WO2000078933A2 - Novel calpains and their use - Google Patents

Abstract

The invention relates to a novel mammalian calpain CAPN11, to the synthesis and to the use thereof.

Description

New calpaine and their use

The invention relates to a new mammalian calpain CAPN11, its synthesis and its use.

Calpains are a superfamily of related proteins, some of which have been shown to act as calcim dependent cysteine proteases. Eight different calpains have been identified in mammals.

Calpains form a family of intracellular calcium-dependent cysteine proteases. An increasing number of mammalian cal-pain homologs have been identified and the individual members can be divided into four classes based on the physical structure and the predicted properties. Class A, the "classic Calpaine" CAPNl, CAPN2, CAPN3 (p94); CAPN8 (nCL-2) and CAPN9 (nCL-4) are probably all protease-active and Ca ²⁺ dependent. They consist of a variable large (80 kDa) and an invariant small subunit (30 kDa). Class B and D calpains, CAPN5 (6.15) and CAPN7 (8) are protease-active, but most likely Ca ²⁺ -independent, the class C calpain, CAPN6, is unlikely to have any protease activity. The calpains can also be divided into categories based on their expression pattern, with CAPN3, CAPN6, CAPN8 and CAPN9 having some tissue specificity. The function of calpains is not known, although they have been associated with a large number of physiological processes and pathological conditions (overview in reference 17). To elucidate their function and evolutionary history, the identification of the entire spectrum of the Calpain family members is necessary.

The invention relates to the new polypeptide CAPN11 with the SEQ. - ID. -No. 2 disclosed amino acid sequence.

The new calpain protein CAPN11 has the characteristics typical of calpains, including potential protease and calcium binding domains. It has a very restricted tissue distribution and is mainly expressed in the testis. Radiation hybrid mapping was used to locate the gene on chromosome 6 in a region that is assigned to pl2. Phylogenetic analysis shows that CAPN11 is most closely related to CAPN1 and CAPN2 in mammals.

However, the predicted CAPNII sequence has the greatest homology to the chick calpain μ / m of the available calpain sequences. Thus CAPN11 can be the human orthologon of the μ / m calpain his. The discovery of this new calpain emphasizes the complexity of the calpain family, whose members can be distinguished on the basis of protease activity, calcium dependency and tissue expression.

The cDNA nucleotide sequence of the CAPNII gene contains 2338 nucleotides (SEQ. ID. No. 1). The cDNA sequence comes from a single mRNA by successfully amplifying the entire putative coding region from human testicular cDNA using flanking primers. Several cDNA clones were fully sequenced to rule out any PCR artifacts.

There is a large open reading frame encoding a 702 amino acid protein (Mr 80 kDa) (Fig. 1). The amino acid sequence resembles the large subunit of members of the calpain family (Fig. 1). The protein can be divided into the four domains typical of calpain. Domain II has the properties of a protease domain and the predicted amino acid sequence has the three amino acid residues (Cysl02, His259 and Asn283) which are part of the active center of cysteine proteases (2). The amino acid sequence of all five Ca ²⁺ -binding sequences (4, 12) described for CAPN2 are somewhat conserved (FIG. 1). This protein is likely to have protease and calcium binding properties. A comparison of the predicted amino acid sequence with that of all others

Calpaine gave the largest sequence homology (57.5%) for the chick cal pain μ / m. The mammalian calpaines were most similar to the human CAPNl (54.3% homology). The least similar human calpain with only 18.7% homology was CAPN6. The gene corresponding to this cDNA was designated CAPN11 by the Human Gene Nomenclature Committee.

The complete amino acid sequence of all identified human calpains was analyzed phylogenetically. The results enable the human calpains to be classified into four main evolution groups (FIG. 2). The first group is represented by CAPN5, CAPN6, CAPN7 and CAPN8, the second by CAPN1 and CAPN2, the third group by CAPN3 and CAPN9, and the fourth group comprises CAPN11. The phylogenetic analysis thus suggests that CAPNII is a separate calpain subfamily.

