GB2382078A

GB2382078A - Protease polypeptide

Info

Publication number: GB2382078A
Application number: GB0224687A
Authority: GB
Inventors: Barbara Susan Ink
Original assignee: Glaxo Group Ltd
Current assignee: Glaxo Group Ltd
Priority date: 2001-10-26
Filing date: 2002-10-23
Publication date: 2003-05-21
Also published as: GB0224687D0

Abstract

The present invention provides an isolated cysteine protease polypeptide, HIPHUM 113, having the amino acid sequence of SEQ ID Nos 2 or 4, or a variant or fragment thereof which is capable of cleaving SUMO from a target protein and/or cleaving the precursor form of SUMO to release its active form. The invention also provides polynucleotides encoding HIPHUM 113, and the use of the polypeptide in screening for compounds capable of cysteine protease modulation. Such compounds may be useful in the treatment of male and female reproductive diseases, cancer, brain diseases and inflammatory diseases such as rheumatoid arthritis.

Description

Protein Field of the Invention The present invention relates to Cysteine Protease polypeptides.

Background of the Invention There are 4 main catalytic types of peptidases ; serine (which includes threonine peptidases) cysteine, aspartic and metallo. The serine, threonine and cysteine peptidases are catalytically very different from the aspartic and metallopeptidases in that the nucleophile of the catalytic site is part of an ammo acid, whereas it is an activated water molecule m the other groups.

Peptidases m which the nucleophile IS the sulphydryl group of a cysteine residue are known as cysteine-type peptidases. The catalytic mechanism is similar to that of the serinetype peptidases in that a nucleophile and a proton donor or general base is required, and the proton donor in all cysteine peptidases (in which it has been identified) is a histidme residue.

Typical examples of cysteine peptidases include the cathepsins (excluding cathepsin G) and caspases.

Human ubiquitin-like protein specific protease (ULP). Ulp is a deconjugating enzyme of SUMO/Sentrin, a ubiquitin-like protein, and its target protein. It is a cysteine

protease that is unrelated to other known deubiquitinating enzymes, but shows similarity to certain viral proteases. The Ulps cleave at a glycine-glycine-X cleavage site that is similar to the consensus sites of adenovirus, African swine fever virus, and certain poxviruses. Ulps also cleave the SUMO precursor to generate the mature form of SUMO.

The human ULPs show homology to the yeast ULP1 and ULP2 genes. Yeast ULP1 cleaves the yeast homolog of SUMO, SMT3, from target proteins. ULP 1 is important throughout the cell cycle and is essential for the G2/M phase. ULP2 also cleaves SMT3 from target proteins, although the target proteins of ULP2 appear to be different from ULP 1. ULP2 is important for the recovery of cells from checkpoint arrest induced by DNA damage, inhibition of DNA replication, or defects in spindle assembly. Deletions of ULP2 in yeast show a phenotype of temperature-sensitive growth, abnormal cell morphology, decreased plasmid and chromosome stability, and a severe sporulation defect.

Summary of the Invention A novel cysteine protease, referred to herein as HIPHUMI13A, is now provided.

Also provided is a splice variant of HIPHUM 113A called herein HIPHUM 113B.

Throughout this specification, where the term HIPHUM 113fis used, it is intended to

refer to both HIPHUM 113A and the splicc variant HIPHUM 113B.

HIPHUM 113A is cxpressed in al1 tissues at various levels. It is highly expressed in cerebellum, lung, thymus, tonsil, and fetal brain. It is also expressed at significant levels in adrenal. myomctrium, omentum. ovary, and testis.

HIPPHUM I 13A expression Ice\'els are elevated in lung tumor, tissues from patients with brain diseases (Alzheimer's, Parasupranuclear palsey (PSP) ), T cells, peripheral blood mononuclear cells (PBMCs), dendritic cells, and RA synovium.

HIPPIHUM] t3A expression) cve ! s arc down regulated in breast tumor and differentiated osteoblasts The novel cysteme protease is a screening target for the identification and development of novel pharmaceutical agents. includmg modulators ofcysteine protease activity. These agents may be used m the treatment and. or prophylaxis of disorders such as male and female reproductive diseases : oncology diseases such as breast or lung cancer, brain diseases such as alzheimers or PSP: inflammatory diseases such as multiple sclerosis, lupus. psoriasis. psoratic arthritis, exzema. asthma and rheumatoid arthrisis or inflammatory musculoskeletal diseases such as osteoporosis. osteopetrosis and pendontal disease Accordingly, the present invention provides an isolated cysteine protease polypeptide comprising (1) the ammo acid sequence of SEQ ID NO: 2 or 4; (n) a variant thereof which is capable ofcleaving SUMO from a target protein and or cleaving the precursor form of SUMO to release the active form of SUMO : or (Ill) a fragment of (]) or (ii) which is capable of cleaving SUMO from a target

protein and. or cleaving the precursor form of SUMO to release the active Z7 form of SUMO.

According to another aspect of the invention there is provided a pol,,, nucleotide encoding a polypeptide of the invention which polynucoeotide includes a sequence comprising : (a) the nucleic acid sequence of SEQ ID NO: I or 3 and/or a sequence complementarythereto; (b) a sequence whic hydridises under stringent conditions to a sequence as defined in (a) :

(c) a sequence that is degenerate as a result of the genetlc code to a sequence as defined in (a) or (b) : or

(d) a sequence having at least 95% identity to a sequence as defined in (a), (b) or (c).

The invention also provides : an expression vector which comprises a polynucleotide of the invention and which is capable of expressing a polypeptide of the invention; a host cell comprising an expression vector of the invention; a method of producing a polypeptide of the invention which method comprises maintaining a host cell of the invention under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide; an antibody specific for a polypeptide of the invention ; a method for identification of a substance that modulates cysteine protease activity and/or expression, which method comprises contacting a polypeptide, polynucleotide, expression vector or host cell of the invention with a test substance and determining the effect of the test substance on the activity and/or expression of the said polypeptide or the polypeptide encoded by the said polynucleotide, thereby to determine whether the test substance modulates cysteine protease activity and/or expression ; a compound which or modulates cysteine protease activity and which is identifiable by the method referred to above; a method of treating a subject having a disorder that is responsive to cysteine protease stimulation or modulation, which method comprises administering to said subject an effective amount of substance of the invention; and use of a substance that stimulates or modulates cysteine protease activity in the manufacture of a medicament for the treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of cysteine protease activity.

Preferably the disorder is selected from male and female reproductive diseases; oncology diseases such as breast or lung cancer; brain diseases such as alzheimers or PSP ; inflammatory diseases such as multiple sclerosis, lupus, psoriasis, psoriatic arthritis, exzema, asthma and rheumatoid arthrisis or inflammatory musculoskeletal diseases such as osteoporosis, osteopetrosis and peridontal disease.

Particularly preferred is when the disorder is an inflammatory disease.

Brief Description of the Sequences SEQ ID NO: I shows the nucleotide sequence of human protein HIPHUMI 13A.

SEQ ID NO: 2 is the amino acid sequence alone of HIPHUMI ISA.

SEQ ID NO : 3 shows the nucleotide sequence of human protem HIPHUM 113B SEQ ID NO : 4 shown the amino acid sequence alone of HIPHUM 113B Detailed Description of the Invention Throughout the present specification and the accompanying claims the words "comprise" and "include" and variations such as "comprises". "comprising", "includes" and "including"are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not spectficall\ recited, where the context shows the present invention relates to a human eysteme protease. referred to herein as

HIPHUM 113. and variants thereof. Sequence information for HIPHUM ! 13A is provided m SEQ ID NO : 1 and in SEQ ID NO : 2, and sequence information for HIPHL'M 113B is provided m SEQ ID N0 : 3 and in SEQ ID N0 : 4 A poh/peptide of the invention thus consists essentially of the ammo acid sequence of SEQ ID NO: 2 or 4, or of a variant of those sequences, or of a fragment of either sequence or variant thereof.

Polypeptides of the invention may he in a substantial'isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypept1de and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form. m which case it will generally comprise the polypeptide in a preparation in \\hich more than

501, e. (-,. more than 80'/ (), 90", ('), 95'o or 99., o, bv 50%. e. g. more than 80%. 90%). 95% or 99%. by weight of the polypeptide in the preparation is a polypeptide of the invention. Routine methods can be employed to purify and'or synthesise the proteins according to the invention. Such methods are we ! ! understood by

persons skilled in the art, and include techniques such as those disclosed in Sambrook et al, Molecular Cloning : a Laboratory Manuai. 2" dition. CSH Laboratory Press, 1989, the disclosure of which is included herein in its entirety by way of reference.

