WO1998011236A1 - Aspartic protease - Google Patents

Abstract

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is believed to be a novel aspartic protease. The invention also relates to inhibiting the action of such polypeptides.

Description

ASPARTTC PROTEASE

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides More particularly, the polypeptide of the present invention is believed to be a novel aspartic protease The invention also relates to inhibiting the action of such polypeptides

There are currently five known human aspartic proteases, namely pepsin, gastπcsin, cathespin D, cathespin E and renin, and these have widely varying functions Pepsin and gastπcsin are involved in nutritive processes in the stomach, cathepsin D is involved in protein turnover in nearly all cell types, and renin has the highly specific function of angiotensin production from its precursor form, angiotensmogen The precise role of cathepsin E remains to be confirmed, although its location in some epithelial cells types has indicated a role in antigen processing It mav also be involved in certain inflammatory conditions, e g Hehcobacter pylori infection in the stomach

There is the possibility that a novel aspartic protease could have an extracellular function There are at present a number of such processes which are thought to involve aspartic proteases, but where the exact enzyme involved remains to be identified Important functions implicated are the processing of endothelin and pro-opiomelanocortin prohormones An aspartic protease is also thought to be involved in the processing of the serum amyloid A protein

Aspartic proteases are so called because of the pair of aspartic acid (Asp) residues that are required for the hydrolvtic cleavage of a peptide bond The catalytic Asp residues are normally located within a -Hyd-Hyd-Asp-Thr-Gly- active site motif sequence (w here Hyd can be any hydrophobic amino acid) Eucaryotic aspartic proteases usuallv possess two such sequences, which have been shown by crystallography to lie adjacent to each other in the active site of the enzyme Another highly conserved part of these enzymes comprises a structural beta-hairpin ioop (often termed the "flap") which lies over the active site and may assist in substrate binding A novel aspartic protease was described by Jordan Tang et al O alaho a Medical

Research Foundation during a presentation at the VII International Conference on Aspartic Protemases Oct 22-27 1996

In accordance with one aspect of the present invention, there is provided a nov el polypeptide which comprises the ammo acid sequence given in SEQ ID NO 1 or SEQ ID NO 15, or a fragment analog or derivative thereof The polypeptide of the present invention is of human origin The polypeptide is believed to be an aspartic protease

Polypeptides of the present invention further include a polypeptide having the amino acid sequence contained in SEQ ID NO 1 or 1 5, and a polypeptide comprising an ammo acid sequence which has at least 80%, preferably at least 90%, more preferably 95%, still more preferably at least 97 to 99% identity to any of the amino acid sequences SEQ ID NO 1 or 15 over its entire length. Also included are polypeptides having the amino acid sequence which have at least 80%, preferably at least 90%, more preferably 95%, still more preferably at least 97 to 99% identity to any of the amino acid sequences SEQ ID NO 1 or 15 over its entire length. Polypeptides of the present invention also include a polypeptide comprising an amino acid sequence encoded by a polynucleotide that have at least 80% identity to that of SEQ ID NO:2 or 16 over its entire length.

The polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.

Fragments 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 amino acid sequence of the aforementioned polypeptides. Such 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 polypeptide of SEQ ID NO 1 or 15. 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 SEQ ID NO 1 or 15, 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 terminus. 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 turn-forming regions, coil and coil- forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate biological activity, for instance aspartyl protease 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 precursor polypeptide, 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.

Polypeptides of the present 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. In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides.

In accordance with a preferred aspect of the present invention, there is provided a polynucleotide which encodes for the polypeptide having the amino acid sequence of SEQ ID NO 1 or SEQ ID NO 15.

In particular, the invention provides a polynucleotide having the DNA sequence given in SEQ ID NO 2 or SEQ ID NO 16. The invention further provides a polynucleotide encoding a polypeptide which comprises the DNA sequence given in SEQ ID NO 2 or SEQ

ID NO 16. Polynucleotides of the present invention further include a polynucleotide comprising a nucleotide sequence that has at least 80%, preferably at least 90%, more preferably at least 95%) identity over its entire length to a nucleotide sequence encoding a polypeptide of SEQ ID NO 1 or SED ID NO 15, and a polynucleotide comprising a nucleotide sequence that is at least 80%, preferably at least 90%, more preferably at least 95% identical to that of SEQ ID NO:2 or SEQ ID NO 16 over its entire length. 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. cDNA molecules (ESTs) showing extended identity sections with the cDNA of SEQ

ID NO 2 and SEQ ID NO 16 have been identified in cDNA libraries of human origin from a wide variety of sources. These ESTs are given in SEQ ID NO 3 to 14.

Sequence No: Library:

SEQ ID NO 3 (EST 176432) Raji cells, cyclohexamide treated

SEQ ID NO 4 (EST 424772) Human b cell lymphoma

SEQ ID NO 5 (EST 443275) Breast lymph node cDNA library SEQ ID NO 6 (EST 567394) Raji cells, cyclohexamide treated

SEQ ID NO 7 (EST 685578) Human activated monocytes

SEQ ID NO 8 (EST 928138) Fetal Liver, subtraction II

SEQ ID NO 9 (EST 947785) Breast lymph node cDNA library

SEQ ID NO 10 (EST 1000163): Spinal chord M .Q ID NO I I (I S I 12 I H021 ) Spleen, chionic lymphocytic leukemia SEQ ID NO 12 (LS I I 120419) Human tonsils, lib 2 SEQ ID NO 13 (LS I 7 16478) cd34 depleted buffy coat cord blood SEQ ID NO 14 (I ,S 1' 857644) I liiinan adult testis, large inserts Accυutingly, in a liulhcr aspect, the present invention provides for a polynucleotide which encodes loi an aspniiic piolcnsc characterised by one or more partial DNA sequences selected lrom SEQ ID NO.s 3 lo 14