The expression of CAPN11 in human tissues was examined by Northern and RNA dot blot analysis. Of the 50 tissue RAs examined, the CAPNII mRNA was most strongly expressed in the testis (FIG. 3A). The specificity of this signal was confirmed by Northern blot analysis and corresponded to an approximately 3 kb mRNA (FIG. 3D). In the thymus and in the mammary gland much weaker signals detected. However, the significance of this finding is unclear, since a further examination of Thymus RNA with Northern blot analysis did not produce any signal despite long exposure times (FIG. 3D and data not shown). A possible explanation would be that this weak signal is due to cross hybridization with related mRNAs. Thus, the testis is the major expression site of CAPNII, although we cannot rule out the possibility that the gene may be expressed in other, unexamined tissues.

We have determined which chromosome the human CAPNII gene is located on. Using PCR with primers that are specific for the human CAPN11 nucleotide sequence, we assigned the gene to chromosome 6 (Correll Cell Repositories) using a somatic human / rodent cell hybrid mapping panel. Radiation hybrid mapping using the Stanford G3 medium resolution panel (Research Genetics Inc.) and the database at the Stanford Human Genome Center (shgc-www.stanford.edu) removed the Gen 5 Centiray from the marker SHGC-32834 this chromosome localized (LOD score 12.87). This marker is located in the space between the microsatellite markers D6S1616 (59.6 cM) and D6S427 (73.9 cM) (7), and a marker in this space, D6S269, has been cytogenetically assigned to 6pl2 (5). Thus CAPN11 is located on chromosome 6 near pl2. No other calpain gene has been located on this chromosome.

The chick μ / m calpain was the first member of the calpain family to be cloned (16). It was originally called m-calpain, but was reclassified after identifying other chick calpains that are most likely orthologous to the μ- and m-calpaines from mammals (18). A mammalian μ / m calpain has yet to be determined. However, since CAPN11 has a greater homology to the chick μ / m calpain than to other mammalian calpains, this may be its orthologue.

So far there are 5 calpains that have a certain degree of tissue specificity - CAPN3 (skeletal muscle), CAPN6 (placenta), CAPN8 (possibly smooth muscles), CAPN9 (stomach and small intestine) and CAPN11 (testes). Numerous proteases in the testis have been identified and are believed to be involved in processes such as tissue reorganization (20), regulation of spermatogenesis (14), sperm penetration of the zona pellucida (10) and fertility (13). However, many of these activities are dependent on secreted proteases and CAPN11 is likely to be located intracellularly. In the testicle, it could be involved in processes in which calpains are involved in other tissues, such as germ cell apoptosis (3) or regulation of testicular-specific

Transcription factors.

Another aspect of this invention relates to the use of the polypeptide CAPNII for the identification of substances which can inhibit the enzymatic activity of this polypeptide, so-called calpain inhibitors, in particular those calpain inhibitors which are selective for CAPNII. Selectivity means that such calpain inhibitors inhibit the activity of CAPNII more than the activity of the other calpains mentioned above, preferably at least 10 times, more preferably 25 times more. The enzyme activity of CAPNII is a Ca-dependent protease activity.

Another aspect of the invention relates to a method for identifying compounds which inhibit the enzyme activity of a polypeptide according to claim 1, comprising:

(a) comparing the level of enzymatic activity of CAPNII in the presence of the compound with the level of enzymatic activity of CAPNII in the absence of the compound and

(b) Selecting compounds that alter the level of CAPNII enzymatic activity versus CAPNII enzymatic activity in the absence of the compound.

The inhibitory compounds identified by the above-mentioned method are suitable for the treatment of diseases which are associated with or associated with an unphysiologically increased CAPNII activity, such as infertility in men.

The dosage and treatment regimen of these inhibitors must be determined by routine procedures known from other protease inhibitors.

LITERATURE

1. Altschul, SF, Gish, W., Miller, W., Myers, EW and Lipman, DJ (1990). Basic local alignment sequencing tool. J Mol. Biol. 215: 403-410. 2. Berti, P.J and Storer, AC (1995). Alignment / phylogeny of the papain superfamily of cysteine proteases. J. Mol. Biol. 246: 273-283. 3. Billig, H., Chun SY, Eisenhauer K. and Hsueh, AJ (1996). Gonadal cell apoptosis. hormone-regulated cell demise. Hu. Reprod. Update 2: 103-117.