The term"variant"refers to a polypeptide which has a same essential character or basic biological functionalitv as HIPHUM 113. The essential character ofIIIPII'M 113 can be defined as follows : HIPHUM H3 is a cystine protease. Preferably a variant poiypeptide is peptide is one which binds to the same target protein as HIPHUM 113, Preferably the polypeptIde is 1-7. peptide is capable of cleaving SUMO from a target protein and/or cleaving the precursor form of SUMO to release the active form of SUMO. Preferably the polypeptide comprises a catalytic domain which comprises a catalytic triad. The catalytic triad is typically composed of a cysteme residue, histidine residue, aspartate residue and a glutamine residue which together form the oxyanion hole at the active site. Preferably the polypeptide comprises a nuclear

localisation signal. Particularly preferably, the nuclear localisation signal comprises the motif KRRK at amino aCId 215, RRHK at amino acid 798 or RHKR at ammo acid 799. A polypeptide having a same essential character as HIPHUM 113 may be identified by monitoring for a function the cysteine protease selected from cleavage of SUMO from a target protein, activation of SUMO and alteration in activity or subcellular localisation of a target protein. Typical target proteins include transcription factors, such as 11d3a/NF-KB, p53, c-jun and HIPK2, proteins involved in intracellular transport, such as RanGAPl, GLUT1 and GLUT4, proteins involved in the intracellular response to DNA damage and repair, such as Topoisomerase 1, Topoisomerase 2, MDM2 and WRN, proteins involved in nuclear localisation, such as PML and SplOO, and viral proteins such as bovine papilloma virus El and human cytomegalovirus E2. Other target proteins may be identified, for example using the yeast-2-hybrid assay. Such target proteins that have been identified using the yeast-2-hybrid screen include TNF and FAS receptors, CEN, and rad 52.

SUMO binds to various transcription factors and is involved in their regulation. Removal of SUMO from IKBa would allow for activation of NF-KB and produce an inflammatory response. SUMO conjugation increases the transcriptional activity of p53, therefore, deconjugation of SUMO may lower the transcriptional activity ofp53. C-jun modification by SUMO decreases its transcriptional activity.

Therefore, Ulp proteases may regulate tumorsuppressor activity with a role in cell cycle arrest, apoptosis, and cellular responses to mitogens, stress, or inflammatory stimuli.

SUMO conjugates to a nuclear kinase, HIPK2, and is needed for the formation of nuclear bodies containing HIPK2. HIPK2 can act as a transcriptional corepressor for homeoproteins. Therefore, deconjugation of SUMO by the protease may regulate the activity ofhomeodomain transcription factors and play a role in development.

SUMO modification of RanGAPl targets the complex to the nuclear pore complex from the cytosol. Ulp protease may be involved in the regulation of the levels of the Ran GAP I within the cytosol and pore complex.

SUMO also modifies GLUT1 and GLUT4 that are involved in insulin stimulated glucose uptake. Deconjugation of SUMO would regulate the amount of glucose uptake in the cell.

The Ulp protease may regulate the cellular response to DNA damage and DNA repair. SUMO conjugates to TOPI and TOP2 when treated with DNA damaging agents.

MDM2 is also conjugated to SUMO to prevent self-ubiquitination of mdm2, thus enhancing E3 hgase activity of p53. A Ulp protease may regulate the conjugation of SUMO to MDM2,

thereby affecting stability ofp53. SUMO also conjugates to WRN that functions as a DNA heliease, exonuclease. and ATPase. Deletions of WRN which are found in Werner's syndrome impair its nuclear localization. It is possible that cleavage of SUMO from WRN would alter the localization of WRN and contribute to its dysregulation during disease.

SUMO modification of PML is needed for its localization into nuclear bodies containing DAXX. PML is involved in many cellular functions such as the interferonresponse, immune surveillance, apoptosis, and as a tumor suppressor. It appears that the function of PML is to meditate the regulation of transcription. Mice lacking PML have shown altered transcription in cellular differentiation by way of nuclear receptors. apoptosis mediated by DAXX and p53. inhibition of growth mediated by pRb and p53, and the immune response through interferons. Therefore, regulating the conjugation- deconjugation of SUMO to PML through Ulp protease may affect these pathways.

Although the function of the interferon-induced protein. Sp I 00. IS unknown, conjugation to SUMO takes place in the nucleus. The Ulp protease may affect the activity of SplOO in response to interferon.

SUMO conjugates to the human cytomegalovirus protein, IE2 IE2 functions as a strong transactivator of both viral and cellular promoters. Overexpression of SUMO reduces the transactivation activity of IE2. Thereofre, the Ulp protease may function to control the level of IE2 sumoylation. thus. affecting both the virus life cycle and host interactions to virus infection.

SUMO conjugates to the bovine papalomavirus E1 protein which functions as the initiator of replication. Mutants which cannot conjugate SUMO are not able to localise to the correct nuclear subdomam. The Ulp protease may control the level ofsumylation of El, thus affecting both the virus life cycle and host reactions to viral infection.

Preferably a polypeptide of the invention is located in the endoplasmic reticulum when expressed in a cell. More preferably a polypeptide of the invention is capable of cycling between the endoplasmic reticulum and other intracellular compartments.

Typically, at least 95% for example at least 96%. 97%. 98% or at least 99%

identity, with the ammo acid sequences of SEQ ID NO : 2 or 4. are considered as variants of the proteins. Such variants may include allelic variants and the deletion, modification or addition of single ammo acids or groups of ammo acids within the protein sequence, as long as the peptide maintains a basic biological functJOnal1ty of the HIPHUM 113 receptor.

Ammo acid substitutions may be made. for example from 1. 2 or 3 to 10,20 or 30 substitutions. The modified polypeptide generally retains activity as a cysteine protease.

Conservative substitutions may be made. for example according to the following Table.

Amino acids m the same block in the second column and preferably in the same line in the third column may be substituted for each other.

ALIPHATIC Non-polar GAP ILV Polar-uncharged C S T M NQ Polar-charged DE KR AROMATIC H F W Y

Shorter polypeptide sequences are within the scope of the invention. For example, a peptide of at least 20 amino acids or up to 50, 60, 70, 80, 100, 150, 200, 300 or 400 amino acids in length is considered to fall within the scope of the invention as long as it demonstrates a basic biological functionality of HIPHUM 113. In particular, but not exclusively, this aspect of the invention encompasses the situation when the protem is a fragment of the complete protein sequence and may represent a catalytic domain or substrate binding domain. Preferred fragments include the C-terminal catalytic domain, the Nterminal domain which is not conserved between different members of the Ulp family of proteases, the leucine zipper domain or the nuclear localisation fragment. Such fragments can be used to construct chimeric proteases preferably with another protease, more preferably with another member of the family of cysteine proteinases. Such chimeric proteases may comprise different domains from different cysteine proteinases. For example, a fragment comprising an N-terminal domain of a polypeptide of the invention may be fused to a C-terminal catalytic domain of a different cysteine proteinase.

Fragments of HIPHUM 113 or a variant thereof can also be used to raise antiHIPHUM 113 antibodies. In this embodiment the fragment may comprise an epitope of the HIPHUM 113 polypeptide and may otherwise not demonstrate the catalytic, substrate binding or other properties of HIPHUM 113.

Polypeptides of the invention may be chemically modified, e. g. post-translationally modified. For example, they may be glycosylated or comprise modified amino acid residue.

They may also be modified by the addition of histidine residues to assist their purification or

by the addition of a signal sequence to promote insertion into the cell membrane. Such modified polypeptides fall wlthm the scope of the term "polypeptIde" of the invention. p The invention also includes nucleotide sequences that encode for HIPHUM] 13 or variant thereof as well as nucleotide sequences which are complementary thereto. The nucleotide sequence may be RNA or DNA including genomic DNA. synthetic DNA or

cDNA. Preferably the nucieotide sequence is a DNA sequence and most preferably. a cDNA sequence, Nucleotide sequence information is provided in SEQ ID NO : 1 and SEQ ID NO : 3.

Such nucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sambrook et al, 1989.

Typically a polynucleotide of the invention comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1 or SEQ ID NO:3.

A polynucleotide of the invention can hy, dndlzc to the coding sequence or the complement of the coding sequence of SEQ ID NO 1 or SEQ II) NO : 3 at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA hbrary. The slgnal level generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of the

coding sequence of SEQ ID NO : I or SEQ ID N0 : 3 is typically at least 10 fold. preferably at d fe 11 least 100 fold. as intense as interactions between other polynuc1eotldes and the coding sequence of SEQ ID NO : I or SEQ ID NO 3. The intensity of interaction may be measured, for example, by radiolabelhng the probe, e.g. with 32P. Selective hydbridsation may typically be achieved using conditions of medium to high stringency. However, such hybridisation may be carried out under any suitable conditions known 10 the art (see Sambrook et al. 1989.

For example, if high stringency is required suitabte conditions include from 0. 1 to 0. 2 x SSC at 60 "C up to 65"C. If lower stringency is required suitable conditions include 2 x SSC at 60 C.