The polyiHicicolides ol SEQ ID NO 2 and SEQ ID NO 16 are structurally related to the aspartic protease family SEQ ID NO 2 and 16 have 1376 and 1347 nucleotides, respectively The first (oi N-Terminal) active site motif is believed to be -Ala-Phe-Asp-Thr- Gly- and is encoded by at least two ESTs (SEQ ID NO 1 1 and 12), by the identical DNA sequence in each case (-GCC-TTT-GAC-ACT-GGC-). The second (or C-Terminal) active site motif is believed to be -lie-Leu- Asp-Thr-Gly and is encoded (by the DNA sequence - ATC-CTG-GAT-ACA-GGC-) by at least one EST (SEQ ID NO 13) The conserved flap region of the enzyme is believed to be -Tyr-Gly-Thr-Gly- and is encoded by at least fouF

ESTs (SEQ ID NO 9, 10, 5 and 12) with the same DNA sequence (-TAT-GGA-ACT-GGG-) in all cases The sequences SEQ ID NO 1 and SEQ ID NO 15 are believed to contain the entire length of the mature form of the novel aspartic protease, with a further approximately 60 amino acids comprising the propart (or prosegment) The initiating Met residue may also be identified in SEQ ID NO 1, the ORF starting at nucleotide 26 Simple blast sequence analysis reveals that the polypeptide of SEQ ID NO 15 is most homologous to Prepro- Cathepsin D from chicken. It is however believed that polypeptides of SEQ ID NO 1 and SEQ ID NO 15 will be, at a functional level, most like Cathepsin E due to the presence of a Cathepsin E specific glycosylation site (-Asn-Phe-Thr-)just before the sequence encoding the N-Termtnal active site motif The widespread distribution in many cell types of ESTs associated with the polypeptide may indicate a non-specific hydrolytic function (similar to cathepsin E and cathepsin D) for the encoded poypetide

The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand The coding sequence which encodes the polypeptide may be identical to the coding sequence shown in SEQ ID NO 2 or SED ID NO 16 or may be a different coding sequence which, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide as the DNA of SEQ ID NO 2 or SED ID NO 16 The present invention includes variants of the heremabove described polynucleotides which encode fragments, analogs and derivatives of the polypeptide having the amino acid sequence ol SEQ ID NO I or SED ID NO 15 The variant of the polynucleotide may be a naturally occurring allclic variant ol the polynucleotide or a non-naturally occurring variant of the polynucleotide Thus, the present invention includes polynucleotides encoding the same polypeptide as shown in SEQ ID NO 1 or SED ID NO 15, as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

The polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence of SEQ ID NO 2 or SED ID NO 16. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.

The polynucleotide which encodes for the polypeptide of SEQ ID NO 1 or SED ID NO 15 may include: only the coding sequence for the polypeptide; the coding sequence for the polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' a^~nd/or 3' of the coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence. The present invention therefore includes polynucleotides, wherein the coding sequence for the polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains. Thus, for example, the polynucleotide of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence).

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)). The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50%> and preferably 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the polypeptide of SEQ ID NO 1 or SED ID NO 15. The terms "fragment," "derivative" and "analog" when referring to the polypeptide of

SEQ ID NO 1 or SED ID NO 15, means a polypeptide which retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. As used herein, the term "identity" is a measure of the identity of nucleotide ^— sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity" er 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 Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER, Gribskov, 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 (Carillo, H., and Lipton, D., SIAM J Applied Math ( 1988) 48: 1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J Applied Math ( 1988)

48: 1073. Methods to determine identity and similarity are codified in 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., J Molec -3/o/ ( 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: 2/16 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 .idi 100 midcotulcs ol llic it leicπ e nucleotide sequence of SEQ ID NO 2/16 In other wotds, lo ohliiin a polyiuideoluic Iniving a nucleotide sequence at least 95% identical to a rclerencc nuclcolidc sequence, up lo 5% ol the nucleotides in the reference sequence may be deleted oi substituted wil nnol ei inicleolidc, or a number of nucleotides up to 5% of the total nucleotides in the lof ienie sequence may be inserted into the reference sequence

These munitions ol ( e iclcrentc sequence may occur at the 5 or 3 terminal positions of the rclerencc nucleotide sequence oi anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the icleien e sequence Similarly, by a polypeptide having an ammo acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence SEQ ID NO 1/15 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 amino acid alterations per each 100 amino acids of the reference ammo acid sequence In other words, to obtain a polypeptide having an ammo acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the ammo acid residues in the reference sequence may be deleted or substituted with another amino acid or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence These alterations of the reference sequence may occur at the ammo or carboxy terminal positions of the reference 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.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide The fragment, derivative or analog of the polypeptide of SEQ ID NO 1 or or SED ID

NO 15 mav be (I) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (n) one in which one or more of the amino acid residues includes a substituent group, or (in) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional ammo acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity

I he term "isolated" means that the material is removed from its original environment (c g , the natural cnviionmciu il it is naturally occurring) For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. The polypeptide is preferably in purified form. By purified form is meant at least 80%, more preferably 90%, still more preferably 95% and most preferably 99% pure with respect to other protein contaminants.

The DNA of the present invention also makes possible the development by homologous recombination or "knockout" stategies (Kapecchi, Science, 244,: 1288- 1292 ( 1989) of animals that fail to express, or express a variant form of this enzyme

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. In accordance with yet a further aspect of the present invention, there is therefore provided a process for producing the polypeptide of the invention by recombinant techniques by expresssing a polynucleotide encoding said polypeptide in a host and recovering the expressed product. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers. Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a cosmid, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and wili be apparent to the ordinarily skilled artisan.

