC CAGCTATTGC TGCTGTTGGT CCCATTAAGC AACTACCAGA 1440

TTTTAAACAG ATACGCAGTA ACATGTGTTG GCTTCGTGAT TAAAATGCTC CTAATCAAGA 1500

TTGTTTGAAC ACATGTATTT ATAAAACAGA GCTAGAGAAA AATAAAAATG AACATGTATA 1560 TACATTTGGA AATTTGAATT AAATACTGTA TCATACTTTC AAAGGATAAA AAGACTACCC 1620

CTCTGAAGGT TGTTTTGTAT TAATGGTCAG TCTTTGTTCT CTGAGAAATT ATGTTGGAAG 1680

CAGCATACTT TCAAATTATT ACCATGAGTA TAATTTTAAG AATGAAAATG TTTACAGTAT 1740

TTTCAGTTTT ATTATAAAAA TGCACACACA A 1771

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 489 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Ala Ala Ala Ala Ala Arg Val Val Leu Ser Ser Ala Ala Arg Gly

1 5 10 15

Gly Leu Trp Gly Phe Ser Glu Ser Leu Leu lie Arg Gly Ala Ala Gly 20 25 30

Arg Ser Leu Tyr Phe Gly Glu Asn Arg Leu Arg Ser Thr Gin Ala Ala

35 40 45

Thr Gin Val Val Leu Asn Val Pro Glu Thr Arg Val Thr Cys Leu Glu 50 55 60 Ser Gly Leu Arg Val Ala Ser Glu Asp Ser Gly Leu Ser Thr Cys Thr 65 70 75 80

Val Gly Leu Trp lie Asp Ala Gly Ser Arg Tyr Glu Asn Glu Lys Asn

85 90 95

Asn Gly Thr Ala His Phe Leu Glu His Met Ala Phe Lys Gly Thr Lys 100 105 110

Lys Arg Ser Gin Leu Asp Leu Glu Leu Glu lie Glu Asn Met Gly Ala

115 120 125

His Leu Asn Ala Tyr Thr Ser Arg Glu Gin Thr Val Tyr Tyr Ala Lys

130 135 140 Ala Phe Ser Lys Asp Leu Pro Arg Ala Val Glu lie Leu Ala Asp lie

145 150 155 160 lie Gin Asn Ser Thr Leu Gly Glu Ala Glu lie Glu Arg Glu Arg Gly

165 170 175

Val lie Leu Arg Glu Met Gin Glu Val Glu Thr Asn Leu Gin Glu Val 180 185 190

Val Phe Asp Tyr Leu His Ala Thr Ala Tyr Gin Asn Thr Ala Leu Gly

195 200 205

Arg Thr lie Leu Gly Pro Thr Glu Asn lie Lys Ser He Ser Arg Lys 210 215 220

Asp Leu Val Asp Tyr He Thr Thr His Tyr Lys Gly Pro Arg He Val

225 230 235 240

Leu Ala Ala Ala Gly Gly Val Ser His Asp Glu Leu Leu Asp Leu Ala 245 250 255

Lys Phe His Phe Gly Asp Ser Leu Cys Thr His Lys Gly Glu He Pro

260 265 270

Ala Leu Pro Pro Cys Lys Phe Thr Gly Ser Glu He Arg Val Arg Asp

275 280 285 Asp Lys Met Pro Leu Ala His Leu Ala He Ala Val Glu Ala Val Gly

290 295 300

Trp Ala His Pro Asp Thr He Cys Leu Met Val Ala Asn Thr Leu He

305 310 315 320

Gly Asn Trp Asp Arg Ser Phe Gly Gly Gly Met Asn Leu Ser Ser Lys 325 330 335

Leu Ala Gin Leu Thr Cys His Gly Asn Leu Cys His Ser Phe Gin Ser

340 345 350

Phe Asn Thr Ser Tyr Thr Asp Thr Gly Leu Trp Gly Leu Tyr Met Val

355 360 365 Cys Glu Ser Ser Thr Val Ala Asp Met Leu His Val Val Gin Lys Glu

370 375 380

Trp Met Arg Leu Cys Thr Ser Val Thr Glu Ser Glu Val Ala Arg Ala

385 390 395 400

Arg Asn Leu Leu Lys Thr Asn Met Leu Leu Gin Leu Asp Gly Ser Thr 405 410 415

Pro He Cys Glu Asp He Gly Arg Gin Met Leu Cys Tyr Asn Arg Arg

420 425 430

He Pro He Pro Glu Leu Glu Ala Arg He Asp Ala Val Asn Ala Glu

435 440 445 Thr He Arg Glu Val Cys Thr Lys Tyr He Tyr Asn Arg Ser Pro Ala

450 455 460

He Ala Ala Val Gly Pro He Lys Gin Leu Pro Asp Phe Lys Gin He

465 470 475 480

Arg Ser Asn Met Cys Trp Leu Arg Asp 485

Claims

What is claimed is:

1. An isolated polypeptide selected from the group consisting of:

(i) an isolated polypeptide comprising an amino acid sequence selected from the group having at least:

(a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity to the amino acid sequence of SEQ ID NO:2 over the entire length of SEQ ID

NO:2; (ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2 or (iii) an isolated polypeptide which is the amino acid sequence of SEQ ID NO:2.

2. An isolate polynucleotide selected from the group consisting of:

(i) an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide that has at least

(a) 70% identity;

(b) 80% identity; (c) 90% identity; or

(d) 95% identity; to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ED NO:2; (ii) an isolated polynucleotide comprising a nucleotide sequence that has at least:

(a) 70% identity (b) 80% identity;

(c) 90% identity; or

(d) 95% identity; over its entire length to a nucleotide sequence encoding the polypeptide of SEQ ID NO:2; (iii) an isolated polynucleotide comprising a nucleotide sequence which has at least:

(a) 70% identity;

(b) 80% identity;

(c) 90% identity; or

(d) 95% identity; to that of SEQ ID NO 1 over the entire length of SEQ ID NO 1 , (iv) an isolated polynucleotide comprising a nucleotide sequence encodmg the polypeptide of SEQ ID NO.2,

(vi) an isolated polynucleotide which is the polynucleotide of SEQ ID NO 1, or (vi) an isolated polynucleotide obtamable by screemng an appropπate hbrary under stringent hybπdization conditions with a labelled probe havmg the sequence of SEQ ID NO 1 or a fragment thereof , or a nucleotide sequence complementary to said isolated polynucleotide

3 An antibody lrnmunospecific for the polypeptide of claim 1

4 A method for the treatment of a subject

(l) m need of enhanced activity or expression of the polypeptide of claim 1 compnsing

(a) administering to the subject a therapeutically effective amount of an agomst to said polypeptide, and/or

(b) providing to the subject an isolated polynucleotide compnsmg a nucleotide sequence encoding said polypeptide in a form so as to effect production of said polypeptide activity m vivo . or

(n) havmg need to inhibit activity or expression of the polypeptide of claim 1 compnsing

(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 a 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 ligand, substrate , or receptor

5 A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of the polypeptide of claim 1 m a subject comprising

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

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

6 A method for screemng to identify compounds which stimulate or which inhibit the function of the polypeptide of claim 1 which compnses a method selected from the group consisting of

(a) measuring the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes bearmg the polypeptide) or a fusion protem thereof by means of a label directly or indirectly associated with the candidate compound,

(b) measunng the bmdmg of a candidate compound to the polypeptide (or to the cells or membranes bearmg the polypeptide) or a fusion protem thereof m the presence of a labeled competitor, (c) testing whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, usmg detection systems appropnate to the cells or cell membranes bearmg the polypeptide, (d) mixing a candidate compound with a solution containing a polypeptide of claim 1, to form a mixture, measunng activity of the polypeptide m the mixture, and comparmg the activity of the mixture to a standard, or

(e) detectmg the effect of a candidate compound on the production of mRNA encodmg said polypeptide and said polypeptide m cells, usmg for mstance, an ELISA assay

7 An agonist or an antagomst of the polypeptide of claim 1

8 An expression system compπsmg a polynucleotide capable of producmg a polypeptide of claim 1 when said expression system is present in a compatible host cell

9 A process for producing a recombmant host cell compπsmg transforming or transfectmg a cell with the expression system of claim 8 such that the host cell, under appropnate culture conditions, produces a polypeptide compnsmg an ammo acid sequence havmg at least 70% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2

10 A recombinant host cell produced by the process of claim 9

11 A membrane of a recombinant host cell of claim 10 expressing a polypeptide comprising an ammo acid sequence havmg at least 70% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2

12. A process for producing a polypeptide comprising culturing a host cell of claim 10 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.