The coding sequence of SEQ ID NO : I or SEQ ID NO : 3 may be modified by

nucleotide substitutions. for example from 1. 2 or 3 to 10, 25, 50 or 100 substitutions. The polynucleotide of SEQ II) NO : 1 or SEQ ID N0 : 3 may alternatively or additionally be modified by one or more insertions and or deletions and or by an extension at either or both ends. A polynucleotide may mclude one or more introns. for example may comprise genomic DNA. Additional sequences such as signal sequences which may assist in insertion of the polypeptide in a cell membrane may also be included. The modified polynucleotide generally encodes a polypeptide which has a HIPHUM 113 activity. Alternatively, a polynucleotide encodes a catalytic or substrate-binding portion of a polypeptide or a polypeptide which inhibits HIPHUM 113 activity.Degenerate substitutions may be made

and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table above.

A nucleotide sequence which is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO: 1 or SEQ ID NO : 3 will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the coding sequence of SEQ ID NO: 1 or SEQ ID NO : 3 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably at least 100 contiguous nucleotides or most preferably over the full length of SEQ ID NO : I or SEQ ED NO : 3.

For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984)

Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul (1993) J. Mol. Evol. 36 : 290-300 ; Altschul et al (1990) J. Mol. Bio1.

215: 403-10.

Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http ://www. ncbi. nlm. nih. qov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, 1990). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them.

The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl.

Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two

sequences ; see e. g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90 : 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability

(P (N))., which provides an indication of the probabihty by which a match between two nucleotide or ammo acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probabihty in comparison of the first sequence to the second sequence is less than about l. preferably less than about 0. 1, more preferably less than about 0. 01. and most preferably less than about 0. 001 Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention, with the more stringent

combinations (I. e. higher sequence identity over longer lengths) being preferred Thus, for example a polynucleotide which has at least 90% sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 40 nucleotides The nucleotides according to the invention have futility in production of the proteins according to the invention, which may take place in vitro, in vivo or ex vivo. The nucleotides may be involved in recombmant protein synthesis or indeed as therapeutic agents in their own right, utilised in gene therapy techniques. Nucleotides complementary to those encoding HIPHUM 113. or antisense sequences, may also be used in genre therapy Pol) l1ucleotides of the invention may be used as a primer, e. g. a PCR primer, a

primer for an alternative amplification reaction, a probe e. g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors.

Such pnmers. probes and other fragments will preferably be at least 10. preferably at least 15 or at least 20. for example at least 25. at least 30 or at least 40 nucleotides in length.

They will typically be up to 40. 50. 60. 70. 100 or 150 nucleotides in length. Probes and fragments can be longer than 150 nucleotides in length, for example up to 200. 300. 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 or 1600 nucleotides in length, or even up to a few nucleotides. such as five or ten nucleotides. short of the coding sequence of SEQ ID NO: 1 or SEQ 10 NO : 3.

The present invention also includes expression vectors that comprise nucleotide sequences encoding the proteins or variants thereof of the invention. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements. such as for example polyadenylation signals which may be necessary). and which are positioned in the correct orientation, in order to allow for protein expression. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. 1989

Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides may also be produced by synthetic means. Such antisense polynucleotides may be used as test compounds in the assays of the invention or may be useful in a method of treatment of the human or animal body by therapy.

Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i. e. the vector is an expression vector. The term"operably linked"refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence, such as a promoter,"operably linked"to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

The vectors may be for example, plasmid, virus or phage vectors provided with a origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistence gene in the case of a bacteria ! plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example in a method of gene therapy.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, yeast promoters include S. cerevisiae GAL4 and ADH promoters, S. pombe nmt 1 and adh promoter. Mammalian promoters include the metallothionein promoter which can be induced in response to heavy metals such as cadmium. Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may also be used. All these promoters are readily available in the art.

Mammalian promoters, such as ss-actin promoters, may be used. Tissue-specific promoters are especially preferred. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter. adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR). Viral promoters are readily available in the art.

The vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences,

preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombmation. In particular. a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the pol nucleotides of the invention to a mammalian cell. Other examples of suitable virla vectors include herpes simplex viral vectors and retroviruses. including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. Gene transfer techniques using these viruses are known to those skilled in the art Retrovirus vectors for example may be used to stably integrate the poiynuclcotide giving rise to the poly-nucleotide into the host genome. Rephcation-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

The invention also includes cells that have been modified to express the HIPHUM

113 polypeptide or a variant thereof. Such cells include transient, or preferably stable higher 113 poly eukaryotic cell lines, such as mammalian cells or msect cells. usmg for example a baculovirus expression system. lower eukaryotic cells, such as yeast or prokaryotic cells such as bacterial cells. Particular examples of cells which may be modified by insertion of vectors encoding for a polypeptide according to the invention include mammalian HEK293T. CHO. Bela and COS cells. Preferably the cell line selected will be one which is not only stable. but also allows for mature glycosylation of a polypeptide. Expression may be achieved in transformed oocytes A polypeptide of the invention may be expressed m cells of a transgenic non-human animal, preferably a mouse. A transgemc non-human animal expressing a polypeptide of the invention is included withm the scope of the

invention. A polypeptide of the invention may also be expressed in A < ?/ : o'm'/gw oocytes.

A polypeptide of the invention may be overexpressed 1n bactenal cells. such as E.Coli. and isolated from the bacterial culture.

According to another aspect, the present invention also relates to antibodies, specific for a polypeptide of the invention. Such antibodies are for example useful in purification. isolation or screening methods involving immunoprecipitation techniques or, indeed. as therapeutic agents in their own right.

Antibodies may be raised against specific epitopes of the polypeptides according to p ing to the invention. Such antibodies may be used to block substrate binding to the receptor. An antibody, or other compound,"specifically binds"to a protein when it binds with preferential

or high affinity to the protein for which it is specific but does substantially bind not bind or binds with only low affinity to other proteins. A variety of protocols for competitive binding or immunoradiometnc assays to determine the specific binding capability of an antibody are well knows m the art (see for example Maddox et al, J. Exp. Med 158. 1211-1226, 1993).

Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.

Antibodies of the invention may be antibodies to human polypeptides or fragments thereof. For the purposes of this invention, the term"antibody", unless specified to the contrary, includes fragments which bind a polypeptide of the invention. Such fragments include Fv, F (ab') and F (ab') 2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.

Antibodies may be used in a method for detecting polypeptides of the invention in a biological sample, which method comprises: I providing an antibody of the invention; II incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and III determining whether antibody-antigen complex comprising said antibody is formed.

A sample may be for example a tissue extract, blood, serum and saliva. Antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions, etc. Antibodies may be linked to a revealing label and thus may be suitable for use in methods of in vivo HIPHUM 113 imaging.

Antibodies of the invention can be produced by any suitable method. Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (1988)"Antibodies : A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising antibody in a host animal against the whole polypeptide or a fragment thereof, for example an antigenic epitope thereof, herein after the"immunogen".

A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the animal's serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified.

A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256,495-497).

An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by i ivf immunisation of human lymphocytes, followed by transformation of the tymphocytes with Epstem-Barr virus.

For the production of both monoclonal and polyclonal antibodies, the experimental

animal is suitably a goat, rabbit, rat or mouse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the ammo acid residues. to a suitable carrier. The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and. If desired, purified.

An important aspect of the present invention is the use of polypeptides according to the invention in screening methods. The screening methods may be used to identify substances that bind to cysteine protease and in particular which bind to HIPHUM 113 such as a substrate for the enzyme. Screening methods may also be used to identify agonists or antagonists which may modulate cysteine protease activry, inhibitors or activators of HIPHUM 113 activity, and/or agents which up-regualte or down-regualte HIPHUM 113 expression.

Any suitable format may be used for the assa\. In general terms such screening methods may involve contacting a polypeptide of the invention with a test substance and monitoring for binding of the test substance to the polypeptide or measuring protease

activity. A polypeptide of the invention may be incubated with a test substance. Modulation of cysteine protease activity may be determined. In a preferred aspect, the assay is a cellbased assay. Preferably the assay may be carried out in a single well of a microtitre plate.

Assay formats which allow high throughput screening are preferred.

A typical assay for determining whether a test substance acts as an inhibitor or A typical assa\, for cictei-mininly activator of HIPHUM 113 activity comprises contacting a fluorescent or colounmetnc substrate with a polypeptide of the invention and a test substance and monitoring protease activity by monitoring any change in the fluorescence or light emission. Any changes in the fluorescence of a substrate as a result of its proteolytic degradation by a polypeptide of the invention may be detected using a fluorescence plate reader. Colounmetic changes may be measured using a spectrophotometer. The inhibitory or stimulatory activity of a test substance may be determined by comparing any fluorescent or colounmetnc changes observed in the presence of a test substance to any changes observed in the absence of a test substance and or in the presence of a known inhibitor of HIPHUM 113 activity.