Suitable expression vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda PL promoter and other promoters known to control expression ol genes m piok. II VOI IL t)i eukaryotic cells or their viruses The expression vector also contains a ribosoinc binding site lor translation initiation and a transcription terminator The vector may also include appropriate sequences for amplifying expression

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phcnotypic trait for selection of transformed host cells such as dihydrololale reductasc or neomycin resistance for eukaryotic cell culture, or such as tetracyclme or ampicillin resistance in E colt

The gene can be placed under the control of a promoter, πbosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction The coding sequence mav or may not contain a signal peptide or leader sequence The protein sequences of the present invention can be expressed using, for example, the E colt tac promoter or the protein A gene (spa) promoter and signal sequence Leader sequences can be removed by the bacterial host in post-translational processing Promoter regions can be selected from any desired gene using CAT (chloramphe col transferase) vectors or other vectors with selectable markers Two appropriate vectors are PKK232-8 and PCM7 Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp Eukaryotic promoters include CMV immediate early, HSV thymidme kinase, early and late SV40, LTRs from retrovirus, and mouse metallothιoneιn-I Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art

In addition to control sequences, it may be desirable to add regulatory sequences which allow for regulation of the expression of the protein sequences relative to the growth of the host cell Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound Other types of regulatory elements may also be present in the vector, for example, enhancer sequences

An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i e , RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence) Modification of the coding sequences may be desirable to achieve this end For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation, I e , to maintain the reading frame The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site Modification of the coding sequences may also be performed to alter codon usage to suit the chosen host cell, for enhanced expression.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampiciliin resistance gene of £. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. ^—

Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage λ (E. coli), pBR322 (£. coli), pACYCl 77 (E. coli), pKX230 (gram-negative bacteria), pGVl 106 (gram-negative bacteria), pLAFRl (gram- negative bacteria), pME290 (non-£. coli gram-negative bacteria), pHV14 (£. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), a baculovirus insect cell system, , YCpl9 (Saccharomyces). See, generally, "DNA Cloning": Vols. I & II, Glover et al. ed. IRL Press Oxford ( 1985) ( 1987) and; T. Maniatis et al. ("Molecular Cloning" Cold Spring Harbor Laboratory (1982).

In some cases, it may be desirable to add sequences which cause the secretion of the polypeptide from the host organism, with subsequent cleavage of the secretory signal. Yeast expression vectors are also known in the art. See, e.g., U.S. Patent Nos. 4,446,235; 4,443,539; 4,430,428; see also European Patent Applications 103,409; 100,561 ; 96,491. pSV2neo (as described in J. Mol. Appl. Genet. 1 :327-341 ) which uses the SV40 late promoter to drive expression in mammalian cells or pCDNA l neo, a vector derived from pCDNA l(Mol. Cell Biol. 7:4125-29) which uses the CMV promoter to drive expression.

Both these latter two vectors can be employed for transient or stable(using G418 resistance) expression in mammalian cells. Insect cell expression systems, e.g., Drosophila, are also useful, see for example. PCT applications WO 90/06358 and WO 92/06212 as well as EP 290.261 -B 1. Polypeptides can be expressed in host cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Samhrook, cl al.. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring 1 larbor. N.Y., ( 1 89), the disclosure of which is hereby incorporated by reference.

Transcription ol^'lhc DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers arc cis-acling elements ol^" DNA, usually about from 10 to 300 bp that act on a promoter to increase ii transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 1 0 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. in a further aspect, the present invention relates to host cells containing the above- described vectors. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. As representative examples of appropriate hosts, there may be mentioned: prokaryotes for example bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium and eukaryotes for example fungal cells, such as yeast, insect cells such as Drosophila and Spodoptera frugiperda, mammalian cells such as CHO, COS or Bowes melanoma, plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation. disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23: 175 ( 1981 ), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK. cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Depending on the expression system and host selected, the polypeptide of the present invention may be produced by growing host cells transformed by an expression vector described above under conditions whereby the polypeptide of interest is expressed The polypeptide is then isolated from the host cells and purified. If the expression system secretes the polypeptide into growth media, the polypeptide can be purified directly from the media. If the polypeptide is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction Where the polypeptide is localized to the cell surface, whole cells or isolated membranes can be used as an assayable source of the desired gene product Polypeptide expressed in bacterial hosts such as E coli may require isolation from inclusion bodies and refolding The selection of the appropriate growth conditions and recovery methods are within the skill of the art

The polypeptide can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography arid lectin chromatography Protein refolding steps can be used, as necessary, in completing configuration of the mature protein Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps

Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated Polypeptides of the invention may also include an initial methionine ammo acid residue

The polypeptide of the present invention is also useful for identifying other molecules which have similar biological activity An example of a screen for this is isolating the coding region of the aspartic protease gene bv using the known DNA sequence to synthesize an oligonucleotide probe or as a probe itself Labeled ohgonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to

The polypeptides may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy "

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide Such methods are well-known in the art For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention

Similarly, cells may be engineered tn vivo for expression of a polypeptide in vivo by, for example, procedures known in the art As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the poiypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in \ t\ n I liesc IIIHI ihci methods lot ailniinisicπng a polypeptide of the present invention by such method should be tinpiiient to those skilled in the art from the teachings of the present invent ion I 01 example, the expression vehicle for engineering cells may be other than a ret virus, loi example, an adenovirus which may be used to engineer cells in vivo after combinat ion with a suitable delivery vehicle

"Recombinant" polypeptides refer to polypeptides produced by recombinant DNA techn iques, i c . pi duccd Irom cells transformed by an exogenous DNA construct encoding the desired polypeptide "Synthetic" polypeptides are those prepared by chemical synthesis

A "replicon" is any genetic element (e g , plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i e , capable of replication under its own control

A "vector" is a replicon, such as a plasmid, phage, or cos id, to which another DNA segment mav be attached so as to bring about the replication of the attached segment

A "double-stranded DNA molecule" refers to the polymeric form of deoxyribonucleotides (bases adenine, guanme, thymine, or cytosine) in a double-stranded helix, both relaxed and supercoiled This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms Thus, this term includes double-stranded DNA found, inter a a, in linear DNA molecules (e g , restriction fragments), viruses, plasmids, and chromosomes In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the sense strand of DNA

A DNA "coding sequence of or a "nucleotide sequence encoding" a particular protein, is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate reguiatorv sequences

A "promoter sequence" is a DNA reguiatorv region capable ot binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S I ), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes

DNA "control sequences" refers collectively to promoter sequences, πbosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the expression (l e , the transcription and translation) of a coding sequence in a host cell