4. Blanchard, H., Grochulski, P., Li, Y., Arthur, J.S.C., Da-

5 vies, PL, Elce, JS and Cygler, M. (1997). Structure of a Ca ²⁺ -binding domain reveals a novel EF-hand and Ca ²⁺ -induced conformational changes. Nature structural Biology 4: 532-538.

5. Bray-Ward, P., Menninger, J., Lieman, T., Desai, T., Mokady, N., Banks, A. and Ward, D.C. (1996). Integration of the cyto-

10 genetic, genetic and physical maps of the human genome by FISH mapping of CEPH YAC clones. Genomics 32: 1-14.

6. Dear, N., Matena, K., Vingron, M. and Boehm, T. (1997). A new subfamily of vertebrate calpains lacking a calmodulin-like domain: Implications for calpain regulation and evolution.

15 Genomics 45: 175-184.

7. Deloukas, P. et al. (1998). A physical map of 30,000 human genes. Science 282: 744-746.

8. Franz, T., Vingron, N., Boehm, T. and Dear, T.N. (1999). Capn7: A highly divergent vertebrate calpain with a novel

20 C-terminal domain. Mom. Genomes, in press.

9. Frohman, M.A., Dush, M.K. and Martin, G.R. (1988). Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Be. USA 85: 8998-9002.

25 10. Kohno, N., Yamagata, K., Yamada, S., Kasiwabara, S., Sakai, Y. and Baba, T. (1998). Two novel testicular serine proteases, TESPl and TESP2, are present in the mouse sperm. acro- some. Biochem. Biophys. Res. Commun. 245: 658-665.

11. Kozak, M. (1996). Interpreting cDNA sequences: some insights 30 from studies on translation. Mom. Genome 7: 563-574.

12. Lin, G.-D., Chattopadhyay, D., Maki, M., Wang, K.K.W. , Carson, M., Jin, L., Yuen, P.-W., Takano, E., Hatanaka, M., DeLucas, L.J. and Narayana, S.V.L. (1997). Crystal structure of calcium bound domain VI of calpain at 1.9 A resolution and

35 its role in enzyme assembly, regulation, and inhibitor binding. Nature Structural Biology 4: 539-547.

13. Mbikay, M., Tadros, H., Ishida, N., Lerner, C.P., De Lami- rande, E., Chen, A., El-Alfy, M., Clermont, Y., Seidah, N.G. , Chretien, N., Gagnon, C. and Simpson, E.M. (1997). Impaired

40 fertility in mice deficient for the testicular germ-cell protease PC4. Proc. Natl. Acad. Be. USA 94: 6842-6846.

14. Monsees, TK, Gornig, N., Schill, WB and Miska, W. (1998). Possible involvement of proteases in the regulation of spermatogenesis. Andrologia 30: 185-191. 15. Mugita, N., Ki ura, Y., Ogawa, M., Saya, H. and Nakao, M.

(1997), Identification of a novel, tissue-specific calpain htra-3; a human homologue of the Caenorhabditis elegans sex determination gene. Biochern. Biophys. Res. Comm. 5 239: 845-850.

16. Ohno, Ξ., Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M. and Suzuki, K. (1984). Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein? Nature 312: 566-570.

10 17. Ono, Y., Sorimachi, H. and Suzuki, K. (1998). Structure and physiology of calpain, an enigmatic protease. Biochern. Biophys. Res. Commun. 245: 289-294.

18. Sorimachi, H., Tsukahara, T., Okada-Ban, M., Sugita, H., Ishiura, S. and Suzuki, K. (1995). Identification of a third

15 ubiquitous calpain species - chicken muscle expresses four distinct calpains. Biochim. Biophys. Acta 1261: 381-393.

19. Thompson J.D. , Higgins D.G. and Gibson T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap 0 penalties and weight matrix choice. Nucl. Acids Res. 22: 4673-4680.

20. Tohonen, V., Osterlund, C. and Nordqvist, K. (1998). Testatin: A cystatin-related gene expressed during early testis development. Proc Natl. Acad. Be. USA 95: 14208-14213. 5

CHARACTERS

FIG. 1. Grouping the predicted amino acid sequence of CAPNII with other human calpains. The multiple grouping of the amino acid sequences was carried out using CLUSTAL W (19). The putative start methionine for CAPNII (GGAatgG) corresponds to the minimal consensus sequence for the translation start site (RNNatgG, where R is a purine, ref. 11). Amino acids that are identical to those of CAPNII 5 in the other proteins are shaded. Dashes indicate gaps that have been added to maximize alignment. Arrowheads indicate the three conserved amino acids that are part of the active center of the Calpaine. The potential EF hand calcium binding domains of CAPN8 are underlined and numbered consecutively. The arbitrary domains of Calpain are specified. The sequences for CAPN4 and CAPN7, which were only identified in rats and mice, are not shown. The published CAPN6 sequence was not included because it is only a partial sequence. The alternative names and accession numbers 5 for the grouped Calpaine are given in the legend of FIG. 2. FIG. 2. Rootless phylogenetic family tree of the family of the large subunit of human calpaine. The analysis was carried out with the PAUP program and the family tree with CLUSTREE from the HUSAR server of the German Cancer Research Center, Heidelberg (www.dkfz-heidelberg.de) was arranged. The lengths of the horizontal lines are proportional to the derived phylogenetic distances; the vertical lines have no meaning. 1000 bootstrapping retries were performed and the values are shown on the inner nodes. The CAPN7 sequence is from the mouse because there is little human nucleotide and protein sequence available. However, the human orthologue exists (see ref. 8), so the use of the mouse sequence for this comparison is justified. The partial human CAPN8 sequence is the predicted translation of the EST clone AA026030 (Hillier et al., 1995, The WashU-Merck EST project, unpublished results). An amino acid translation of this clone shows high similarity to the rat CAPN8 sequence. No bootstrapping was done with this sequence because it is much shorter than the others. The bootstrapping value can therefore not be meaningfully compared with the values from comparisons of full-length sequences. The nomenclature specified by the Human Gene Nomenclature Committee is used. Previous names for the different calpains are: CAPNl - m-Calpain, CAPN2 - m-Calpain, CAPN3 - p94, nCl-1, CAPN8, nCL-2, CAPN9, nCL-4. The EMBL accession numbers for the calpain sequences used are: CAPNl (P17655), CAPN2 (P07384), CAPN3 (P20807), CAPN5 (Y10656), CAPN6 (Y12582), CAPN7 (AJ012475) and CAPN9 (AF022799).

Figure 3. Expression of CAPNII. A ³² P-labeled DNA probe with an 800 base pair segment of the coding sequence of the human CAPNII cDNA was attached to a master blot (A), a nylon filter with dot blots of RNAs from 50 different human tissues or a Clontech Multi-tissue Northern blot (D) hybridizes. The filters were washed with high stringency (6x SSC, 65 ° C). The exact location of the different RNAs on the dot blot filter is shown schematically (C). The RNAs on the Northern blot are indicated above the corresponding lanes. Dot blot and Northern blot were re-hybridized with human ubiquitin (B) and b-actin DNA probes to determine the amounts of poly (A +) RNA applied. The positions of the size markers (in kilobases) are given for the Northern blot. The exposure times were: A, 72 hours; B, 24 hours; D, 48 hrs. PBL = peripheral blood leukocytes.

Claims

claims

1. Polypeptide with the amino acid sequence SEQ. -ID. -No. Second

2. polynucleotide sequence encoding a polypeptide according to claim 1.

3. Polynucleotide sequence with the sequence SEQ. -ID. -No. 1.

4. Use of a polypeptide according to claim 1 for the identification of substances which can inhibit the enzymatic activity of this polypeptide.

A method of identifying compounds that inhibit the enzymatic activity of a polypeptide according to claim 1, comprising:

(a) comparing the level of enzymatic activity of CAPNII in the presence of the compound with the level of enzymatic activity of CAPNII in the absence of the compound and

(b) Selecting compounds that alter the level of CAPNII enzymatic activity versus CAPNII enzymatic activity in the absence of the compound.

6. Use of compounds identified by the method of claim 5 for the treatment of fertility disorders in men.