Modulator activity can be determined by contacting cells expressing a polypeptide of the invention with a substance under investigation and by monitoring an effect mediated by the polypeptide. The cells expressing the polypeptide may be in vitro or in vivo. The polypeptide of the invention may be naturally or recombinantly expressed. Preferably, the assay is carried out in vitro using cells expressing recombinant polypeptide. Preferably, control experiments are carried out on cells which do not express the polypeptide of the invention to establish whether the observed responses are the result of activation of the polypeptide.

The binding of a test substance to a polypeptide of the invention can be determined directly. For example, a radiolabelled test substance can be incubated with the polypeptide of the invention and binding of the test substance to the polypeptide can be monitored.

Typically, the radiolabelled test substance can be incubated with cell membranes containing the polypeptide until equilibrium is reached. The membranes can then be separated from a non-bound test substance and dissolved in scintillation fluid to allow the radioactive content to be determined by scintillation counting. Non-specific binding of the test substance may also be determined by carrying out a competitive binding assay.

Substances that inhibit the interaction of a polypeptide of the invention with a HIPHUM 113 substrate or with another protease may also be identified through a yeast 2hybrid assay or other protein interaction assay such as a co-immunoprecipitation or an ELISA based technique.

Assays may be carried out using cells expressing HIPHUM 113, and incubating such cells with the test substance optionally in the presence of a HIPHUM 113 substrate. The results of the assay are compared to the results obtained using the same assay in the absence of the test substance. Cells expressing HIPHUM 113 constitutively may be provided for use in assays for HIPHUM 113 function. Additional test substances may be introduced in any assay to look for inhibitors or activators of substrate binding or inhibitors or activators of protease activity.

Assays may also be carried out to identify substances which modify HIPHUM 113 expression, for example substances which up-or down-regulate expression. Such assays may be earned out for example by using antibodies for HIPHUM 113 to monitor levels of HIPHUM 113 expression. Other assays which can be used to monitor the effect of a test substance on HIPHUM 113 expression include using a reporter gene construct driven by the HIPHUM 113 regulatory sequences as the promoter sequence and monitoring for expression of the reporter polypeptide.

Additional control experiments may be carried out.

Suitable test substances which can be tested in the above assays include

combinatorial libraries, defined chemical entities and compounds, peptide and peptide mimetics. oligonucleotides and natural product libraries. such as display (e. g. phage display libraries) and antibody products.

Typically, organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 daltons. Candidate products can be biomolecules including, saccharides, fatty acids, steroids, purines,

''d*nes, der'-ati,,, es, structura I I I pyrimidines, derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

Test substances may be used in an initial screen of. for example, ] 0 substances per reaction, and the substances of these batches which show inhibition or activation tested individually. Test substances may be used at a concentration of from InM to I 0mM. preferably from. 10nM to 10 M or from 1 M to 100 M, more preferably from lu. M to I Ou. M. Preferably, the activity of a test substance is compared to the activity shown by a known activator or inhibitor. A test substance which acts as an inhibitor may produce a 50% inhibition of activity of the receptor Alternatively a test substance which acts as an activator may produce 50% of the maximal acti\ity produced using a known activator.

Another aspect of the present invention is the use ofpolynucleotides encoding the HIPHUM 113 polypeptides of the invention to identify mutations m IIPHL'M ! 13 genes which may be implicated m human disorders. Identification of such mutations may be used to assist m diagnosis or susceptibility to such disorders and lI1 assessing the physiology of such disorders. Polynucleotides may also be used m hybridisation studies to monitor for upor down-regualtion of HIPHUM 113 expression. Polynucleotides such as SEQ ID NO: I or SEQ ID N0 : 3 or fragments thereof may be used to identify a11ehc \'anants. genomic DNA and species variants.

The present invention provides a method for detecting variation in the expressed products encoded by HIPHUM] 13 genes. This may comprise determining the level of an HIPHUM 113 expressed in cells or determining specific alterations in the expressed product.

Sequences of interest for diagnostic purposes include, but are not limited to. the conserved portions as identified by sequence similarity and conservation ofmtron'exon structure. The

diagnosis may be performed in conjunction with kindred studies to determine whether a dia-h mutation of interest co-segregates with disease phenotype in a family.

Diagnostic procedures may be performed on polynucleotides isolated from an individual or alternatively, may be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Appropriate procedures are described in, for example, Nuovo, GJ., 1992, "PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY). Such analysis techniques include, DNA or RNA blotting analyses, single stranded conformational polymorphism analyses, in situ hybridization assays, and polymerase chain reaction analyses. Such analyses may reveal both quantitative aspects of the expression pattern of a HIPHUM 113 and qualitative aspects of HIPHUM 113 expression and/or composition.

Alternative diagnostic methods for the detection of HIPHUM 113 nucleic acid molecules may involve their amplification, e. g. by PCR (the expenmental embodiment set forth in U. S. Patent No. 4,683, 202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad.

Sci. USA 88: 189-193), self sustained sequence replication (Guatelh et al., 1990, Proc. Natl.

Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. 15 USA 86: 1173-1177), Q-Beta Replicas (Lizardi et al., 1988, Bio/Technology 6: 1197) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art.

These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

Particularly suitable diagnostic methods are chip-based DNA technologies such as those described by Hacia et al., 1996, Nature Genetics 14: 441-447 and Shoemaker et al., 1996, Nature Genetics 14: 450-456. Briefly, these techniques involve quantitative methods for analyzing large numbers of nucleic acid sequence targets rapidly and accurately. By tagging with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization.

Following detection, the results seen in a given patient may be compared with a statistically significant reference group of normal patients and patients that have HIPHUM 113 related pathologies. In this way, it is possible to correlate the amount or kind of HIPHUM 113 encoded product detected with various clinical states or predisposition to clinical states.

Another aspect of the present invention is the use of the substances that have been identified by screening techniques referred to above in the treatment of disease states, which are responsive to regulation of cysteine protease activity. The treatment may be therapeutic

or prophylactic. The condition of a patient suffering from such a disease state can thus be Improved In particular. such substances may be used in the treatment of male and female reproductive diseases, oncology diseases such as breast or lung cancer : bram diseases such as alzheimers or PSP : inflammatory diseases such as multiple sclerosis, lupus, ssonasis, psonatic arthritis, exzema. asthma and rheumatoid arthnsis or intlammatory musculoskeltal diseases such as osteoporosis. osteopetrosis and pendontal disease. Preferably, the disorder to be treated is an inflammatory disease.

Substances that act as inhibitors ofHIPHUM 113 activity may be used in the treatment of disease states in which HIPI UM 1 ! 3 expression is up-regulated such as oncology diseases such as lung cancer, braind diseases such as alzheimers and PSP. inflammatory autoimmune diseases such as multiple sclerosis, lupus, psoriasis and psonatic arthritis, exzema and rheumatoid arthritis. Substances that act as activators ofHIPHUM 113 activity may be used in the treatment of disease states in which expression of HIPHUM 113 is down-regulated such as oncology diseases such as breast cancer, and musculoskeletal diseases such as osteoporosis. osteopetrosis. and pendontal disease.

Substances identified according to the screening methods outlined above may be formulated with standard pharmaceutically acceptable carriers and or excipients as is routine in the pharmaceutical art. For example, a suitable substance may be dissolved in physiological saline or water for injections. The exact nature of a formulation ill depend upon several factors including the particular substance to be administered and the desired route of administration. Suitable types of formulation are full\ described m Remington's Pharmaceutical Sciences. Mack Publishing Company, Eastern Pennsylvanta, 17th Ed. 1985, the disclosure of which is included herein of its entirety by way of reference.

The substances may be administered by enteral or parenteral routes such as via oral. buccal, anal. pulmonary. intravenous, mtra-artenal. intramuscular. intrapentoneal, topical or other appropriate administration routes.

A therapeutically effective amount of a modulator is administered to a patient. The dose of a modulator may be determined according to various parameters, especially according to the substance used: the age, weight and condition of the patient to be treated : the route of administration : and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0. 1 to 50 mg per kg of body weight, according to the activity of the

specific modulator, the age. weight and conditions of the subject to be treated, the type and if Z--, pe and severity of the degeneration and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.

Nucleic acid encoding HIPHUM 113 or a variant thereof which inhibits HIPHUM 113 activity may be administered to the mammal. In particular, a nucleic acid encoding a polypeptide with HIPHUM 113 activity may be administered to a subject suffering from a condition in which HIPHUM 113 expression is down-regulated, such as oncology diseases such as breast cancer, and musculoskeletal diseases such as osteoporosis, osteopetrosis, and peridontal disease. A nucleic acid encoding a variant of HIPHUM 113 that inhibits HIPHUM 113 activity may be administered to a patient suffering from a condition in which HIPHUM 113 expression is up-regulated such as oncology diseases such as lung cancer, braind diseases such as alzheimers and PSP, inflammatory autoimmune diseases such as multiple sclerosis, lupus, psoriasis and psonatic arthritis, exzema and rheumatoid arthritis. Nucleic acid, such as RNA or DNA, and preferably, DNA, is provided in the form of a vector, such as the polynucleotides described above, which may be expressed in the cells of the mammal.