A control sequence "directs the expression" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA which is then translated into the polypeptide encoded bv the coding sequence Λ "host cell" is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous DNA sequence

A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell In prokaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid With respect to eukaryotic cells, a stably transformed or transfected cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cell containing the exogenous DNA

A "clone' is a population of cells derived from a single cell or common ancestor by mitosis A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations Two DNA or polypeptide sequences are "substantially homologous" or "substaπtiallv the same" when at least about 85% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule and includes allelic variations As used herein, substantially homologous also refers to sequences showing identity to the specified DNA or polypeptide sequence DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system Defining appropriate hybridization conditions is within the skill of the art See, e g , "Current Protocols in Mol Biol " Vol I & II, Wiley Interscience Ausbel et al (ed ) ( 1992) Protein sequences that are substantially the same can be identified by proteolvtic digestion, gel electrophoresis and microsequencing

The term "functionally equivalent" intends that the amino acid sequence of the subject protein is one that will exhibit enzymatic activity of the same kind as that of the aspartic protease

A "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature

The polypeptides of the present invention may be be of use in therapy According!} , in a further aspect, the present invention provides a polypeptide having the ammo acid sequence given in SEQ ID NO 1 or SED ID NO 15, and fragments, analogs or derivative thereof, for use in therapy Suitably, such polypeptides may play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, hypertension inflammation, asthma and cardio-pulmonary conditions

The polypeptides and polynucleotides of the present invention may be employed in combination with a suitable pharmaceutical carrier Such compositions comprise a thcrapeulically effective amount of the active agent, and a pharmaceutically acceptable carrier or excipient Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof The formulation should suit the mode of administration The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention Associated with such contamer(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration In addition, the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds

The pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, lntrapeπtoneal, intramuscular, subcutaneous, intranasal or intradermal routes The polypeptides or polynucleotides of the present invention is administered in an amount which is effective for treatment and/or prophylaxis of the specific indication The amounts and dosage regimens of active agent administered to a subject will depend on a number of factors such as the mode of administration, the nature of the condition being treated and the judgment of the prescribing physician.

The sequences of the present invention are also valuable for chromosome identification The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome Moreover, there is a current need for identifying particular sites on the chromosome Chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries I luoi es en e in situ hybridization (FISH) of a cDNA clones to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step This technique can be used with cDNA as short as 500 or 600 bases, however, clones larger than 2.000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection FISH requires use of the clones from which the EST was derived, and the longer the better For example 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time For a review of this technique, see Verma et al , Human Chromosomes a Manual of Basic Techniques, Pergamon Press, New York (1988) 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, Mendehan Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheπtance of physicallv adjacent genes) ^—

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes (This assumes 1 megabase mapping resolution and one gene per 20 kb)

Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence Ultimately, complete sequencing of genes from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymorphisms

The polypeptides of the invention or cells expressing them can be used as an immunogen to produce antibodies thereto These antibodies can be for example, poiyclonal or monoclonal antibodies The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library Various procedures known in the art mav be used for the production of such antibodies and fragments Antibodies generated against the polypeptides of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the po peptides to an animal, preferably a nonhuman The antibodv so obtained will then bind the polvpeptides itself In this manner, even a sequence encoding onlv a fragment of the polvpcptidcs can be used to generate antibodies binding the whole native polypeptides Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples include the hybπdoma technique (Kohler and Milstein, 1975, Nature, 256 495-497), the tπoma technique, the human B-cell hybπdoma technique (Kozbor et al . 1983, Immunology Today 4 72), and the EBV-hvbπdoma technique to produce human monoclonal antibodies (Cole, et al , 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc , pp 77-96)

Techniques described for the production of single chain antibodies (U S Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention

This invention further provides a method of screening compounds to identify those compounds which inhibit the polypeptide comprising contacting isolated polypeptide with a test compound and measuring the rate of turnover of an enzyme substrate as compared with the rate of turnover in the absence of test compound The invention also relates to ^~ compounds identified thereby

This invention also provides transgenic non-human animals comprising a polynucleotide encoding a polypeptide of the invention Also provided are methods for use of said transgenic animals as models for mutation and SAR (structure/activity relationship) evaluation as well as in drug screens

The present invention is also directed to inhibitor molecules of the polypeptides of the present invention, and their use in reducing or eliminating the function of the poK peptide

An example of an inhibitor is an antibody or in some cases, an oligonucleotide which binds to the polypeptide

An example of an inhibitor is an antisense construct prepared using antisense technology Antisense technology can be used to control gene expression through tπple- helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA For example, the 5' coding portion of the polynucleotide sequence, which encodes for the polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al , Nucl Acids Res , 6 3073 ( 1979), Cooney et al, Science, 241 456 ( 1988), and Dervan et al , Science, 251 1360 ( 1991 )), thereby preventing transcription and the production of polypeptide The antisense RNA oligonucleotide hybridizes to the mRNA tn vivo and blocks translation of the mRNA molecule into the polypeptide (Okano, J Neurochem , 56 560 (1991), Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression CRC Press, Boca Raton, TL ( 1988)) The o gonucleotides ilesu ibul above can also be ddivcicd lo cel ls such that the antisense RNA or DNA may be cxpi cssc ///

o to inhibit piodu lion ol polypeptide

Another example ol an inhibitor is a small molecule which binds to and occupies the catalytic site ol the polypeptide thereby making the catalytic site inaccessible to substrate such that noi m l biological activity is prevented Examples of small molecules include but arc not limited to small peptides or peptide-like molecules

When used in therapy, the inhibitors of the invention are formulated in accordance with standard phai macoutical practice

The inhibitoi which ai e active when given orally can be formulated as liquids, for example syrups, suspensions or emulsions, tablets, capsules and, lozenges

A liquid foi mulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carπer(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol. oils or water with a suspending agent, preservative, flavouring or colouring agent A composition in the form of a tablet can be prepared using any suitable ^~~ pharmaceutical carπer(s) routinely used for preparing solid formulations Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose

A composition in the form of a capsule can be prepared using routine encapsulation procedures For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule, alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carπer(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule

Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil for example polyethylene glycol, polyvinyl pyrrolidone, lecithin arachis oil or sesame oil Alternatively, the solution can be lyophihsed and then reconstituted with a suitable solvent just prior to administration

A typical suppository formulation comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycois, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats

Preferably the composition is in unit dose form such as a tablet or capsuie Each dosage unit for oral administration contains preferably from 1 to 250 mg (and for parenteral adm inistration contains preferably from 0 1 to 25 mg) of an inhibitor of the invention

The daily dosage regimen for an adult patient may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0 1 mg and 100 mg, preferably between 0 1 mg and 25 mg, of the compound of the formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day. Suitably the compounds will be administered for a period of continuous therapy.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

Sequence Data

SI X NO I I) 1

MSPPPLLLPLLLLLPLLNVEPAGATLIRIPLRQVHPGRRTLNLLRGWGKP AELPKLGAPSPGDKPΛSVPLSKFLDAQYFGEIGLGTPPQNFTVAFDTGSS ULWVPSRRCHFFSVPCWFHHRFNPNASSSFKPSGTKFAIQYGTGRVDGIL SEDKLTIGGI GASVIFGEALWESSLVFTVSRPDGILGLGFPILSVEGVR PPLDVLVEQGLLDKPVFSFYFNRDPEVADGGELVLGGSDPAHYIPPLTFV PVTVPAYWQIHMERVKVGSRLTLCAQGCAAILDTGTPVIVGPTEEIRALH AAIGGIPLLAGEYIIRCSEIPKLPAVSLLIGGV FNLTAQDYVIQFAQGD VRLCLSGFRALDIASPPVPVWILGDVFLGAYVTVFDRGDMKSGARVGLAR ΛRPRGADLGRRETAQAQYRGCRPGDAHAHRVARASATQ

(438aa)

SEQ ID NO 2

TGGGTTCACACCCGGCTCCCCAGCGaiSTCTCCACCACCGCTGCTGCTACCCTTGCTGCTGCTG TGCC TCTGCTGAATGTGGAGCCTGCTGGGGCCACACTGATCCGGATCCCTCTTCGTCAAGTCCACCCTGGACG CAGGACCCTGAACCTACTGAGGGGATGGGGAAAACCAGCAGAGCTCCCCAAGTTGGGGGCCCCATCCCC TGGGGACAAGCCTGCCTCGGTACCTCTCTCCAAATTCCTGGATGCCCAGTATTTTGGGGAAATTGGGCT GGGAACGCCTCCACAAAACTTCACTGTTGCCTTTGACACTGGCTCCTCCAATCTCTGGGTCCCGTCCAG GAGATGCCACTTCTTCAGTGTGCCCTGCTGGTTCCACCACCGCTTCAATCCCAATGCCTCCAGCTCCTT CAAGCCCAGTGGGACCAAGTTTGCCATTCAGTATGGAACTGGGCGGGTAGATGGAATCCTGAGTGAGGA CAAGCTGACTATTGGTGGAATCAAGGGTGCATCCGTGATTTTCGGGGAAGCTCTGTGGGAATCCAGCCT GGTCTTCACTGTTTCCCGCCCCGATGGGATATTGGGCCTCGGTTTTCCCATTCTGTCTGTGGAAGGAGT TCGGCCCCCGCTGGATGTACTGGTGGAGCAGGGGCTATTGGATAAGCCTGTCTTCTCCTTTTACTTCAA CAGGGACCCTGAAGTGGCTGATGGAGGAGAGCTGGTCCTGGGGGGCTCAGACCCGGCACACTACATCCC ACCCCTCACCTTCGTGCCAGTCACAGTCCCCGCCTACTGGCAGATCCACATGGAGCGTGTGAAGGTGGG CTCACGGCTGACTCTCTGTGCCCAGGGCTGTGCTGCCATCCTGGATACAGGCACACCTGTCATCGTAGG ACCCACTGAGGAGATCCGGGCCCTGCATGCAGCCATTGGGGGAATCCCCTTGCTGGCTGGGGAGTACAT CATCCGGTGCTCAGAAATCCCAAAGCTCCCCGCAGTCTCACTCCTCATTGGGGGGGTCTGGTTTAATCT CACGGCCCAGGATTACGTCATCCAGTTTGCTCAGGGTGACGTCCGCCTCTGCTTGTCCGGCTTCCGGGC CTTGGACATCGCTTCGCCTCCAGTACCTGTGTGGATCCTCGGCGACGTTTTCTTGGGGGCGTATGTGAC CGTCTTCGACCGCGGGGACATGAAGAGCGGCGCACGAGTGGGACTGGCGCGCGCTCGCCCTCGCGGAGC GGACCTGGGAAGGCGCGAGACCGCGCAGGCGCAGTACCGCGGGTGCCGCCCAGGTGATGCGCATGCGCA CCGGGTAGCc/aGAGCTAGCGCTACTCAGTAAAAATCCAATATTTCCATTGAAAAAAAAAAAAAAA

c/a=eιther a C or an A at this position, unknown at present initiating ATG underlined, stop codon unknown

SEQ ID NO 3 (EST 176432)

NAATTCGGCAllAGAAGGAAAACTAGGAAGCCTGGGTTCACACCCGGCTCCCCAGCAATGT CTCCACCACTGCTGCTGCTACCCTTGACTGCTGCTGCTGCCTCTGCTGAATGTGGAGCCT GCTGGGGCCACACTGATCCGGΛTCCCTCTTCGTCAAGTCCACCCTGGACGCAGGCCCCTG AAACCTACTGAGGGGATGGGGAAAACCAGCAGAGCTCCCCAAGTTGGGGGGCCCCATCCC CTGGGGACAAGCCTGCCTCCGTACCTCTNTCCAAATTCCTGGGTGGCCCATTATTTTGGG GGAAATTGCGGTGGGGAACGGCTTCCACAAAATTTCATTGTTGGCTTTGNACATGGGTTN CTTCCAATN_TNTGGGTCCCGTTCCAGGAGANGGCCATTTTTTTNATGTTGGCCCTGNTGG GTTCCACCACNGTTTAANTTCCAATGGNNTCCAATTTCTTTAAAGCCCATGGGGNCCAAN TTTNGCCATTNΛΛTNTGGGA