Nucleic acid encoding the polypeptide may be administered by any available technique. For example, the nucleic acid may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particlemediated gene delivery. The nucleic acid may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration.

Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the nucleic acid to be

administered can be altered. Typically the nucleic acid is administered in the range of 1 pg to 1 mg, preferably to 1 pg to 1 OJ... lg nucleic acid for particle mediated gene delivery and IOVG to Img for other routes.

The following Examples illustrate the invention.

Example 1: Characterisation of the sequence A cysteine protease, designated as HIPHUM 113 has been identified. The nucleotide and ammo acid sequences of the receptor and a particular splice varian have been determined. These are set out below in SEQ ID NOs: 1, 2 3 and 4. The closest known homologue to HIPHUM 113 is SUMO-1-specific protease (human): EMBL=AF196304 Protein Acc. No. =Q9UJV5 Suitable primers and probes were designed and used to analyse tissue expression.

HIPIHUM 113 is expressed in all tissues at various levels. It is highly expressed cerebellum, lung, thymus, tonsil, and fetal brain. It is also expressed at significant

levels in adrenal, myometrium, omenturn, ovary, and testis Therefore, modulation of activity of HIPP[HUM 113 may be useful in the treatment of male and female reproductive diseases.

HIPHUM I 13 expression evels are ele\ atcd in lung tumor, brain diseases (Alzheimer's. Parasupranuclear palsey), T ceHs. penpheral blood mononuclear cells (PBMCs), dendritic cells, and RA s\T) o\'ium. Therefore, modulation of activity of HIPHUM 113 may be useful in the treatment of oncology diseases (lung. solid tumors), brain diseases Alzheimer's, PSP), inflammatory autoimmune diseases (Multiple Sclerosis, Lupus, Psoriasis and psoriatic arthritis, exzema. and rheumatoid arthritis).

HIPHUM 113 expression levels are down regualted in breasttumorand differentiated

osteoblasts. Therefore. modulation of activity of HIPHUM !] 3 mavbe usefu] in the treatment of oncology diseases (breast) and musculoskeletal diseases (osteoporosis, osteopetrosis, and peridontal disease).

The Chromosomal localisation was also mapped. H [PHUM 113 mapped to 3ql2q 13 in humans. This region shows chromosomal linkage at a putative susceptibility locus for RA. The adjoining chromosome of3q21 mapped clone to a region associated with psoriasis and joint complaints In the mouse this region mapped to chromosome 16, locus Cyprl, that is associated with production of cytokine IL-4.

Therefore, modultion of HIPHUM 113 could be used for the treatment of inflammatory diseases such as rheumatoid arthritis, psoriasis, and allergic diseases such as asthma..

Example 2: Screening for substances which exhibit protein modulating activity Preparations of a purified polypeptide of the invention are generated for screening purposes. 96 and 384 well plate, high throughput screens (HTS) are employed using fluorescence or colourimetnc indicator moelcules, including but not limited to metalloprotease substrates such as G1 1426 12A (DNP-Por-Cha-Gly-Cys(Me)-His-

Als-Lys (NMA)-NH) and TES substrate (Ac-Pro-Leu-G ! y-SCH [CH CH (CH3) 2] COLeu-Gly-OC2H-.) (Bachem). Secondary screening involves the same technology. Tertiary screens involve the study of modulators in rat, mouse and guinea-pig models of disease relevant to the target.

A brief screening assay protocol is as follows :A polypeptide of the invention is expressed in E. Coli, purified and refolded

by direct dilution in assay buffer (200mM NaCl, 50mM Tris, 5mM CaC12, ICuM ZnS04, 0. 01% Brij 35, pH 7.5). Test substances are provided in pools of 10 at 5mM for each test substance for high throughput screening or are serially diluted in dose response assays. The screening assay is run on an automated system incorporating an OCRA rail to move the plates, a Tecan liquid handler, a Multimek liquid handler and a Titertek liquid handler. (The assay may also be run on manually or in combination with any suitable liquid handling equipment.)

60 ul of serially diluted test substance is added to 96 well plates (4 control wells with no inhibitor), 60 Ill of a solution of a polypeptide of the invention (0. 5 u. M to 150 uM) is then added to each well and 50 mM EDTA (20 ul of 0. 5M EDTA) is added to 4 inhibited control wells. The plates are then incubated at room temperature for 20 minutes before initiating with substrate (60 u. I GI 142612A for a fluorescence assay or 10 l of DNTB (5,5'-dithio-bis (2-nitrobenzoic acid) at 5mM in 150mM Tris base) followed by 10 J. l TES substrate for a colourimetric assay). In a fluorescence assay, the plates are read in a Fluostar (SLT) fluorescence plate reader or equivalent at an excitation of 343 nm and an emission of 450 nm. In a colourimetric assay the plates are read continuously in a SLT spectrophotometer at 405 nm for 3 minutes.

Percentage inhibition is calculated for each concentration (unknown values = U) in the dose response based on the range determined using the value of the difference of the control (no test substance) (mean of 4 = Cl) and the EDTA treated wells (mean of 4 = C2) using the equation 100* (1- (U-C2)/ (CI-C2).

SEQ ID 1 GAGGGGTCCTGAGGTGACAGCGCCTGCAACTGAAATTTCAGCAGCGGGAGAAGATGGACAAG AGAAAGCTCGGGCGACGGCCATCTTCATCCGATGGAGTCTCGCTCTGTCGCCAGGCTGGAGT GCAGTGACGTGATCTCGGCTCACTGCGACCTCTGCCTCCTGGGTTCAAGCGATTCTCCTGCA TCAGCCTCCTGAGTAGCTGGGACTACAGAAATCATCACAGAAGGAAAAAGGAAAAAGTCATC TTCTGATTTATCGGAGATAAGAAAGATGTTAAATGCAAAACCAGAGGATGTCCATGTTCAAT

CACCACTGTCCAAATTCAGAAGCTCAGAACGCTGGACTCTCCCTTTGCAGTGGGAAAGAAGC CTAAGGAATAAAGTCATCTCTCTAGACCATAAAAATA/\AAAACATATCCGAGGGTGTCCTGT TACTTCCAAGTCATCACCAGAAAGGCAACTCAAAGTTATGTTGACGAATGTCCTATGGACGG ATTTAGGACGAAAATTCAGAAAGACCCTACCTAGAAACGATGCTAATTTATGTGATGCCAAC

AAGGTGCAATCAGACTCATTGCCTTCGACATCTGTTGACAGCCTAGAGACATGTCAAAAATT AGAACCTCTTCGCCAAAGCCTTAATTTATCTGAAAGGATACCCAGAGTTATATTGACGAATG TCCTGGGAACGGAGTTAGGAAGAAAATACATAAGGACCCCACCTGTAACTGAGGGAAGTTTG AGTGATACAGACAACTTGCAATCAGAGCAACTTTCTTCATCATCTGATGGCAGCCTAGAATC TTATCAAAATCTAAACCCTCACAAGAGCTGTTATTTArCTGAAAGGGGCTCACAACGAAGTA AGACAGTAGATGACAATTCTGCAAAGCAGACTGCGCACAATAAAGAAAAACGAAGAAAGGAT

GATGGCATTTCTCTTTTAATATCTGATACTCAGCCTG1GACCTTACAGTGGAAGTAGAGG TTGTGATCATCTCGAACAGGAAAGCAGAAACAAGGATGTTAAAIATTCTGATTCAAAAGTGG AACTCACTCTGATTTCCAGGAAGACAAAGAGAAGGCTTAGAAATAATTTACCTGATTCTCAA TATTGTACTTCTTTGGATAAGTCAACAGAACAGACAAJ'AAAACAAGAAGATGACTCAACAAT ATCCACTGAGTTTGAAAAGCCAAGTGAAAACTATCATCAGGATCCAAAACTGCCTGAAGAAA TTACAACTAAACCTACAAAAAGTGATTTTACTAAACTATCCTCACTTAACAGTCAGGAGTTG ACTTTGAGTAATGCCACCAAAAGTGCCTCTGCCGGTTCAACCACTGAAACTGTTGAGAACTC TAATTCCATTGATATTGTGGGGATTTCTTCCCTGGTTGAGAAGGATGAGAATGAGTTGAATA CCATAGAAAAGCCTATTCTAAGAGGACATAATGAAGGGAACCAATCACTGATCTCAGCTGAA CCAATTGTTGTTTCCAGTGATGAAGAAGGACCTGTTGAACATAAAAGTTCAGAAATTCTTAA GTTACAATCTAAGCAAGACCGTGAGACAACTAATGAAAATGAGAGTACTTCTGAATCAGCAT TGTTAGAACTACCATTGATTACATGTGAATCTGTACAGATGTCATCTGAGTTATGCCCATAT AATCCTGTCATGGAGAACATTTCCAGTATTATGCCTAGTAATGAGATGGATCTACAACTGGA TTTTATATTTACTTCTGTTTATATTGGTAAAATAAAAGGAGCTTCTAAAGGTTGTGTTACAA TCACAAAAAAATATATTAAGATCCCATTTCAAGTGTCCCTGAATGAGATTTCATTGCTAGTG GATACCACACATTTAAAGCGGTTTGGGTTATGGAAAAGTAAGGATGATAATCACAGTAAAAG GAGTCATGCTATTCTTTTCTTCTGGGTCTCTTCAGATTATCTTCAAGAGATTCAGACCCAAT TAGAACACTCTGTATTAAGCCAGCAATCAAAATCTAGTGAATTCATTTTCCTTGAACTACAC AATCCTGTTTCACAAAGAGAAGAATTGAAGCTGAAAGATATTATGACGGAAATAAGTATAAT CAGTGGAGAATTAGAGCTTTCTTACCCGTTGTCTTGGGTTCAGGCATTTCCTTTGTTTCAGA ACCTCTCTTCAAAAGAAAGTTCTTTTATTCATTATTACTGTGTTTCAACTTGTTCTTTCCCT GCTGGTGTTGCTGTTGCTGAAGAAATGAAGCTGAAATCAGTATCTCAGCCCTCAAACACAGA TGCGGCCAAGCCTACTTACACCTTCCTGCAGAAGCAAT'GTAGCGGTTGCTACTCCCTTTCTA TTACATCTAATCCAGATGAAGAATGGCGAGAAGTCAGGCACACTGGACTTGTTCAGAAGTTG

ATTGTATATCCTCCACCACCTACTAAGGGGGGATTAGGAGTAACTAATGAAGATCTGGAGTG TTTAGAAGAAGGAGAGTTTCTTAATGATGTAATCATTGATTTTTACCTTAAGTATCTTATAT TGGAGAAGGCATCAGATGAACTTGTTGAACGAAGTCACATTTTTAGTAGCTTTTTCTATAAA TGCTTGACAAGAAAGGAAAATAATTTAACAGAAGATAA. TCCAA. z\TCTTTCAATGGCACAGAG AAGACATAAAAGAGTAAGAACATGGACTCGTCACATAAACATTTTTAATAAAGATTACATCT TTGTACCTGTAAATGAGTCGTCTCACTGGTATCTCGCAGTCATTTGTTTTCCATGGTTAGAA GAAGCTGTGTATGAAGATTTTCCACAAACTGTATCCCAGCAGTCCCAGGCTCAGCAGTCCCA AAATGACAACAAAACAATAGATAATGATCTACGTACTACTTCGACACTGTCTTTGAGTGCAG AGGATTCCCAAAGTACCGAGTCGAATATGTCAGTACCAAAGAAAATGTGTAAAAGGCCATGT ATTCTTATACTAGACTCCTTGAAAGCTGCTTCTGTACPAAACACAGTTCAGAATTTACGAGA GTATTTAGAGGTAGAGTGGGAAGTTAAACTAAAAACTCATCGTCAATTCAGCAAAACAAACA TGGTGGATCTATGCCCTAAAGTTCCTAAACAGGACAATAGCAG7GATTGTGGAGTATATTTA TTGCAGTATGTGGAAAGCTTCTTCAAGGATCCTATTGTTAACTTTGAACTTCCAATTCATTT GGAGAAGTGGTTTCCTCGTCATGTAATAAAGACCAAACGGGAAGATATTCGAGAGCTCATCT

TGAAACTTCATTTACAGCAACAGAAGGGCAGCAGTAGCTAGTTAATCTGTACAAACATGACA CAGATGTTCTCTAAGATTACTGGAAAGCCTCTTACCAGCATTTGTGTTAGCCAGCTCACAGA GAAGAAAATAACTTGCAGTAGTTTTATAATAAGTCATTGGAACATTATTTAAAATATGTAGG ACACATTATTAGAATTGTTGGGATCTCATAGATGGAATGGGAATGGGGGTGATATAGATAAA CTTACTAGATATAAATTAAAATTTTATAAATATTTCATATTTTTCTGAGTAAATATGATTGG ATTATGCAACAGCATATGTAATATGGGAATGTTTTGTAGATAATAAAACTTACATGATCTGT ACTTCCACGTGACTGGGTGCTGAGGGGAGTTAAAGCCTCCCTGGTGCCAGCCCCAGTGCTTG TCAAATTTGCTGACAGGTCACATCATATTGTAATTCTATTCTTTGCAGCTCAAGCATGCAGT ATGAATACTGTGTATTTTTTAAAAAAATAATTTAGTATCAAGGCTTCAGAAAATGCCATTTA CGGCATCCCTTCTGTATGTAACAAAAAGACATTCATAATGTTAGGAAGATGATAAAAATTCG CTCTTTTAAAGTGCAGCTTATTATTCTCAATTGCTAAATACGATTACTCTGCTTTATTTTTT TTTCATTTCTTTTGATGTCATATGTGAGTATCTTATAATTTAGTTCATTTGTTCAGGGTAAA ATTTGAAACAAAAAATTTTACCTGTGCAAAATAGTTTTTTAAAAATTATACATGTAGCTCAA CTTGAGGTACTGCTATATAAATATTCACTCACATTATCACGGAATTTATGTATAGTTTCTCT AATATAGAAGATAAAATTGGTGTCCTCATAACTTTAACAAAGAAAACCCTCAGTCTTATTTA TTAATGGGTAGAATTAAATATATAATTTTATAGCTCAGTTTACCCAGTATTCATCTGCAAAG CCAGATTGCTCTCATTGCTTTTATATTTTTAAATTGTAGCTTTTAGAGACCTATGATCCTCA TGGAACTTAATTTTTTATTAAATATTCAGGTAACAGTTCTGAATTCATGTGATAATGGTGGC ATTATATATGATTAAACACTTCAGAACTTTCTAATGTTATCAGGAGTATTTTGAGGGAGATA TGATTATATTGTATTTTCTCAGATAAGAAAAATGTTTTTTAACAATATTATTTTAATCTGTT TTAAGCATCTCTTAGATTTACATTATAACTACATAAAGCAGTGAAGCAAAGGCAAATTAAGA TAAAGCTAGAAAGTCTGAACATTTTATTTCAAAATCATATGAATCGGGGTCAGTTAAGCCTC AGTATTCTTAGCTTTTGTTGATTTTGGCACTATCTTTATATTATTAAATATATTTGTTGTTT GGATATTTCATATAAAGATGGCTATAATTACATATTTCATTCCCAATTTGTGTGTGTTGGGG GGTACTTTTAAAGGTGACTATTGTTTTGTACATCTAATTTTGGGAAAGCAAGTCTATAAGAC ATCTTGTGATTTCTTAATGTTTTTGTTTGTATGTTTTTCAAAGATATCACTGTCCTTTATCA TGTTTTGAAGATTGTTTAAAATTCATTTTCCTAAATTAATGTGCAAGTAATGTTTTGAGGAT ATCAGTGTTTTATATTAAACATATTTCCAATTCATTAAATTGAGGTGTAATTTTTTTCCTTA AGAAACATGAGCTACTGTTAGAAATAAATTTCCAGTTGTATGCTGAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID 2 MLNAKPEDVHVQSPLSKFRSSERWTLPLQWERSLRNKVISLDHKNKKHR GCPVTSKSSPERQLKVMLTN'LWTDLGRKFRKTLPRNDANLCDAKVQSD SLPSTSVDSLETCQKLEPLRQSLNLSERIPRVILTNVLGTELGRKYIRTP PVTEGSLSDTDNLQSEQLSSSSDGSLESYQNLNPHKSCYLSERGSQRSKT VDDNSAKQTAHNKEKRRKDDGISLLISDTQPEDLNSGSRGCDHLEQESRN KDVKYSDSKVELTLISRKTKRRLRNNLPDSQYCTSLDKSTEQTKKQEDDS TISTEFEKPSENYHQDPKLPEEITTKPTKSDFTKLSSLNSQELTLSNATK SASAGSTTETVENSNSIDIVGISSLVEKDENELNTIEKPILRGHKE8NQS LISAEPIVVSSDEEGPVEHKSSEILKLQSKQDRETTNENESTSESALLEL PLITCESVQMSSELCPYNPVMENISSIMPSNEMDLQLDFIFTSVYI8KIK GASKGCVTITKKYIKIPFQVSLNEISLLVDTTHLKRFGLWKSKDDNHSKR SHAILFFWSSDYLQEIQTQLEHSVLSOQSKSSEFIFLELHNPVSQREEL KLKDIMTEISIISGELELSYFLSWVQAFPLFQNLSSKESSFIHYYCVSTC SFPAGVAVAEEMKLKSVSQPSNTDAAKPTYTFLQKQSSGCYSLSITSNPD EEWREVRHTGLVQKL : VYPPPPTKGGLGVTNEDLECLEEGEFLNDVIZDF YLKYLILEKASDELVERSH ; FSSFFYKCLTRKENNLTEDNPNLSr4AQRRH KRVRTWTRHINIFNKDYIFVPVNESSHWYLAVICFPWLEEAVYEDFPQTV SQQSQAQQSQNDNKT : DNDLRTTSTLSLSAEDSQSTESNMSVPKKMCKRP CILILDSLKAASVQNTVQNLREYLEVEWEVKLKTHRQFSKTNMVDLCPIO PKQDNSSDCGVYLLQYVESFFKDPIVNFELPIHLEKWFPRHVIKTKREDI RELILKLHLQQQKGSSS*