SEQ ID NO 4 (EST 424772):

GGAATCCTGAGTGAGGACAAGCTGACTGTTGGTGGANTCAAGGGTNCATCCGTGAATTTT

CGGGGAAGCTCTGTGGGAATCCAGCCTGGTCTTCACTGTTTCCCGNCCCGATGGGNATAT

TGGGCCTCGGTTTTCCCATTCTGTCTGTGGAAGGAGTTCGGCCCCCGCTGGGATGTACTG GTGGAGCAGGGGCTATTGGATAAGCCTGTCTTCTCCTTTTACTTCAACAGGGACCCTGNA

AGTGGCTGATGGAGGAGAGCTGGTCCTGGGGGGCTCAGACCCGGCACACTACATCCCACC

CCTCACCTTCGTGNCCAGTTCACAGTTCCCCGGCTACTGGCAGATTCCACATGGTGCGTG

TTGAAGGTNGGGGTCAAGGGTGAANTNTNTGTGGCCCAGGGTGTGTTGGCCATCCTGGAT

AACAGCACAATTTTCATNGTAGGGACCCATTG

SEQ ID NO 5 (EST 443275):

TGGTTCCACCACCGCTTCAATCCCAATGCCTCCAGCTCCTTCAAGCCCAGTGGGACCAAG TTTGCNATTCAGTATGGAACTGGGCGGGTAGATGGAATCC GAAGTAAGGACAAGCTGAC TATTGGTGGGAATCAAGGGTGCATCCGTGAATTTTGGGGGAAGCTCTGTGGGAATCCAGC CTGGTCTTCACTGTTTCCCGCCCCGATGGGATATTGGGCCTCGGTTTTCCCATTCTGTCT GTGGGAAGGAGTTCGGCCCCCGNTGGATGTTACTGGTGGGAGCANGGGGCTATTGGGTNA AGCCCGTCTTCTNCCTTTTAANTTCAACAGGGGACCCTGAAAGTGGGTT

SEQ ID NO 6 (EST 567394): HACCACTGCTGCTGCNACCCTTCCTGCTGCTGCTGCCTCTCCTGAATG/TGGAGCCTGCTG

GGGCCACACTNATCCGNATCCCTCTTCGTNAAGTCCACCCTGGACGCAGGACCCTNAACC

TACTGAGGGGATGGGGAAAACCAGCAGAGCTCCCCAAGTTGGGGGCCCCATCCCCTGGGG

ACAAGCCTGCNTCGGTACCTCTNTCCAAATTCCTGGATGCCCAGTATTTTGGGGAAATTG

GGCTGGGAACGCCTCCACAAAACTTCACTGTTGCCTTTGAANACTGGCTCCTCCAATCTT TGGGTCCCGTCCAGGTGTTGCCACTTGTTNCAGTGTGGCCCTGATTGGTTTNCACCCACC

NTTTTCAATTCCCATGNCCTTNCAG

SEQ ID NO 7 (EST 685578):

TCCACCACTGCTGCTGCTACCCTTNCTGCTGCTGCTGCCTCTNCTGAATGTGGAGCCTGC TGGGGCCACACTGATCCGGNNCCCTCTTCGTNAAGTCCACCCTGGACGCAGGACCCTGAA CCTACTGAGGGGATTGGGNAAA CAGCAGAGCTCGCCAAGTTGGGGGTCCNATNCCCTNG GGACAAGGCTGGC

SEQ ID NO 8 (EST 928138):

GGGCAGNGGNTCTCCACCACTGCTGCTGCTACCCTTGCTGCTGCTGCTGCC CTGCTGAA TGTGGAGCCTGCTGGGGCCACACTGATCCGGATCCC CTTCGTCAAGTCCACCCTGGACG CAGGACCCTGAACCTACTGAGGGGATGGGGAAAACCAGCAGAGCTCCCCAAGTTNGGGGC CCCATCCCCTNGGGACAAGCCTGCCTCGGTACCTCTCTTCAAATTCCTGGATGCCCAGTA TTTTTGGGAAATTTGGCTTGGAACGCTTCACAAAACTTCACTGTTGCTTTGACAAT

SEQ ID NO 9 (EST 947785): occv ^■( IΛ( i( "π II i i"π 'i 'Λi ^•< 'ΛI ^•( ^•< K "i^,-π -ΛΛ'I'( ^•CΓΛΛTCICCTCCΛGCTCCTTCAAGCCCAGT

GGGΛCCΛΛCITTTC It 'I'Λ'ITI -Λ< ITΛT( l< IΛΛCTϋOUCααGTΛGATGGAATCCTGAGTGAGGAC AAGCTGACTATT< IUTCKIAATl7VAU(l<miCATCCaTaATTTTCGGGGAAGCTCTGTGGGAA TCCAGCCTGGTC'I'TCAl-nrn-Tt.-t-t.XlNfC UATGGGATATTGGGCCTCGGTTTTCCCATT CTGTCTGTCGAACHlAcrn'C-lKlCCrc'l'trrrGaATGTACTGGTGGAGCAAGGGCTATTGGAT

AAGCCTGTCTTCTTCCTTTTAAr rCAACAGGGNCCNGAAGTGGTTGANGGAGGAGAGCTG GTCCCGGGGGCGTTCAAGACCCGGGAAATNACANCCCACCCT

SEQ ID NO 10 (EST 1000163): GGCACGAGGAGATGCCACTTCTTCAGTGTGCCCTGCTGGTTCCACCACCGCTTCAATCCC AATGCCτCCAGCTCCTTCΛΛGCCCΛGTGGGACCAAGTTTGCCATTCAGTATGGAACTGGG CGGGTAGATGGAATCCTGAGTGAGGACAAGCTGACTATTGGTGGAATCAAGGGTGCATCC GTGATTTTCGGGGAAGCTCTGTGGGAATCCAGCCTGGTCTTCACTGTTTCCCGGCCCGAT GGGATATTGGGNCTCGGTTTTCCCATTCTGTCTGTGGAAGGAGTTCGGNCCCCGCTGGAT GTACTGGTGGAGCAGGGGNTATTGGATAAGCCTGTNTTCTTCTTTTAATTCAACAAGGAC CCTGAAGTGGNTTAATGGAGGAGAGCTTGTCCTNGGGGGGNT

SEQ ID NO 11 (EST 1218021):

ACCCΛCGCGTCCGCACCACTGCTGCTGCTACCCTTGCTGCTGCTGCTGCCTCTGCTGAAT GTGGAGCCTGCTGGGGCCACACTGATCCGGATCCCTCTTCGTCAAGTCCACCCTGGACGC AGGACCCTGAACCTACTGAGGGGATGGGGAAAACCAGCAGAGCTCCCCAAGTTGGGGGCC CCATCCCCTGGGGACAAGCCTGCCTCGGTACCTCTCTCCAAATTCCTGGATGCCCAGTAT TTTGGGGAAATTGGGCTGGGAACGCCTCCACAAAACTTCACTGTTGCCTTTGACACTGGC TCCTCCAATC CTGGGTCCCGTCCAGGAGATGCCACTTCTTCAGTGTGCCCTGCTGGTTC CAACAACGCTTCAATCCCAATGCCTCCAGCTCCTTCAAGCCCAGTGGGAACCAAGTNTGC CATTCAGTATGGAACTNGGCCGGGTAGATGGGAATCCTGAATGANGACNAAGCT

SEQ ID NO 12 (EST 1320439):

GGGTCGACCCACGCGTCCGGGGCTGGGAACGCCTCCACAAAACTTCACTGTTGCCTTTGA CACTGGCTCCTCCAATCTCTGGGTCCCGTCCAGGAGATGCCACTTCTTCAGTGTGCCCTG CTGGTTCCACCACCGCTTCAATCCCAATGCCTCCAGCTCCTTCAAGCCCAGTGGGACCAA GTTTGCCATTCAGTATGGAACTGGGCGGGTAGATGGAATCCTGAGTGAGGACAAGCTGAC TATTGGTGGAATCAAGGGTGCATCCGTGATTTTCGGGGAAGCTCTGTGGGAATCCAGCCT GGTCTTCACTGTTTCCCGCCCCGATGGGATATTGGGCCTCGGTTTTCCCATTCTGTCTGT GGAAGGAGTTCGGCCCCCGCTGGATGTACTGGTGGAGCAAGGGCTATTGGATAAGCCTGT CTTCTCCTTTTACTTCAACAGGGACCCTGAAAGTGGCTGATTGAAGAGAACTTGTCCTGG G

SEQ ID NO 13 (EST 716478):

ACAGTCCCCNCCTACTGGCAGATCCACATGGAGCGTGTGAAGGTGGGCTCACGGCTGACT CTCTGTGCCCAGGGCTGTGCTGCCATCCTGGATACAGGCACACCTGTCATCGTAGGACCC ACTGAGGAGATCCGGGCCCTGCATGCAGCCATTGGGGGAATCCCCTTGCTGGCTGGGGAG TACATCATCCGGTGCTCAGAAATCCCAAAGCTCCCCGCAGTCTCACTCCTCATTGGGGGG CTCTσGTττ,V,TCτCΛCGGCCCΛGGΛTTΛCGTCATCCAGTTTσcτCAGGGTGACGTCCGC CTCTGCTTGTCCGCCTTCCGGGCCTTGGACATCGCTTGGCTNCAGTACCTGTGTGGATCC

- 77 - TCGGCGΛl 'I 1( H !( i( '(iΛTGTGΛCCGTTTTGACCGGGGGACATNAAGAGCGGCG AACOAGTNGAC'l"n .'(l(lCU10TTGCCT-lOCGAGGACCTTGNAGCGGAACGGAAGGAGNCCG GGGTCCCCAGTATCOA'I'GACGGNGAAGTGGTCTAGAAACATATATTAAAAAAAAAAAAAA ΛΛCTGGGNTTNGCCG

SEQ I D NO 14 (I -ST 857644):

GGCACGAGGCAAGGGTGCATCAGTGATTTTCGGGGAGGCTCTCTGGGAGCCCAGCCTGGT CTTCGCTTTTGCCCATT_TTGATGGG_ATATTGGGCCTCGGTTTTCCCATTCTGTCTGTGGA

AGGAGTTCGGCCCCCGATGGATGTACTGGTGGAGCAGGGGCTATTGGATAAGCCTGTCTT CTCCTNTTACCTCAACAGGGACCCTGGAAGAGCCTGATGGAGGAGAGCTGGTCCTGGGGG GCTCGGACCCGGCACACTΛCATNCCACCNCTCAACTTCGTGCCAGTCACGGTCCCGGNCT ACTGGCAGATCCACATGGAGCGTGTGAAGGTGGGGNCCCAGGCTGACTTTNTNGTGGCCA AGGGTGTGCTGNCCATC

SEQ ID NO. 15

XFGXEGKLGSLGSHPAPQQCLHHCCCYPS LLPLLNVEPAGATLIRIPLRQVHPGRRTLN LRGV.-GKPAELPKLGAPSP GDKPASVPLSKFLDAQYFGEIGLGTPPQNFTVAFDTGΞSNLWPΞRRCHFFSVPCWFHHRFNPNASSSFKPSGTKFAIQY GTGRVDGILSEDK TIGGI KGASVIFGEA ESSLVFTVSRPDGILG GFPILSVEGVRPPLDVL'/ΞOGLLDKPVFSFYF NRDPEW GGELVLGGSDPAHYIPPLNFVPVTVPAYWQIHMERVKVGPRADSLCQGCAAILDTGTY VITGPTEEIRA H AAIGGIPLLAGEYI IRCSEIPKLPAVSL IGGV FN TAQDYVIQTTRKGDλ LC SGFRALDIA EGPVWILGEVF G

ICDRFDRGT . RAANPS . LAGVALRGPXSGTEGXRGPQYR . RXSG ETYI

446 amino acids (+ 3 additional stop codons)

SEQ ID NO 16 tlAATTCGGCAN'AGAAGGAAAACTAGGAAGCCTGGGTTCACACCCGGCTCCCCAGCAATGTCTCCACCACTGCTGCTGCTA

ACCCT GACGCAGGACCCTGAACC7ACTGAGGGGATGGGGAAAACCAGCAGAGCTCCCCAAG7TC GGGCCCCATCCCCT

ACAAAACTTCACTGTTGCCTTTGACACTGGCTCCTCCAATCTCTGGGTCCCGTCCAGGAGATGCCACTTCTTCAGTGTGC CCTGCTGGTTCCACCACCGCTTCAATCCCAATGCCTCCAGCTCCTTCAAGCCCAGTGGGACCAAGTTTGCCATTCAGTAT GGAACTGGGCGGGTAGATGGAATCCTGAGTGAGGACAAGCTGACTATTGGTGGAATCAAGGGTGC TCCGTGATTTTCGG GGAAGCTCTGTGGGAATCCAGCCTGGTCTTCACTGTTTCCCGCCCCGATGGGATATTGGGCCTCGGTTTTCCCATTCTGT CTGTGGAAGGAGTTCGGCCCCCGCTGGATGTACTGGTGGAGCAAGGGCTATTGGATAAGCCTGTCTTCTCCTTTTATTTC AACAGGGACCCTGAAGTGGTTAATGGAGGAGAGCTGGTCCTGGGGGGCTCGGACCCGGCACACTACATCCCACCCCTCAA CTTCGTGCCAGTCACGGTCCCCGCCTACTGGCAGATCCACATGGAGCGTGTGAAGGTGGGGCCCAGGGCTGACTCTCTGT GCCAAGGGTGTGCTGCCATCCTGGATACAGGCACGTACCTGGTCATCACAGGACCCACTGAGGAGATCCGGGCCCTGCAT GCAGCCATTGGGGGAATCCCCTTGCTGGCTGGGGAGTACATCATCCGGTGCTCAGAAATCCCAAAGCTCCCCGCAGTCTC ACTCCTCATTGGGGGGGTCTGGTTTAΛTCTCACGGCCCAGGATTACGTCATCCAGACTACTCGAAAGGGTGACGTCCGCC TCTGCTTGTCCGGCTTCAGGGCCTTGGACATCGCTCGGGCTGAAGGACCTGTCTGGATCCTCGGCGAAGTTTTTTGGGGA ATATGTGACCGTTTTGΛCCGGGGGΛCΛTGΛAGAGCGGCGAACCCGAGTTGACTTGCGGGGGTTGCCTTGCGAGGACCTTG NΛGCGGΛΛCGGΛΛGGΛGNCCGGGGTCCCCΛGTATCGATGACGGNGAAGTGGTCTAGAAACATATAT?

Claims

Claims

1 An isolated polynucleotide comprising a nucleotide sequence that has at least 80% identity over Us enliie length to a nucleotide sequence encoding the polypeptide comprising i c a mo acid sequence of SEQ ID NO 1 or SEQ ID NO 15 or a fragment, analog or derivative ol said polypeptide

2 The polynucleotide of claim 1 w hich comprises the nucleotide sequence contained in SEQ ID NO 2 or SEQ ID NO 16

An isolated polynucleotide which comprises a nucleotide sequence that is at least 80% identical to that of SEQ ID N02 or SEQ ID NO 16 over its entire length

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

5 The polynucleotide of any one of ciaims 1 to 4 wherein the polynucleotide is DNA

6 The polynucleotide of any one of claims 1 to 4 wherein the polynucleotide is RNA

7 The polynucleotide of claim 5 w herein the polynucleotide is genomic DNA

8 A vector containing the DNA of any one of claims 2, 4, 5, 6, or 7

9 A host cell geneticalK engineered with the vector of claim 8

10 A process tor producing a poK peptide comprising expressing from the host cell of claim 9 the polypeptide encoded by said DNA

1 1 A process for producing cells capable of expressing a polypeptide comprising geneticalK engineering cells with the vector of claim 8

12 A polynucleotide hybπdizable to the polynucleotide of any one of claims I to 7 and encoding a polypeptide having substantially the same biological function or activity as the polypeptide of SEQ I D NO 1 or SEQ ID NO 15

13 A polypeptide which is at least 80% identical over its enire length to the amino acid sequence of SEQ I D NO 1 or SLQ ID NO 15 and fragments analogs and derivatives thereof

14 A polypeptide having the amino acid sequence of SEQ ID NO 1 or SEQ ID NO 15 and fragments, analogs and derivatives thereof

15. The polypeptide of Claim 14 wherein the polypeptide has the amino acid sequence of SEQ ID NO 1 or SEQ ID NO 15

16. The polypeptide of claim 13, 14 or 15 in isolated form.

17. The polypeptide of any one of claims 13 to 16 for use in therapy.

18. A method of screening compounds to identify those compounds which inhibit the polypeptide of claim 13, 14 or 15 comprising contacting isolated polypeptide with a test compound and measuring the rate of turnover of an enzyme substrate as compared with the rate of turnover in the absence of test compound

19. A compound identified by the method of claim 18.

20. An inhibitor of the polypeptide of claim 13, 14 or 15.

21. An inhibitor according to claim 20 which is an antibody to the polypeptide of claim 13, 14 or 15

22. A pharmaceutical composition comprising the polynucleotide of claim 1 or 12, a polypeptide of claim 13 or 14, a compound of claim 19 or an inhibitor of claim 20 and a pharmaceutically acceptable carrier

23. A method for the treatment of a patient having need to inhibit the polypeptide of claim 13, 14 or 15 comprising, administering to the patient a therapeutical ly effective amount of the compound of claim 19 or inhibitor of claim 20

24. The use of a compound of claim 19 or inhibitor of claim 20 for the manufacture of a medicament for use in therapy.