SEQ ID NO : 3 GAGGGGTCCTGAGGTGACAGCGCCTGCAACTGAAATTTCAGCAGCGGGAGAAGATGGACAAG AGAAAGCTCGGGCGACGGCCATCTTCATCCGAAATCATCACAGAAGGAAAAAGGAAAAAGTC ATCTTCTGATTTATCGGAGATAAGAAAGATGTTAAATGCAAAACCAGAGGATGTCCATGTTC AATCACCACTGTCCAAATTCAGAAGCTCAGAACGCTGGACTCTCCCTTTGCAGTGGGAAAGA AGCCTAAGGAATAAAGTCATCTCTCTAGACCATAAAAATAAAAAACATATCCGAGGGTGTCC TGTTACTTCCAAGTCATCACCAGAAAGGCAACTCAAAGTTATGTTGACGAATGTCCTATGGA CGGATTTAGGACGAAAATTCAGAAAGACCCTACCTAGAAACGATGCTAATTTATGTGATGCC AACAAGGTGCAATCAGACTCATTGCCTTCGACATCTGTTGACAGCCTAGAGACATGTCAAAA ATTAGAACCTCTTCGCCAAAGCCTTAATTTATCTGAAAGGATACCCAGAGTTATATTGACGA ATGTCCTGGGAACGGAGTTAGGAAGAAAATACATAAGGACCCCACCTGTAACTGAGGGAAGT TTGAGTGATACAGACAACTTGCAATCAGAGCAACTTTCTTCATCATCTGATGGCAGCCTAGA ATCTTATCAAAATCTAAACCCTCACAAGAGCTGTTATTTATCTGAAAGGGGCTCACAACGAA GTAAGACAGTAGATGACAATTCTGCAAAGCAGACTGCGCACAATAAAGAAAAACGAAGAAAG GATGATGGCATTTCTCTTTTAATATCTGATACTCAGCCTGAAGACCTTAACAGTGGAAGTAG AGGTTGTGATCATCTCGAACAGGAAAGCAGAAACAAGGATGTTAAATATTCTGATTCAAAAG TGGAACTCACTCTGATTTCCAGGAAGACAAAGAGAAGGCTTAGAAATAATTTACCTGATTCT CAATATTGTACTTCTTTGGATAAGTCAACAGAACAGACAAAAAAACAAGAAGATGACTCAAC AATATCCACTGAGTTTGAAAAGCCAAGTGAAAACTATCATCAGGATCCAAAACTGCCTGAAG AAATTACAACTAAACCTACAAAAAGTGATTTTACTAAACTATCCTCACTTAACAGTCAGGAG TTGACTTTGAGTAATGCCACCAAAAGTGCCTCTGCCGGTTCAACCACTGAAACTGTTGAGAA CTCTAATTCCATTGATATTGTGGGGATTTCTTCCCTGGTTGAGAAGGATGAGAATGAGTTGA

ATACCATAGAAAAGCCTATTCTAAGAGGACATAATGAAGGGAACCAATCACTGATCTCAGCT GAACCAATTGTTGTTTCCAGTGATGAAGAAGGACCTGTTGAACATAAAAGTTCAGAAATTCT TAAGTTACAATCTAAGCAAGACCGTGAGACAACTAATGAAAATGAGAGTACTTCTGAATCAG CATTGTTAGAACTACCATTGATTACATGTGAATCTGTACAGATGTCATCTGAGTTATGCCCA TATAATCCTGTCATGGAGAACATTTCCAGTATTATGCCTAGTAATGAGATGGATCTACAACT GGATTTTATATTTACTTCTGTTTATATTGGTAAAATAAAAGGAGCTTCTAAAGGTTGTGTTA CAATCACAAAAAAATATATTAAGATCCCATTTCAAGTGTCCCTGAATGAGATTTCATTGCTA GTGGATACCACACATTTAAAGCGGTTTGGGTTATGGAAAAGTAAGGATGATAATCACAGTAA AAGGAGTCATGCTATTCTTTTCTTCTGGGTCTCTTCAGATTATCTTCAAGAGATTCAGACCC AATTAGAACACTCTGTATTAAGCCAGCAATCAAAATCTAGTGAATTCATTTTCCTTGAACTA CACAATCCTGTTTCACAAAGAGAAGAATTGAAGCTGAAAGATATTATGACGGAAATAAGTAT AATCAGTGGAGAATTAGAGCTTTCTTACCCGTTGTCTTGGGTTCAGGCATTTCCTTTGTTTC AGAACCTCTCTTCAAAAGAAAGTTCTTTTATTCATTATTACTGTGTTTCAACTTGTTCTTTC CCTGCTGGTGTTGCTGTTGCTGAAGAAATGAAGCTGAAATCAGTATCTCAGCCCTCAAACAC AGATGCGGCCAAGCCTACTTACACCTTCCTGCAGAAGCAAAGTAGCGGTTGCTACTCCCTTT CTATTACATCTAATCCAGATGAAGAATGGCGAGAAGTCAGGCACACTGGACTTGTTCAGAAG TTGATTGTATATCCTCCACCACCTACTAAGGGGGGATTAGGAGTAACTAATGAAGATCTGGA GTGTTTAGAAGAAGGAGAGTTTCTTAATGATGTAATCATTGATTTTTACCTTAAGTATCTTA TATTGGAGAAGGCATCAGATGAACTTGTTGAACGAAGTCACATTTTTAGTAGCTTTTTCTAT AAATGCTTGACAAGAAAGGAAAATAATTTAACAGAAGATAATCCAAATCTTTCAATGGCACA GAGAAGACATAAAAGAGTAAGAACATGGACTCGTCACATAAACATTTTTAATAAAGATTACA TCTTTGTACCTGTAAATGAGTCGTCTCACTGGTATCTCGCAGTCATTTGTTTTCCATGGTTA GAAGAAGCTGTGTATGAAGATTTTCCACAAACTGTATCCCAGCAGTCCCAGGCTCAGCAGTC CCAAAATGACAACAAAACAATAGATAATGATCTACGTACTACTTCGACACTGTCTTTGAGTG CAGAGGATTCCCAAAGTACCGAGTCGAATATGTCAGTACCAAAGAAAATGTGTAAAAGGCCA TGTATTCTTATACTAGACTCCTTGAAAGCTGCTTCTGTACAAAACACAGTTCAGAATTTACG AGAGTATTTAGAGGTAGAGTGGGAAGTTAAACTAAAAACTCATCGTCAATTCAGCAAAACAA ACATGGTGGATCTATGCCCTAAAGTTCCTAAACAGGACAATAGCAGTGATTGTGGAGTATAT TTATTGCAGTATGTGGAAAGCTTCTTCAAGGATCCTATTGTTAACTTTGAACTTCCAATTCA TTTGGAGAAGTGGTTTCCTCGTCATGTAATAAAGACCAAACGGGAAGATATTCGAGAGCTCA TCTTGAAACTTCATTTACAGCAACAGAAGGGCAGCAGTAGCTAGTTAATCTGTACAAACATG ACACAGATGTTCTCTAAGATTACTGGAAAGCCTCTTACCAGCATTTGTGTTAGCCAGCTCAC

AGAGAAGAAAATAACTTGCAGTAGTTTTATAATAAGTCATTGGAACATTATTTAAAATATGT AGGACACATTATTAGAATTGTTGGGATCTCATAGATGGAATGGGAATGGGGGTGATATAGAT AAACTTACTAGATATAAATTAAAATTTTATAAATATTTCATATTTTTCTGAGTAAATATGAT TGGATTATGCAACAGCATATGTAATATGGGAATGTTTTGTAGATAATAAAACTTACATGATC TGTACTTCCACGTGACTGGGTGCTGAGGGGAGTTAAAGCCTCCCTGGTGCCAGCCCCAGTGC TTGTCAAATTTGCTGACAGGTCACATCATATTGTAATTCTATTCTTTGCAGCTCAAGCATGC AGTATGAATACTGTGTATTTTTTAAAAAAATAATTTAGTATCAAGGCTTCAGAAAATGCCAT TTACGGCATCCCTTCTGTATGTAACAAAAAGACATTCATAATGTTAGGAAGATGATAAAAAT TCGCTCTTTTAAAGTGCAGCTTATTATTCTCAATTGCTAAATACGATTACTCTGCTTTATTT TTTTTTCATTTCTTTTGATGTCATATGTGAGTATCTTATAATTTAGTTCATTTGTTCAGGGT AAAATTTGAAACAAAAAATTTTACCTGTGCAAAATAGTTTTTTAAAAATTATACATGTAGCT

CAACTTGAGGTACTGCTATATAAATATTCACTCACAT'ATCACGGAATTTATGTATAGTTTC TCTAATATAGAAGATAAAATTGGTGTCCTCATAACTTTAACAAAGAAAACCCTCAGTCTTAT TTATTAATGGGTAG-kATTAAATATATAATTTTATAGC"CAGTTTACCCAGTATTCATCTGCA AAGCCAGATTGCTCTCATTGCTTTTATATTTTTA. ATTGTAGCTTTTAGAGACCTATGATCC TCATGGAACTTAATTTTTTATTAAATATTCAGGTAACAGTTCTGAATTCATGTGATAATGGT GGCATTATATATGATTAAACACTTCAGAACTTTCTAATGTTATCAGGAGTATTTTGAGGGAG ATATGATTATATTGTATTTTCTCAGATAAGAAAAATGTTTTTTAACAATATTATTTTAATCT GTTTTAAGCATCTCTTAGATTTACATTATAACTACATAAAGCAGTGAAGCAAAGGCAAATTA AGATAAAGCTAGAAAGTCTGAACATTTTATTTCAAAATCATATGAATCGGGGTCAGTTAAGC

CTCAGTATTCTTAGCTTTTGTTGATTTTGGCACTATCTTTATATTATTAAATATATTTGTTG 1'TTGGATATTTCATATAAAGATGGCTATAATTACATATTTCATTCCCAATTTGTGTGTGTTG GGGGGTACTTTTAAAGGTGACTATTGTTTTGTACATCTAATTTTGGGAAAGCAAGTCTATAA GACATCTTGTGATTTCTTAATGTTTTTGTTTGTATGTTTTTCAAAGATATCACTGTCCTTTA TCATGTTTTGAAGATTGTTTAAAATTCATTTTCCTAAATTAATGTGCAAGTAATGTTTTGAG GATATCAGTGTTTTATATTAAACATATTTCCAATTCATTAAATTGAGGTGTAATTTTTTTCC

TTAAGAAACATGAGCTACTGTTAGAAATAAATTTCCACTTGTATGCTGAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO : 4 MDKRKLGRRPSSSEIITEGKRKKSSSDLSEIRKMLNAKPEDVHVQSPLSK FRSSERWTLPLQWERSLRNKVISLDHKNKKHIRGCPVTSKSSPERQLKVM LTNVLWTDLGRKFRKTLPRNDANLCDANKVQSDSLPSTSVDSLETCQKLE PLRQSLNLSERIPRVILTNVLGTELGRKYIRTPPVTEGSLSDTDNLQSEQ LSSSSDGSLESYQNLNPHKSCYLSERGSQRSKTVDDNSAKQTAHNKEKRR KDDGISLLISDTQPEDLNSGSRGCDHLEQESRNKDVKYSDSKVELTLISR KTKRRLRNNLPDSQYCTSLDKSTEQTKKQEDDSTISTEFEKPSENYHQDP KLPEEITTKPTKSDFTKLSSLNSQELTLSNATKSASAGSTTETVENSNSI DIVGISSLVEKDENELNTIEKPILRGHNEGNQSLISAEPIWSSDEEGPV EHKSSEILKLQSKQDRETTNENESTSESALLELPLITCESVQMSSELCPY NPVMENISSIMPSNEMDLQLDFIFTSVYIGKIKGASKGCVTITKKYIKIP FQVSLNEISLLVDTTHLKRFGLWKSKDDNHSKRSHAILFFWVSSDYLQEI QTQLEHSVLSQQSKSSEFIFLELHNPVSQREELKLKDIMTEISIISGELE LSYPLSWVQAFPLFQNLSSKESSFIHYYCVSTCSFPAGVAVAEEMKLKSV SQPSNTDAAKPTYTFLQKQSSGCYSLSITSNPDEEWREVRHTGLVQKLIV YPPPPTKGGLGVTNEDLECLEEGEFLNDVIIDFYLKYLILEKASDELVER SHIFSSFFYKCLTRKENNLTEDNPNLSMAQRRHKRVRTWTRHINIFNKDY IFVPVNESSHWYLAVICFPWLEEAVYEDFPQTVSQQSQAQQSQNDNKTID NDLRTTSTLSLSAEDSQSTESNMSVPKKMCKRPCILILDSLKAASVQNTV QNLREYLEVEWEVKLKTHRQFSKTNMVDLCPKVPKQDNSSDCGVYLLQYV ESFFKDPIVNFELPIHLEKWFPRHVIKTKREDIRELILKLHLQQQKGSSS

Claims

CLAIMS I, An Isolated cysteme protease polypeptidc comprising (1) the amino acid sequence of SEQ ID NO : 2 or SFQ ID NO : 4 or (11) a variant thereof which is capable of cleaving SUMO from a target protein and or c1ea\ mg the precursor form of SUMO to release the active form of SUMO or (111) a fragment ot' (i) or (n) which is capable of cleaving SUMO from a target protein and. or cleaving the precursor form of SUMO to release the active form of SUMO.

2. A polypeptide according to claim I wherein the variant (n) has at least 95% identity to the ammo acid sequence of SEQ ID NO: 2 or SEQ ID NO:4.

3. A polynucleotide encoding a polypeptide according to claim 1 or 2.

4. A polynucleotide according to claim 3 which is a cDNA sequence.

5. A po ! ynudcotide encoding a cysteine protease polypeptide which is capable of cleaving SUMO from a target protein and/or cleaving the precursor form of SUMO to release the active form of SUMO which polynucleotide comprises : (a) the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO : 3 and/or a sequence complementary thereto; (b) a sequence which hybridises under stirngent conditions to a sequence as defined in (a) : (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b) : or (d) a sequence having at least 95"-'o identity to a sequence as defined in (a), (b) or (c).

6. An expression vector comprising a polynucleotide according to any one of

claims to claims 3 to 5.

A host cell comprising an expression vector according to ciaim 6.

8. An antibody specific for a polypeptide according to claim 1 or 2.

9. A method for the identification of a substance that modulates cysteine protease TENNIS activity and or expression, which method comprises : (i) contacting a test substance and a po] ypeptide according to claim I or 2. a polynucleotide according to any one of claims 3 to 5, an expression vector according to claim 6 or a host cell according to clam 7, and

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(ii) determining the effect of the test substance on the activity and/or expression of the said polypeptide or the polypeptide encoded by said polynucleotide, thereby to determine whether the test substance modulates cysteme protease activity and/or expression.

10. A method according to claim 9 wherein the polypeptide is in a substantially isolated form.

11. A substance which modulates cysteine protease activity and which is identifiable by a method according to claim 9 or 10.

12. A method of treating a subject having a disorder that is responsive to cysteine protease modulation, which method comprises administering to said subject an effective amount of a substance according to claim 11.

13. A method according to claim 12 wherein the disorder is selected from male and female reproductive diseases; oncology diseases such as breast or lung cancer; brain diseases such as alzheimers or PSP; inflammatory diseases such as multiple sclerosis, lupus, psoriasis, psoriatic arthritis, exzema, asthma and rheumatoid arthrisis or inflammatory musculoskeletal diseases such as osteoporosis, osteopetrosis and pendontal disease.

14. Use of a substance as defined in claim 11 in the manufacture of a medicament for treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of cysteine protease activity.

15. A use according to claim 14 wherein the disorder is selected from male and female reproductive diseases; oncology diseases such as breast or lung cancer; brain diseases such as alzheimers or PSP; inflammatory diseases such as multiple sclerosis, lupus, psoriasis, psoriatic arthritis, exzema, asthma and rheumatoid arthrisis or inflammatory musculoskeletal diseases such as osteoporosis, osteopetrosis and peridontal disease.

16. A method of producing a polypeptide according to claim 1 or 2, which method comprises maintaining a host cell as defined in claim 7 under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide.