EP0942995A1

EP0942995A1 - Novel calpaines, production and use thereof

Info

Publication number: EP0942995A1
Application number: EP97952002A
Authority: EP
Inventors: Thomas Boehm; Neil T. Dear
Original assignee: BASF SE
Current assignee: Abbott GmbH and Co KG
Priority date: 1996-12-04
Filing date: 1997-11-28
Publication date: 1999-09-22
Also published as: CN1245536A; CA2274304A1; CZ9902005A3; KR20000057365A; US6569665B1; WO1998024916A1; HUP0000498A2; AU5557698A; NO992690L; JP2001506846A; BR9713849A; NO992690D0; IL129909A0; HUP0000498A3; AU737504B2

Abstract

The invention relates to novel calpaines and their production. The invention further relates to a screening method for new calpaine inhibitors and the use thereof.

Description

New calpaine, their production and use

description

The invention relates to new calpains and their production. The invention further relates to methods for screening for new calpain inhibitors and their use.

Calpains are intracellular, non-lysosomal

Enzymes from the group of cysteine proteases. They are involved in the Ca ²⁺ -dependent signal transduction in eukaryotic cells, ie they regulate cellular functions depending on the Ca ²⁺ concentration. Calpains occur ubiquitously in animal tissues or cells of, for example, humans, chickens, rabbits or in the rat. Calpains have also been found in lower animals such as Drosophila melanogaster or Caenorhabditis elegans. No calpains have been found in yeasts, fungi or bacteria.

So far, three main isoforms of these ubiquitous calpains are known, which differ in vitro by their calcium-dependent activation. Calpain I (= μCalpain) is activated by μ-molar calcium ion concentrations, while Calpain II (= mCalpain) is only activated by millimolar concentrations of calcium ions. Both Calpaine consist of two subunits, a large subunit with approx. 80 kDa and a small subunit of approx. 30 kDa. Both subunits of the active heterodimer have binding sites for calcium. The large subunit is made up of the following four protein domains (= I - IV): a protease domain (= domain II), a calcium-binding domain (= domain IV) and two other domains (= domain I and III), their function is unclear. The small 30 K subunit consists of a calcium-binding subunit (= IV) and another subunit (= V), the function of which is unclear. In addition to these two types of calpain, a third type of calcium activation (= μ / m 80K) was found in the chicken (Wang KKW, et al., TiPS, Vol. 15, 1994: 412-419, Suzuki, K et al., Biol. Chem. Hoppe-Seyler, vol. 376, 1995: 523-529).

In addition to these ubiquitous calpaines, two new tissue-specific expressed calpains have recently been identified. nCL-1 (= p94) is a muscle-specific calpain found in chickens, rats and humans, which could presumably be active as a monomer and consists only of the 80 kd subunit. In addition to nCL-1, there is a stomach-specific calpain that is divided into two Splicing variants nCL-2 and nC -2 'can occur. nC -2 'differs from nCL-2 by the lack of the calcium-binding region (Sorimachi, HS et al., J. Biol. Chem. Vol. 268, No. 26, 1993: 19476-19482, Sorimachi, H: S: et al., FEBS Lett. 5 343, 1994: 1-5). Also in Drosophila, a calpain-homologous protein (= CalpA), which interacts with actin and is believed to play an important role in embryonic development, was found to have two different splicing variants (Mol. Cell. Biol. Vol. 15, No. 2, 1995: 824-834). Here, too, the shorter 0 variant lacks the calcium binding site.

Calpaine are believed to play important roles in various physiological processes. A large number of cytoskeletal, membrane-binding or regulatory proteins such as protein kinase C, phospholipase C, spectrin, cytoskeleton proteins such as MAP2, muscle proteins, neurofilaments and neuropeptides, platelet proteins, “epidermal growth factor”, NMDA receptor and proteins which are involved in mitosis, as well as other proteins, are calpain substrates (Barrett MJ et al., Life Sei. 48, 1991: 0 1659-69, Wang KK et al., Trends in Pharmacol. Sei., 15, 1994: 412 - 419). The normal physiological function of calpaine has not yet been clearly understood.

Increased calpain levels were measured in various pathophysiological processes and diseases 5, for example in: ischemia of the heart (e.g. heart attack), the kidney or central nervous system (e.g. stroke), inflammation, muscle dystrophies, cataracts of the eyes (cataracts), Central nervous system injuries (eg trauma), Alzheimer's disease, HIV-induced neuropathy, Parkinson's and Huntigton's disease, etc. (see Wang KK above). It is suspected that these diseases are related to an increased and persistent intracellular calcium level. As a result, calcium-dependent processes are overactivated and are no longer subject to physiological regulation. Accordingly, overactivation of calpains can also trigger pathophysiological processes.

Therefore, it has been postulated that inhibitors of calpain enzymes can be useful in the treatment of these diseases. Various studies have confirmed this. For example, Seung-Chyul Hong et al. (Stroke 1994, 25 (3), 663-669) and Bartus RT et al. (Neurological Res. 1995, 17, 249-258) demonstrated a neuroprotective effect of calpain inhibitors in acute neurodegenerative disorders such as those which occur after a stroke. Likewise, after experimental brain trauma, calpain inhibitors improved the recovery of memory deficits and neuromotor disorders (Saatman KE et al., Proc. Natl. Acad. Be. USA, 93, 1996: 3428-3433). Edelstein CL et al. (Proc. Natl. Acad. Sci. USA, 92, 1995, 7662-7666) found a protective effect of calpain inhibitors on kidneys damaged by hypoxia. Yoshida KI et al. (Jap. Circ. J. 59 (1), 5 1995, 40-48) were able to show beneficial effects of calpain inhibitors after cardiac damage which was caused by ischemia or reperfusion. Since calpain inhibitors inhibit the release of β-AP4 protein, a potential use as a therapeutic agent for Alzheimer's disease has been proposed (Higaki J. 0 et al., Neuron, 14, 1995: 651-659). The release of interleukin-lα is also inhibited by calpain inhibitors (Watanabe N. et al., Cytokine, 6 (6), 1994: 597-601). Furthermore, it has been found that calpain inhibitors show cytotoxic effects on tumor cells (Shiba E. et al., 20th Meeting Int. Ass. 5 Breast Cancer Res., Sendai Jp, 1994, September 25-28, Int. J. Onco. 5 (Suppl.), 1994, 381). Calpain also plays an important role in restenosis and arthritis, and calpain inhibitors can have a positive effect on the clinical picture (March K: L: et al. Circ. Res. 72, 1993: 413-423, Suzuki K. et al., Biochem 0 J ., 285, 1992: 857-862).

Further possible uses of calpain inhibitors can be found in Wang K.K (Trends in Pharmacol. Sci., 15, 1994: 412-419).

5 The most potent and selective calpain inhibitor is the naturally occurring intracellular protein calpastatin. It inhibits both calpain I and calpain II, but not other cysteine or thiol proteases such as cathepsin B, L or papain. However, calpastatin, which consists of approximately 700 amino acids, has the disadvantage that it is out of the question for therapeutic options due to the size and impassability of the cell membrane. In addition to low-molecular peptide calpain inhibitors, a number of non-peptide inhibitors have been identified. The disadvantage of these inhibitors is that they are unstable, are rapidly metabolized and are sometimes toxic. Many calpain inhibitors are also characterized by a lack of selectivity, i.e. they not only inhibit calpain I and II but also other cysteine proteases such as papain, chymotrypsin, elastase or cathepsin B and L.

There is therefore still a need for selective, highly effective calpain inhibitors. The screening for these selective, well-effective calpain inhibitors requires highly specific test systems that make it possible to identify selective inhibitors. The screening tests are usually carried out with the ubiquitous Calpainen Calpain I and Calpain II. In order to find selective inhibitors, it is necessary and desirable to provide further calpains for testing, which are expressed as tissue-specifically as possible so that the inhibitors can be tested for their selectivity between the individual calpains.

In addition, other new calpains are sought-after proteins, since they are likely to be expressed differently in the various clinical pictures or diseases and play an important role in these diseases.

It was an object of the present invention to provide means for profiling and identifying calpain inhibitors which make it possible to identify calpain inhibitors which, on the one hand, have an inhibitory effect only on one calpain and, on the other hand, have an inhibitory effect on several calpaines and which serve as a therapeutic target To make available.

The invention relates to a new calpain and its allelic variants, analogs or derivatives.

The invention also relates to a method for the identification of calpain inhibitors, wherein the calpain nCL-3 coded by the sequence SEQ ID NO: 1 or SEQ ID NO: 6 is isolated from tissues or cells in which the enzyme nCL-3 is expressed and the inhibition of the cleavage of a substrate of the enzyme nCL-3 and in at least one further test measures the inhibition of the cleavage of a substrate of the enzymes Calpain I and / or II by test substances and selects the test substances which inhibit at least one of the calpains Show effect or select the test substances which do not inhibit the enzyme nCL-3, but which inhibit the enzymes Calpain I and / or II or which inhibit the enzyme nCL-3, but not the enzymes Calpain I and / or II or the nCL-3 and inhibit the enzymes Calpain I and / or II.

The invention furthermore relates to a method for identifying calpain inhibitors, characterized in that the inhibition of the cleavage of a substrate of the enzyme nCL-3 or of calpains I and / or II is determined by test substances in cellular systems and such test substances are selected which Pass through the cell membrane and inhibit the intracellular activity of the enzyme nCL-3 and / or Calpaine I and / or II.

With the help of the Calpain-specific primers, Calpain-specific were used in the so-called domain fingerprinting (Boehm T., Oncogene 8, 1993: 1385-1390) using genomic DNA Sequence signatures produced by means of PCR technology, which advantageously also contain intron sequences for better differentiation of the calpain sequences.

The redundant PCR primers listed in Table 1 were used in cloning the gene for nCL-3.

Table 1

Redundant PCR primers used to clone nCL-3 (= 2930)

Name sequence

CA 1 5 «- tng gng att gtt ggc tnc t - 3« c c t t

CA 2 5 «- ctn gaa aaa gen tat gen aa - 3« t g g c

CA 3 5 «- ttt ngc ata ngc ttt ttc na - 3« c g c c

CAL4 5 «- gtn aaa ggn cat gen tat ac - 3« g c e

CA 5 5 «- gag tan gca tgn cet ttn ac - 3« ta g c

CAL6 5 «- ttn cgn aat cen tgg gg - 3« c c a

CAL7 5 «- cec can gga ttn cgn aan cg - 3« g g t

CAL8 5 «- gat ggn gaa ttt tgg atg - 3« c g c

CAL9 5 "- gac atc caa aat ten cca tc - 3" et g c g

CA 10 5 «- nag att aca tat ttc na - 3« a g g a c g

With the primer pair Calδ and Cal9 (see Table 1) a clone with the designation 29/30 (= 2930) could be represented. This clone codes for a gene whose gene product was named nCL-3 as the new calpain. The nucleic acid sequence of clone 29/30 (= nCL-3 or 2930) can be found in sequence SEQ ID NO: 1. The derived amino acid sequence of calpain nCL-3 can be found in the sequence SEQ ID NO: 2. The amino acid sequence deduced taking into account an existing intron has one typical Calpain signature, whereby an assignment to the well-known Calpain subfamilies μCalpain, mCalpain, nCL-1 or nCL-2 is not possible due to the low homology (see Table 2). FIG. 3 shows the homology to known calpain subfamilies. The Calpain nCL-3 is a new, previously unknown Calpain.

Sequence analysis showed the typical three amino acid residues (Cys81, His252 and Asn284) of the catalytic center of cysteine proteases. The amino acid residues 75 - 86 (QGQVGNCWFVAA) derived from the gene sequence correspond to the conserved pattern of typical thiol proteases.

Table 2

Homology (%) at the amino acid level between mouse nCL-3 = 2930) and other calpains

Name% homology nematode tra-3 34.5

Drosophila CalpA 31.5

Chicken p94 31.2

Human p94 30.9

Mouse p94 30.5 rat p94 30.0

Chicken µ / m 28.8

Chicken m 27.8

Human m 27.3

Chicken µ 25.4 Pig p94 25.4

Rat m 24.4

Rat nCL-2 23.9

Nematode CPL1 23.6

Human μ 23.1 Schistosoma 21.7

Rabbit m 16.1

Pig m 15.8

Pig μ 15.6

Mouse CAP4 13.7 rabbit μ 12.9

The intron shown in sequence SEQ ID NO: 5 (from nucleotides 109 to 514) was determined by comparison with the cDNA.

Sequence comparisons between the mouse 29/30 sequence (nCL-3) in various databases such as Genbank.nr and Genbank. dbest gave homologies to CalpA, Tra-3 and a human sequence with the loading Drawing EST01106 Homo sapiens cDNA clone HHCPE79 (see Figure 2). The DNA and amino acid sequences were checked for homology with non-redundant nucleic acid, protein and EST databases at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). The amino acid sequence comparisons were carried out with Clustal W (Thompson et al. Nucl. Acids Res. 22, 1994, 4673-468 =).

In comparison to the other calpains (FIG. 2), nCL-3 has a shortened domain I and a modified C-terminal end which has no pronounced homology to domain IV of the other calpains. The consensus sequence of the Ca ²⁺ binding site of the calpains (so-called “EF-hand”) is located in the domain IV. This Ca ²⁺ binding site is absent in the case of nCL-3, which means that possibly no Ca ^{2+ is} bound to domain IV and the protein is activated in another way. It is the only vertebrate calpain that lacks domain IV similar to calmodulin.

CalpA is a tissue-specific expressed calpain homolog from Drosophila (Theopold V. et al., Mol. Cell. Biol., Vol. 15, No. 2, 1995: 824-834). It is expressed in some neurons in the central nervous system, in scattered cells of the midgut and in blood cells from Drosophila. CalpA found two different splicing variants. The shorter variant lacks the calcium binding site typical of Calpain.

The homology at the amino acid level between CalpA and nCL-3 is 31.5% (see Table 2).

Tra-3 is involved in sex determination of Caenorhabditis elegans. In a cascade of several genes and their gene products, tra-3 also decides whether caenorhabditis males or hermaphrodites develop (Kuwabara P.E. et al., TIG, Vol. 8, No.5, 1992: 164-168). Tra-3 appears to be involved in spermatogenesis.

The homology at the amino acid level between tra-3 and nCL-3 is 34.5% (see Table 2). NCL-3 may also be involved in gender determination.

Further homologies between nCL-3 and other calpaines can be found in Table 2.

The greatest homology exists between nCL-3 and the human partial sequence ΞST01106. The partial sequence EST01106 was obtained from a hippocampus library. Nothing is known about the function (Nature 355, 6361, 1992: 632-634). The complete gene sequence and whether the sequence is a calpaingen is also unknown.

The sequence comparisons between CalpA, Tra-3, EST01106 and nCL-3 are shown in FIG. 2.

Starting from human hippocampus marathon-ready cDNA (Clontech), a modified RACE method (= rapid amplification of cDNA ends) according to Frohman et al. (Proc. Natl. Acad.

Be. USA 85, 1988, 8998-9002) and Edwards et al. (Nucl. Acids Res. 19, 1991, 5227-5232) using the above primers (Cal6 and Cal9) the entire sequence of the clone EST01106 can be cloned. The 3 'region of the cDNA could initially not be cloned using the reverse primer of the Clontech kit. This was only possible using a primer that was complementary to the human EST sequence and a reverse primer that was complementary to the cDNA sequence of the last 6 amino acids of the mouse nCL-3 sequence (5 '-tcagacagccgtgagagagg-3') 3 'end to be cloned.

The amino acid sequence (SEQ ID NO: 7) derived from the gene sequence SEQ ID NO: 6 shows 92.2% homology to the mouse nCL-3 sequence (see FIG. 4). This similarity corresponds to the amino acid level homology between human and mouse m-calpain (97%) and human and mouse p94 (93.5%) as our sequencing showed. EST01106 is therefore most likely the human ortholog of the mouse nCL-3 sequence. Figure 4 also shows the sequences of Caenorhabditis tra-3, Drosophila CalpA, mouse p94. Mouse m-calpain, human μ-calpain and rats nCL-2. Amino acids that match between the different calpains and nCL-3 are indicated by dots. Dashes indicate gaps that have been introduced in order to achieve maximum match of the sequences. The C-terminal ends of the alternative splicing products of the nCL-3 and CalpA transcripts are indicated above and below the respective overall sequence, starting with the beginning of the different sequence. Stars indicate the remains conserved between all calpains. The two amino acid sequences corresponding to the oligonucleotides Cal6 and Cal9 were marked with boxes. The "splice sites" of the corresponding mouse nCL-3-DNA are marked by arrows.

Figure 5 shows the phylogenetic family tree of the different Calpaine. The phylogenetic analyzes for the construction of this family tree were carried out using the closest neighboring method (Saitou et al. Mol. Biol. Evol. 4, 1987, 406-425) Exclusion of the gaps carried out. With the help of these phylogenetic analyzes, the vertebrate calpains could be divided into six different groups (Figure 5, right side). The non-vertebrate calpains can be assigned to the nCL-3 group as the nearest neighbors or are in a separate group. The nCL-3 genes thus form a separate group of calpains, which are more similar to invertebrate calpains than to vertebrate calpains. The length of the horizontal lines is proportional to the phylogenetic distance of the different Calpaine. The length of the vertical lines is irrelevant. The sequences used to compile the phylogenetic family tree have the following SWISSPROT and EMBL numbers ("accession numbers"): human m (P17655), μ (P07384), p94 (P20807); Rats m (Q07009), nCL-2 (D14480), p94 (P16259); Mouse p94 (X92523); Chickens m (D38026), µ (D38027), µ / m (P00789), p94 (D38028); Nematodes tra-3 (U12921); Drosophila Calp A (Q11002) and Dm (X78555), Schistosoma (P27730).

The nCL-3 gene structure is shown in Figure 6. The exon / intron splicing transitions within the coding sequence of the gene were determined by comparing the DNA sequences of genomic DNA and cDNA. 11 introns were found within the coding sequence. In Figure 4, the position of the "splicing sites" is marked by arrows. The position of the genomic fragment first amplified with the primers Cal6 and Cal9 is marked by brackets.

Figure 6b shows the location of the "splicing sites" of different Calpaine. Surprisingly, despite the relatively high homology, nCL-3 and tra3 do not have any common "splice sites". The agreement in the position of some "splice sites" between nCL-3 and the vertebrate calpaines indicates a common genesis. The question mark above the last preserved splice site in the chicken μ / m-Calpaingen indicates that the published sequence around this interface is not in agreement with the original cDNA sequence.

In addition to the nCL-3 gene shown in sequence SEQ ID NO: 1, a truncated form, which is shown in sequence SEQ ID NO: 3, was identified. The deduced amino acid sequence of the truncated nCL-3 gene can be found in the sequence SEQ ID NO: 4. This truncated nCL-3 gene, known as nCL-3 ', is believed to result from alternative splicing. Semi-quantitative RT-PCR analyzes with mRNA isolated from dE17 cells using primers that match the flanking Covering regions of the intron (see Figure 6) showed that the unsplit product accounted for approximately 0.5% of the n-CL3 mRNA.

The new calpain nCL-3 according to the invention is expressed in many tissues with different intensities (FIG. 1). In the mRNA analyzes examined, nCL-3 is clearly expressed in the skin, the kidneys, the heart, the lungs, the thymus and in the liver.

The nCL-3 gene is also expressed to different extents in humans. Low expression was detected in all tissues examined. Strong expression was found in the colon, testicles, kidney, liver and trachea.

The nCL-3 gene was located on chromosome 7 in mice and chromosome 11 in humans. It is around 84 cM (= centi acres) on the long arm of the human chromosome. This is 12 - 14 cM from the mapped position of the μ-calpain (llql3). nCL-3 could be catalyzed very close to the glycoprotein A gene (llql3.5 - ql4).

The mouse orthologist nCL-3 gene was localized between 44 and 53 cM on mouse chromosome 7.

For the identification of selective calpain inhibitors, specific methods for identifying the inhibitors are required. It is important that the selected inhibitors only inhibit the desired calpaine or s, but not other cysteine proteases and thus interfere with physiological processes.

The test substances to be tested for their inhibitory activity can be, for example, chemical substances, microbial or plant extracts. They are usually tested in addition to the test for their inhibitory activity towards nCL-3, calpain I and / or II for their activity against cathepsin B or other thiol proteases.

Ideally, good inhibitors should have little or no activity against cathepsin B, L, elastase, papain, chymotypsin or other cysteine proteases, but should have good activity against calpaines I and II. With the novel calpain nCL-3 according to the invention, inhibitors can be identified with the methods according to the invention, which can discriminate their inhibitory effect between the different calpaines Calpain I, II, nCL-1, nCL-2 and / or nCL-3.

The various inhibitor tests were carried out as follows:

Cathepsin B test

Cathepsin B inhibition was determined analogously to a method by S. Hasnain et al., J. Biol. Chem. 1993, 268, 235-240.

To 88 μl cathepsin B (human liver cathepsin B from Calbiochem, diluted to 5 units in 500 μM buffer), 2 μl of an inhibitor solution, prepared from the chemical substance to be tested, a microbial or vegetable extract and DSMO (final concentration: 100 μM up to 0.01 μM) added. This batch is preincubated for 60 minutes at room temperature (= 25 ° C.) and the reaction is then started by adding 10 μl of 10 mM Z-Arg-Arg-pNA (in buffer with 10% DMSO). The reaction is monitored for 30 minutes at 405 nm in a microtiter plate reader. The ICso's are then determined from the maximum gradients.

Calpain I and II test

The activity of the calpain inhibitors was examined in a colorimetric test with casein according to Hammarsten (Merck, Darmstadt) as the substrate. The test was performed in microtiter plates, according to the publication by Buroker-Kilgore and Wang in Anal. Biochem. 208, 1993, 387-392. Calpain I (0.04 U / test) from erythrocytes and Calpain II (0.2 U / test) from kidneys, both from pigs, from Calbiochem, were used as enzymes. The substances to be tested were incubated with the enzyme for 60 minutes at room temperature, a concentration of 1% of the solvent DMSO not being exceeded. After adding the Bio-Rad color reagent, the optical density was measured at 595 nm in the Easy Reader EAR 400 from SLT. The 50% activity of the enzyme results from the optical densities, which were determined for the maximum activity of the enzyme without inhibitors and the activity of the enzyme without the addition of calcium. The activity of calpain inhibitors can also be determined with the substrate Suc-Leu Tyr-AMC. This fluorometric method is described in Zhaozhao Li et al, J. Med. Chem. 1993, 36, 3472-3480.

Because calpains are intracellular cysteine proteases, calpain inhibitors must cross the cell membrane to prevent calpain from breaking down intracellular proteins. Some known calpain inhibitors, such as E 64 and leupeptin, only poorly cross the cell membranes and accordingly show, although they are good calpain inhibitors, only poor activity on cells. It is therefore advantageous to carry out an additional test for the permeability of potential calpain inhibitors such as the human platelet test.

Platelet test to determine the cellular activity of calpain inhibitors

Calpain-mediated protein degradation in platelets has been described by ZhaozhaoLi et al. , J. Med. Chem., 36, 1993, 3472-3480. Human platelets were isolated from fresh sodium citrate blood from donors and adjusted to 10 ⁷ cells / ml in buffer (5 mM Hepes, 140 mM NaCl and 1 mg / ml BSA, pH 7.3).

Platelets (0.1 ml) are pre-incubated for 5 minutes in 1 μl at various concentrations of potential inhibitors (dissolved in DMSO). This was followed by the addition of calcium ionophore A 23187 (1 μM in the test) and calcium (5 mM in the test) and a further incubation of 5 minutes at 37 ° C. After a centrifugation step, the platelets were taken up in SDS-Page sample buffer, boiled for 5 minutes at 95 ° C. and the proteins were separated in an 8% gel. The degradation of the two proteins actin binding protein (= ABP) and talin was followed by quantitative densitometry. After the addition of calcium and ionophore, these proteins disappeared and new bands of less than 200 Kd molecular weight were formed. From this, the half maximum enzyme activity is determined with or as a control without an inhibitor.

Tissue parts such as brain sections or cell cultures are also suitable for testing membrane permeability.

Inhibition of nCL-3 is carried out in cells which express this protein and which can be detected with a specific antibody. Are cells with z. B: Calcium and the corresponding ionophore stimulated, this leads to an activation of nCL-3. Takaomi Saido described in 1992 in J. Bio- chem. Vol. 11, 81-86 the autolytic transition of μ-calpain after activation and the detection with antibodies. Appropriate antibodies are generated for the detection of nCL-3. Calpain inhibitors prevent the autolytic transition and a corresponding quantification is possible with antibodies.

In addition to the in vitro tests described, such as the cellular platelet test, all other calpain tests known to the person skilled in the art, such as the test for inhibition of glutamate-induced cell death on cortical neurons (Maulucci-Gedde M.A. et al.,

J. Neurosci. 7, 1987: 357-368), calcium-mediated cell death in NT2 cells (Squier MKT et al., J. Cell. Physiol., 159, 1994: 229-237, Patel T. et al., Faseb Journal 590 , 1996: 587-597) or the analysis in tissue samples for degradation products of proteins such as Spectrin, MAP2 or Tau (Ami Arai et al., Brain Research, 1991, 555, 276-280, James Brorson et al., Stroke, 1995, 26, 1259-1267).

For the in vitro tests of nCL-3, the calpain nCL-3 or its animal or human homologue is made from tissues or cells in which the enzyme is expressed, such as the kidney, the thymus, the liver, the lung, or from cells or microorganisms which contain at least one gene copy and / or a vector with at least one gene copy of the nCL-3 gene, purified and used as a crude extract or as a pure enzyme.

For the method according to the invention, the various calpain inhibitor tests are advantageously carried out in combination with the test for inhibition of the nCL-3 enzyme activity by potential inhibitors. Inhibitors are selected so that they either only inhibit the enzyme nCL-3 and not the other calpains or, conversely, only the other calpains and not the enzyme nCL-3 or the enzyme nCL-3 and at least one other calpain.

The various inhibitor tests are carried out in such a way that, in addition to the test for the inhibitory effect of the test substance against nCL-3, Calpain I and / or II as a control, the tests are carried out without the test substance. With this test arrangement, the inhibitory effects of the test substances can easily be recognized.

Another method according to the invention uses the enzyme nCL-3 for the screening for new calpain inhibitors, these inhibitors advantageously being able to generally inhibit all calpains or individual calpains such as calpain I, II, nCL-1, nCl-2 or nCL-3. The different test substances can be used individually or tested in parallel in test systems. The test substances are advantageously screened for their inhibitory effect in parallel, automated test systems.

All substances are generally suitable for the inhibitor tests. For example, the substances come from classic chemical synthesis, from combinatorics, from microbial, animal or vegetable extracts. Microbial extracts are, for example, fermentation broths, cell disruption of microorganisms or substances after biotransformation. Cell fractions are also suitable for the tests.

All prokaryotic or eukaryotic expression systems which are suitable for isolating an enzymatically active gene product are suitable for cloning the nCL-3 gene or its animal homologs or its human homologues. Expression systems are preferred which enable expression of the nCL-3 gene sequences in bacterial, fungal or animal cells, very particularly preferably in insect cells. An enzymatically active gene product is to be understood as nCL-3 proteins which, directly after isolation from the expression organism, for example from a prokaryotic or eukaryotic cell, or after renaturation, give an active protein which is capable of at least one known calpain substrate such as those mentioned above or to split yourself through autocatalysis.

All calpain tests known to the person skilled in the art, such as in vitro tests such as the tests described above for calpain I and II, or cellular tests such as the platelet test, are suitable for determining the enzymatic activity. Tests which are based on a colorimetric assay (Buroker-Kilgore M. et al., Anal. Biochem. 208, 1993: 387-392) or on the basis of a fluorescence assay can be used as the detection possibility.

In addition, all partial sequences that are the catalytic

Contain the center of the nCL-3 gene and / or further sequences of the nCL-3 gene and / or other calpain gene sequences and / or other sequences and show enzymatic activity, to be understood by the enzymatic active gene product of nCL-3.

Among host organisms are all prokaryotic or eukaryotic organisms which are suitable as host organisms are, for example, bacteria such as Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptococcus carnosus, yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, fungi, Trypsoptera as such as Aspergillusiperia nod Cells or any other insect cells that are for viral expression is suitable, or to understand animal cells such as CV1, COS, C127, 3T3 or CHO or human cells.

Expression systems are to be understood as the combination of the expression organisms mentioned above by way of example and the vectors which match the organisms, such as plasmids, viruses or phages such as the T7 RNA polymerase / promoter system or vectors with regulatory sequences for the phage λ.

The term expression systems is preferably to be understood as the combination of Escherichia coli and its plasmids and phages or the baculovirus system and the corresponding insect cells such as Spodoptera frugiperda.

Additional 3 'and / or 5' terminal regulatory sequences are also suitable for the advantageous expression of the nCL-3 gene according to the invention.

These regulatory sequences are intended to enable the targeted expression of the nCL-3 gene. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.

The regulatory sequences or factors can preferably have a positive influence on nCL-3 gene expression and thereby increase it. Thus, the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers". But there is also a reinforcement of the

Translation possible, for example by improving the stability of the mRNA.

“Enhancers” are understood to mean, for example, DNA sequences which bring about increased nCL-3 gene expression via an improved interaction between RNA polymerase and DNA.

One or more DNA sequences can be connected upstream and / or downstream of the nCL-3 gene with or without an upstream promoter or with or without a regulator gene, so that the gene is contained in a gene structure.

The gene expression of the nCL-3 gene can also be increased by increasing the nCL-3 gene copy number. To increase the number of copies of the gene, the nCL-3 gene is amplified, for example, in a CHO expression vector. Vectors of the pED series - dicistronic vectors - are also suitable as vectors contain the amplifiable marker gene dihydrofolate reductase. Details can be found in the Current Protocols in Molecular Biology Vol 2, 1994.

An increase in the nCL-3 enzyme activity can be achieved, for example, compared to the starting enzyme by changing the nCL-3 gene or its animal homologues through classic mutagenesis such as UV radiation or treatment with chemical mutants and / or through targeted mutagenesis such as site directed mutagenesis, Achieve deletion (s), insertion (s) and / or substitution (s). An increase in enzyme activity can be achieved, for example, by changing the catalytic center so that the substrate to be cleaved is converted more quickly. In addition to the gene amplification described, increased enzyme activity can also be achieved by eliminating factors which repress enzyme synthesis and / or by synthesizing active instead of inactive nCL-3 proteins. In this way, increased amounts of enzyme can be made available for the in vitro tests.

nCL-3 or its animal homologues can advantageously be derived from genomic DNA or cDNA using, for example, the PCR technique (see Molecular Cloning, Sambrok, Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press, Second Edition 1989, Chapter 14, 1-35, ISBN 0-87969-309-6 and Saiki et al., Science, 1988, Vol. 239, 487ff), preferably nCL-3 can be selected using genomic DNA and particularly preferably using genomic DNA Clone mouse cells or human cells.

Suitable host organisms for cloning are, for example, all Escherichia coli strains, preferably the Escherichia coli strain DH10B. Suitable vectors for cloning are all vectors which are suitable for expression in Escherichia coli (see Molecular Cloning, Sambrok, Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press, Second Edition 1989, ISBN 0-87969-309-6) . For example, vectors which are derived from pBR or pUC or shuttle vectors are particularly suitable. PBluescript is very particularly suitable.

After isolation and sequencing, nCL-3 genes with nucleotide sequences are available which code for the amino acid sequence given in SEQ ID NO: 2 or its allele variants. Allele variants are understood to mean nCL-3 variants which have 60 to 100% homology at the amino acid level, preferably 70 to 100%, very particularly preferably 80 to 100%. Allelic variants include, in particular, functional variants which are obtained by deleting, inserting or substituting nucleotides from the SEQ ID NO: 1 or SEQ ID NO: 6 sequence are available, but the nCL-3 activity is retained.

Analogs of nCL-3 mean, for example, its animal homologs, shortened sequences such as nCL-3 '(see SEQ ID NO: 3), single-stranded DNA or RNA of the coding and non-coding DNA sequence, particularly antisense RNA.

Derivatives of nCL-3 are, for example, those derivatives which cannot be cleaved enzymatically or are difficult to cleave like the nucleic acid - phosphonates or phosphothioates, in which the phosphate group of the nucleic acids has been replaced by a phosphonate or thioate group.

The promoter upstream of the specified nucleotide sequence can also be changed by one or more nucleotide exchanges, by insertion (s) and / or deletion (s), but without the functionality or effectiveness of the promoter being impaired. Furthermore, the effectiveness of the promoter can also be increased by changing its sequence, or it can be completely replaced by more effective promoters, including organisms of other species.

The cal pain inhibitors identified by the methods according to the invention are suitable for the manufacture of medicaments for the treatment of diseases selected from the group of cardiovascular, immunological, inflammatory, allergic, neurological, neurodegenerative or oncological diseases such as restenosis, arthritis, ischemia of the heart or the like Kidney or the central nervous system (e.g. stroke), inflammation, muscular dystrophies, cataracts of the eyes (cataracts), injuries to the central nervous system (e.g. trauma), Alzheimer's disease, HIV-induced neuropathy, Parkinson's and Huntigton's disease.

The nCL-3 gene sequences according to the invention are also advantageously suitable for diagnosing diseases, for example for diagnosing muscular dystrophy or for gene therapy. Examples

Example 1: Cloning of the nCL-3 gene

Genomic DNA from mouse cells ES E14 was used to clone the nCL-3 gene with the sequence SEQ ID NO: 1. The 5 '-3' (= forward) and 3 '-5' (= reverse) sequences of the primers CAL6 and CAL9 (see table 1) and the following PCR conditions (see molecular cloning, Sambrok, Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press, Second Edition 1989, Chapter 14,

1-35, ISBN 0-87969-309-6 and Saiki et al., Science, 1988, Vol. 239, 487ff) are used for the cloning:

250 ng forward primer 250 ng reverse primer

1.5 mM MgC12

0.2mM dNTPs

50 mM KC1

10 mM Tris pH 9.0 1 ug genomic DNA

2 units of Taq polymerase.

35 PCR cycles were carried out, the temperature being maintained at 72 ° C. for 45 seconds at 94 ° C., 45 seconds at 48 ° C. and 2 minutes. The nCL-3 gene was cloned into the vector pBluescript (SK +) with the enzyme EcoRV (see Holton et al., Nucleic Acids Research, Vol. 19, No. 5, 1156ff.). With the pBluescript vector with the cloned-in nCL-3 gene, the Escherichia coli strain DH10B according to Maniatis et al. transformed (see Molecular Cloning, Sambrok, Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press, Second Edition 1989, Volume 1, Chapters 1, 74-84, ISBN 0-87969-309-6 and Saiki et al., Science, 1988 , Vol. 239, 487ff).

The human sequence of the nCL-3 gene with SEQ ID NO: 6 can also be cloned in this way, it being possible to start from 0.1 ng cDNA or 0.5 μg genomic DNA. The cloning batch may also advantageously contain 0.1% Triton X-100.

Example 2: Expression of the nCL-3 gene in different mouse tissues

For expression, mRNA was isolated from dE12 embryos and from mouse, kidney, heart, lung, brain, thymus and small intestine tissue. For the extraction of the mRNA, the tissues were dispersed in liquid nitrogen and in 10 ml of a solution of 4 M guanidinium isothiocyanate, 25 mM Na citrate, 0.5% sarcosyl and 72 ul 2-mercaptoethanol resuspended. Then 1 ml of Na acetate (pH 4.0), 10 ml of water-saturated phenol and 2 ml of chloroform were added with mixing. The samples were centrifuged for 20 minutes (5000 xg, 4 ° C). The precipitate was discarded. The RNA was precipitated from the supernatant with a volume of isopropanol at −20 ° C. (precipitation for at least one hour) and centrifuged again for 30 minutes (19000 × g, 4 ° C.). The precipitate was resuspended in 300 μl and again precipitated in the cold with Na acetate / ethanol and washed with 70% ethanol, centrifuged (19000 × g, 4 ° C.) and dissolved in 300 μl water. The mRNA concentration at 250 nm was then determined in a photometer and in an agarose gel with a 5 μl mRNA sample against a reference.

Via RT-PCR, in which a cDNA copy was first made with the aid of reverse transcriptase, using the isolated mRNA, the following two primers were used: 3 'the expression of the nCL-3 gene in the various mouse tissues was determined (see FIG. 1).

The cDNA was produced according to a protocol from GIBCO as follows:

2 μl oligo (dT) and 12 μl DEPC dH 0 were added to 5 μg mRNA (isolated according to the method described above). This mixture was incubated at 70 ° C. for 10 minutes and then placed on ice. 2 μl 10 × buffer, 2 μl 25 mM MgCl ₂ , 1 μl 10 mM dNTPs, 2 μl 0.1 M DTT were added to the sample in the order given. After 5 minutes of incubation at 23 ° C., 1 μl of SuperScript Reverse Transcriptase was added and the whole incubation continued for 10 minutes at 25 ° C., 50 minutes at 42 ° C. and 15 minutes at 70 ° C. Then 1 μl RNAse H and 79 μl dH0 were added and the reaction was carried out for 20 min at 37 ° C and 15 min at 70 ° C. 1 ul of this cDNA was used for the RT-PCR reaction.

The PCR reaction for the detection of the expression of nCL-3 was carried out as follows:

250 ng forward primer 5 250 ng reverse primer 1.5 mM MgC12 0.2 mM dNTPs 50 mM KC1 10 mM Tris pH 9, 0 0 1 μg CDNA

2 units of Taq polymerase.

35 PCR cycles were carried out, the temperature being kept at 72 ° C. for 45 seconds, 58 ° C. for 45 seconds and the temperature at 72 ° C. for 1 minute 5.

The tissues tested showed that nCL-3 is expressed in the dE12 embryos. nCL-3 is also expressed in the skin, kidney, heart, lungs and thymus. Expression in the brain and small intestine is weaker than in the aforementioned organs (not shown in Figure 1). Hprt (= hypoxanthine phosphorus ribosyl transferase) was used as the internal standard.

3. Example: Cloning of the human sequence of nCL-3

The 3 'end of the mouse or human nCL-3 cDNA was determined using the so-called RACE method (= rapid amplification of cDNA ends), as described by Frohman et al. (Proc. Natl. Acad. Sci. USA 85, 1988, 8998-9002) and Edwards et al. (Nucl. Acids Res. 19, 1991, 5227-5232). Human hippocampus marathon-ready cDNA (Clontech) was used for the human sequence of the 5 'and 3' end. For the mouse sequence, cDNA from day 12 mouse embryos was used as described in Example 2. The human 3 'end could not be isolated with the kit reverse primer. The cloning was carried out with the aid of a forward primer complementary to the human EST sequence and a reverse primer which corresponded to the last 6 amino acids of the mouse nCL-3 sequence (5 '-teagacage-cgtgagagagg-3').

4. Example: Isolation and Characterization of Cosmid Clones

Cosmid clones with the mouse nCl-3 gene were isolated from a cosmid library, which was produced from genomic ES mouse cell DNA by cloning in the Vector pSuperCos (Stratagene). The library was divided into 348 "pools" of 1000 clones. Positive "pools" were identified using PCR analysis using fertilization of nCL-3-specific primers identified. These "pools" were then plated out and screened. Positive clones were identified by colony hybridization with ³² P-labeled mouse nCL-3 cDNA fragments.

5. Example: RNA expression analysis

The expression of the human nCL-3 was examined by hybridizing a human RNA master blot (Clontech) with a ³² P-labeled human nCL-3 fragment (nucleotide 1-928 coding for amino acids 1-295). The hybridization and the stringent washing conditions were carried out according to the manufacturer's instructions.

6. Example: Localization of the nCL-3 gene on the chromosome

The gene was localized in the mouse by PCR analysis of genomic DNA isolated from mouse somatic hamster cell hybrids as described by Williamson et al. (Mamm. Genome 6, 1995, 429-432) using a set of DNAs as described by Schupp et al. (Immunogenet. 45, 1997, 180-187). 5'-tgca-cagcctacagcataag-3 'and 5' -tcagacagccgtgagagagg-3 'were used as primer sequences. With the help of these primers an approximately 2.7 kb mouse fragment and no hamster DNA could be amplified. The PCR reactions were carried out using the "Expand Long Template PCR System (Boehringer Mannheim) according to the manufacturer's instructions at an" annealing "temperature of 58 ° C.

The gene was localized in humans using the NIGMS human / rodent somatic cell hybrid "mapping panels" (Coriell Cell Repositories). The following primers were used as primer sequences for the PCR reaction: 5 '-acttcatcttc-tggcttcttgacttc-3' and 5 '-gctgcatcaaccacaaggacac-3'. The PCR amplification was carried out at an "annealing" temperature of 58 ° C. and resulted in a 600 bp fragment. The results were examined for correspondence between the presence of human chromosomes and the PCR product. The exact localization of the gene in the human chromosome was carried out with the help of the "Genebridge 4 RH Panel" (Research Genetics) and transmission of the PCR results to the localization service the MIF Center for Genome Research (http://www-genome.wi.mit. edu). 7. Example: Cathepsin B test

Cathepsin B inhibition was determined analogously to a method by S.Hasnain et al., J. Biol. Chem. 1993, 268, 235-40.

2μL of an inhibitor solution, prepared from inhibitor and DMSO (final concentrations: 100μM to 0.01μM), are added to 88μL cathepsin B (cathepsin B from human liver (Calbiochem), diluted to 5 units in 500 μM buffer). This mixture is preincubated for 60 minutes at room temperature (25 ° C.) and the reaction is then started by adding 10 μL 10 mM Z-Arg-Arg-pNA (in buffer with 10% DMSO). The reaction is monitored for 30 minutes at 405 nM in a microtiter plate reader. The IC50 is then determined from the maximum gradients.

8. Example: Calpain test

The activity of the calpain inhibitors was examined in a colorimetric test with casein according to Hammarsten (Merck, Darmstadt) as a substrate. The test was performed in the microtiter plate, according to the publication by Buroker-Kilgore and Wang in Anal. Biochemistry 208, 387-392 (1993). 29/30, which was expressed in one of the systems described above and subsequently purified, was used as the enzyme. The substances were incubated with the enzyme for 60 minutes at room temperature, a concentration of 1% of the solvent DMSO not being exceeded. After adding the Bio-Rad color reagent, the optical density was measured at 595 nm in the Easy Reader EAR 400 from SLT. The 50% activity of the enzyme is derived from optical densities that were determined without the enzyme inhibitors and the activity of the En ^¬ zyms without the addition of calcium at the maximum activity.

9. Example: Platelet test for determining the cellular activity of calpain inhibitors

Calpain-mediated protein degradation in platelets has been described by ZhaozhaoLi et al. , J. Med. Chem., 1993, 36, 3472-3480. Human platelets were isolated from fresh sodium citrate blood from donors and adjusted to 10 ⁷ cells / ml in buffer (5 mM Hepes, 140 mM NaCl and 1 mg / ml BSA, pH 7.3).

Platelets (0.1 ml) are preincubated for 5 minutes with 1 μl of various concentrations of inhibitors (dissolved in DMSO). This was followed by the addition of calcium ionophore A 23187 (1 μM in the test) and calcium (5 mM in the test) and a further incubation of 5 minutes at 37 C. After a centrifugation step, the platelets were taken up in SDS-Page sample buffer, boiled for 5 minutes at 95 ° C. and the proteins were separated in an 8% gel. The breakdown of the two proteins actin binding protein (ABP) and talin was monitored by quantitative densitometry, since after the addition of calcium and ionophore these proteins disappeared and a new band in the range of 200 Kd molecular weight was formed. The half maximum enzyme activity is determined from this.

SEQUENCE LOG

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: BASF Aktiengesellschaft

(B) STREET: Carl Bosch Strasse

(C) LOCATION: Ludwigshafen

(D) FEDERAL STATE: Rhineland-Palatinate

(E) COUNTRY: Germany

(F) POSTCODE: D-67056

(ii) APPLICATION TITLE: New Calpaine, their production and use

(iii) NUMBER OF SEQUENCES: 7

(iv) COMPUTER READABLE FORM:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.25 (EPA)

(2) INFORMATION ABOUT SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2459 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNS

(iii) HYPOTHETICAL: NO

(iii) ANTISENSE: NO

(vi) ORIGINAL ORIGIN:

(A) ORGANISM: Mus musculus

(ix) FEATURES:

(A) NAME / KEY: 5 'UTR

(B) LOCATION: 1..193

(ix) FEATURES:

(A) NAME / KEY: 3 'UTR

(B) LOCATION: 2117..2459

(ix) FEATURES:

(A) NAME / KEY: CDS

(B) LOCATION: 194..2116

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CTGAAGCCCG GGGGTCCAAG TTCCAACCCC CGCCTGCGGG CTGCCGGGGT ATCATCTCCC ^{^} 60

CGCAGAGTCC CGGCCGTGGC GCGGGCTGGT CTAGCCTCCG CTCCAGTGCC CGCACTGTGC 120

TCTGCATCCC GGGAGTCCAG CTCCAGCTGC GGCGACGCGG CAGGTGCCTC CCCTTCTTGG 180

GGACGTGGTC ACC ATG TTC TCC TGC GCG AAG GCC TAT GAG GAC CAG AAC 229 Met Phe Ser Cys Ala Lys Ala Tyr Glu Asp Gin Asn 1 5 10

TAC TCG GCG CTG AAG CGG GCC TGC CTG CGC AAG AAG GTG CTG TTC GAG 277 Tyr Ser Ala Leu Lys Arg Ala Cys Leu Arg Lys Lys Val Leu Phe Glu 15 20 25

GAT CCC CTC TTC CCT GCC ACC GAC GAC TCC CTT TAC TAT AAG GGC ACC 325 Asp Pro Leu Phe Pro Ala Thr Asp Asp Ser Leu Tyr Tyr Lys Gly Thr 30 35 40

CCA GGG CCC ACA GTC AGG TGG AAG CGG CCT AAG GAT ATC TGC GAC GAT 373 Pro Gly Pro Thr Val Arg Trp Lys Arg Pro Lys Asp Ile Cys Asp Asp 45 50 55 60

CCC CGG CTC TTC GTA GAT GGC ATC AGC TCC CAT GAC CTG CAC CAG GGC 421 Pro Arg Leu Phe Val Asp Gly Ile Ser Ser His Asp Leu His Gin Gly

65 70 75

CAG GTG GGC AAC TGC TGG TTT GTG GCT GCC TGC TCA TCA CTG GCC TCC 469 Gin Val Gly Asn Cys Trp Phe Val Ala Ala Cys Ser Ser Leu Ala Ser 80 85 90

CGA GAG TCA CTC TGG CAG AAG GTC ATC CCA GAC AAG TGG CAG GAG GAA 517 Arg Glu Leu Ser Trp Gin Lys Val Pro Ile Asp Trp Lys Glu Glu Gin ^'95 100 105

TGG AAC CCC GAG AAG CCT GAC AGC TAT GCT GGC ATC TTC CAC TTC AAC 565 Trp Asn Pro Glu Lys Pro Asp Ser Tyr Ala Gly Ile Phe His Phe Asn 110 115 120

TTC TGG CGC TTT GGG GAG TGG GTG GAC GTA ATC GTC GAT GAC CGG CTG 613 Phe Trp Arg Phe Gly Glu Trp Val Asp Val Ile Val Asp Asp Arg Leu 125 130 135 140

CCC ACA GTC AAC AAC CAG CTC ATT TAC TGC CAT TCC AAC TCC AAA AAT 661 Pro Thr Val Asn Asn Gin Leu Ile Tyr Cys His Ser Asn Ser Lys Asn 145 150 155

GAG TTC TGG TGT GCC CTG GTG GAG AAG GCC TAT GCC AAG CTG GCC GGC 709 Glu Phe Trp Cys Ala Leu Val Glu Lys Ala Tyr Ala Lys Leu Ala Gly 160 165 170

TGT TAC CAG GCC CTG GAC GGA GGC AAC ACG GCC GAT GCA TTG GTG GAT 757 Cys Tyr Gin Ala Leu Asp Gly Gly Asn Thr Ala Asp Ala Leu Val Asp 175 180 185 TTC ACA GGT GGT GTT TCT GAA CCC ^' ATT GAC CTG ACC GAG GGG GAC TTG ^{^} 805 Phe Thr Gly Gly Val Ser Glu Pro Ile Asp Leu Thr Glu Gly Asp Leu 190 195 200

GCC ACT GAC GAG GCT AAG AGG AAT CAG CTC TTT GAG CGA GTG CTG AAG 853 Ala Thr Asp Glu Ala Lys Arg Asn Gin Leu Phe Glu Arg Val Leu Lys 205 210 215 220

GTG CAC AGC AGA GGC GGG CTC ATC AGT GCC TCC ATC AAG GCT GTG ACA 901 Val His Ser Arg Gly Gly Leu Ile Ser Ala Ser Ile Lys Ala Val Thr 225 230 235

GCA GCT GAC ATG GAG GCC CGC CTG GCA TGT GGC CTG GTG AAG GGC CAT 949 Ala Ala Asp Met Glu Ala Arg Leu Ala Cys Gly Leu Val Lys Gly His 240 245 250

GCA TAC GCT GTC ACC GAT GTG CGC AAG GTG CGC CTG GGC CAT GGC CTG 997 Ala Tyr Ala Val Thr Asp Val Arg Lys Val Arg Leu Gly His Gly Leu 255 260 265

CTG GCC TTC TTC AAG TCA GAG AAG CTT GAT ATG ATC CGT CTG AGG AAC 1045 Leu Ala Phe Phe Lys Ser Glu Lys Leu Asp Met Ile Arg Leu Arg Asn 270 275 280

CCC TGG GGC GAG CGG GAG TGG ACG GGG CCC TGG AGT GAC ACG TCA GAG 1093 Pro Trp Gly Glu Arg Glu Trp Thr Gly Pro Trp Ser Asp Thr Ser Glu 285 290 295 300

GAA TGG CAG AAA GTG AGC AAG AGT GAG AGG GAG AAG ATG GGC GTG ACC 1141 Glu Trp Gin Lys Val Ser Lys Ser Glu Arg Glu Lys Met Gly Val Thr 305 310 315

GTG CAG GAT GAT GGG GAA TTC TGG ATG ACC TTT GAG GAC ATG TGC CGG 1189 Val Gin Asp Asp Gly Glu Phe Trp Met Thr Phe Glu Asp Met Cys Arg 320 325 330

TAC TTT ACT GAC ATC ATT AAA TGC CGC CTG ATT AAC ACG TCC TAC CTG 1237 Tyr Phe Thr Asp Ile Ile Lys Cys Arg Leu Ile Asn Thr Ser Tyr Leu 335 340 345

AGC ATC CAT AAG ACA TGG GAG GAG GCC CGG CTG CAT GGT GCC TGG ACG 1285 Ser Ile His Lys Thr Trp Glu Glu Ala Arg Leu His Gly Ala Trp Thr 350 355 360

AGA CAT GAG GAC CCA CAG CAG AAC CGC AGT GGA GGC TGC ATC AAC CAC 1333 Arg His Glu Asp Pro Gin Gin Asn Arg Ser Gly Gly Cys Ile Asn His 365 370 375 380

AAG GAC ACT TTC TTC CAG AAC CCA CAG TAC GTA TTT GAA GTC AAG AAG 1381 Lys Asp Thr Phe Phe Gin Asn Pro Gin Tyr Val Phe Glu Val Lys Lys 385 390 395 CCA GAA GAT GAA GTG TTG ATC AGT ^" ATC CAG CAG CGG CCG AAG CGC TCA 14 ^~ 29 Pro Glu Asp Glu Val Leu Ile Ser Ile Gin Gin Arg Pro Lys Arg Ser 400 405 410

ACT CGC CGG GAG GGC AAA GGC GAG AAT CTG GCC ATT GGC TTC GAC ATC 1477 Thr Arg Arg Glu Gly Lys Gly Glu Asn Leu Ala Ile Gly Phe Asp Ile 415 420 425

TAT AAG GTG GAA GAG AAC CGC CAA TAC CGT ATG CAC AGC CTA CAG CAT 1525 Tyr Lys Val Glu Glu Asn Arg Gin Tyr Arg Met His Ser Leu Gin His 430 435 440

AAG GCC GCC AGC TCC ATC TAC ATC AAT TCC CGC AGC GTT TTT TTG AGG 1573 Lys Ala Ala Ser Ser Ile Tyr Ile Asn Ser Arg Ser Val Phe Leu Arg 445 450 455 460

ACA GAG CTG CCC GAG GGC CGC TAC GTT ATC ATC CCT ACC ACC TTT GAG 1621 Thr Glu Leu Pro Glu Gly Arg Tyr Val Ile Ile Pro Thr Thr Phe Glu 465 470 475

CCA GGC CAC ACT GGC GAG TTC CTG CTC CGA GTC TTC ACA GAT GTC CCC 1669 Pro Gly His Thr Gly Glu Phe Leu Leu Arg Val Phe Thr Asp Val Pro 480 485 490

TCC AAC TGC CGG GAA CTA CGC CTG GAT GAG CCC CCT CGG ACC TGT TGG 1717 Ser Asn Cys Arg Glu Leu Arg Leu Asp Glu Pro Pro Arg Thr Cys Trp 495 500 505

AGT TCC CTC TGT GGC TAC CCT CAG CAG GTG GCC CAG GTA CAT GTC CTG 1765 Ser Ser Leu Cys Gly Tyr Pro Gin Gin Val Ala Gin Val His Val Leu 510 515 520

GGG GCT GCT GGC CTC AAG GAC TCC CCA ACA GGA GCA AAC TCA TAT GTG 1813 Gly Ala Ala Gly Leu Lys Asp Ser Pro Thr Gly Ala Asn Ser Tyr Val 525 530 535 540

ATC ATC AAG TGT GAG GGC GAA AAG GTT CGC TCA GCT GTG CAG AGA GGG 1861 Ile Ile Lys Cys Glu Gly Glu Lys Val Arg Ser Ala Val Gin Arg Gly 545 550 555

ACC TCG ACA CCA GAG TAC AAT GTA AAA GGC ATC TTC TAT CGC AAG AAG 1909 Thr Ser Thr Pro Glu Tyr Asn Val Lys Gly Ile Phe Tyr Arg Lys Lys 560 565 570

CTG GCT CAG CCT ATC ACC GTG CAG GTT TGG AAT CAC CGA GTC CTG AAG 1957 Leu Ala Gin Pro Ile Thr Val Gin Val Trp Asn His Arg Val Leu Lys 575 580 585

GAT GAA TTC CTG GGC CAG GTG CAC CTG AAG ACT GCC CCG GAT GAC CTG 2005 Asp Glu Phe Leu Gly Gin Val His Leu Lys Thr Ala Pro Asp Asp Leu 590 595 600 CAG GAC CTC CAC ACC CTC CAT CTC CAG GAC CGC AGT AGC CGG CAG CCC 2D53 Gin Asp Leu His Thr Leu His Leu Gin Asp Arg Ser Ser Arg Gin Pro 605 610 615 620

AGT GAC CTG CCA GGC ATT GTA GCT GTG CGA GTC CTC TGC AGT GCC TCT 2101 Ser Asp Leu Pro Gly Ile Val Ala Val Arg Val Leu Cys Ser Ala Ser 625 630 635

CTC ACG GCT GTC TGACCCCAGC CTGCCTGTCC TGCCCCACTA GTCCTCACCA 2153

Leu Thr Ala Val 640

CTACTCGCAT GTCCCCACCT TGCCTGGGAC CAGCCTGGGA ACCAGACACT GGGGCCCTTT 2213

CCTCACTCTT CCACTGACCC ACTGTGTGAC CTGAAGAGAG CCCTGCCCTC TCTGAGCCTC 2273

AGTGTTTGGA GGGCCCCAAA GAATTCCCGT CTTGTGGGGG AGTTTTCTTG CCTAAGATTT 2333

AATGCAGTTC TCTCTACCCA GTGGGCGCTG CTGTTAAGGG GCCATCTGCT GAAAACGTTT 2393

CCCCAGGCCC TGCTGTCTGC CAGGAGTGCC AAGTGTCAAC TGTTTACACA CAAACTGCCA 2453

TGTCCC 2459

(2) INFORMATION ABOUT SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) L0NGE: 640 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

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

Met Phe Ser Cys Ala Lys Ala Tyr Glu Asp Gin Asn Tyr Ser Ala Leu 1 5 10 15

Lys Arg Ala Cys Leu Arg Lys Lys Val Leu Phe Glu Asp Pro Leu Phe 20 25 30

Pro Ala Thr Asp Asp Ser Leu Tyr Tyr Lys Gly Thr Pro Gly Pro Thr 35 40 45

Val Arg Trp Lys Arg Pro Lys Asp Ile Cys Asp Asp Pro Arg Leu Phe 50 55 60

Val Asp Gly Ile Ser Ser His Asp Leu His Gin Gly Gin Val Gly Asn 65 70 75 80

Cys Trp Phe Val Ala Ala Cys Ser Ser Leu Ala Ser Arg Glu Ser Leu

85 90 95 Trp Gin Lys Val Ile Pro Asp Trp Lys Glu Gin Glu Trp Asn Pro Glu 100 105 110

Lys Pro Asp Ser Tyr Ala Gly Ile Phe His Phe Asn Phe Trp Arg Phe 115 120 125

Gly Glu Trp Val Asp Val Ile Val Asp Asp Arg Leu Pro Thr Val Asn 130 135 140

Asn Gin Leu Ile Tyr Cys His Ser Asn Ser Lys Asn Glu Phe Trp Cys 145 150 155 160

Ala Leu Val Glu Lys Ala Tyr Ala Lys Leu Ala Gly Cys Tyr Gin Ala 165 170 175

Leu Asp Gly Gly Asn Thr Ala Asp Ala Leu Val Asp Phe Thr Gly Gly 180 185 190

Val Ser Glu Pro Ile Asp Leu Thr Glu Gly Asp Leu Ala Thr Asp Glu 195 200 205

Ala Lys Arg Asn Gin Leu Phe Glu Arg Val Leu Lys Val His Ser Arg 210 215 220

Gly Gly Leu Ile Ser Ala Ser Ile Lys Ala Val Thr Ala Ala Asp Met 225 230 235 240

Glu Ala Arg Leu Ala Cys Gly Leu Val Lys Gly His Ala Tyr Ala Val 245 250 255

Thr Asp Val Arg Lys Val Arg Leu Gly His Gly Leu Leu Ala Phe Phe 260 265 270

Lys Ser Glu Lys Leu Asp Met Ile Arg Leu Arg Asn Pro Trp Gly Glu 275 280 285

Arg Glu Trp Thr Gly Pro Trp Ser Asp Thr Ser Glu Glu Trp Gin Lys 290 295 300

Val Ser Lys Ser Glu Arg Glu Lys Met Gly Val Thr Val Gin Asp Asp 305 310 315 320

Gly Glu Phe Trp Met Thr Phe Glu Asp Met Cys Arg Tyr Phe Thr Asp 325 330 335

Ile Ile Lys Cys Arg Leu Ile Asn Thr Ser Tyr Leu Ser Ile His Lys 340 345 350

Thr Trp Glu Glu Ala Arg Leu His Gly Ala Trp Thr Arg His Glu Asp 355 360 365

Pro Gin Gin Asn Arg Ser Gly Gly Cys Ile Asn His Lys Asp Thr Phe 370 375 380 Phe Gin Asn Pro Gin Tyr Val Phe Glu Val Lys Lys Pro Glu Asp Glu 385 390 395 400

Val Leu Ile Ser Ile Gin Gin Arg Pro Lys Arg Ser Thr Arg Arg Glu 405 410 415

Gly Lys Gly Glu Asn Leu Ala Ile Gly Phe Asp Ile Tyr Lys Val Glu 420 425 430

Glu Asn Arg Gin Tyr Arg Met His Ser Leu Gin His Lys Ala Ala Ser 435 440 445

Ser Ile Tyr Ile Asn Ser Arg Ser Val Phe Leu Arg Thr Glu Leu Pro 450 455 460

Glu Gly Arg Tyr Val Ile Ile Pro Thr Thr Phe Glu Pro Gly His Thr 465 470 475 480

Gly Glu Phe Leu Leu Arg Val Phe Thr Asp Val Pro Ser Asn Cys Arg 485 490 495

Glu Leu Arg Leu Asp Glu Pro Pro Arg Thr Cys Trp Ser Ser Leu Cys 500 505 510

Gly Tyr Pro Gin Gin Val Ala Gin Val His Val Leu Gly Ala Ala Gly 515 520 525

Leu Lys Asp Ser Pro Thr Gly Ala Asn Ser Tyr Val Ile Ile Lys Cys 530 535 540

Glu Gly Glu Lys Val Arg Ser Ala Val Gin Arg Gly Thr Ser Thr Pro 545 550 555 560

Glu Tyr Asn Val Lys Gly Ile Phe Tyr Arg Lys Lys Leu Ala Gin Pro 565 570 575

Ile Thr Val Gin Val Trp Asn His Arg Val Leu Lys Asp Glu Phe Leu 580 585 590

Gly Gin Val His Leu Lys Thr Ala Pro Asp Asp Leu Gin Asp Leu His 595 600 605

Thr Leu His Leu Gin Asp Arg Ser Ser Arg Gin Pro Ser Asp Leu Pro 610 615 620

Gly Ile Val Ala Val Arg Val Leu Cys Ser Ala Ser Leu Thr Ala Val 625 630 635 640

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1743 base pairs

(B) TYPE: nucleic acid (C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO

(vi) ORIGINAL ORIGIN:

(A) ORGANISM: Mus musculus

(B) STEM: balb / c

(ix) FEATURES:

(A) NAME / KEY: 5 'UTR

(B) LOCATION: 1..193

(ix) FEATURES:

(A) NAME / KEY: 3 'UTR

(B) LOCATION: 1736..1743

(ix) FEATURES:

(A) NAME / KEY: CDS

(B) LOCATION: 194..1735

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CTGAAGCCCG GGGGTCCAAG TTCCAACCCC CGCCTGCGGG CTGCCGGGGT ATCATCTCCC 60

CGCAGAGTCC CGGCCGTGGC GCGGGCTGGT CTAGCCTCCG CTCCAGTGCC CGCACTGTGC 120

TCTGCATCCC GGGAGTCCAG CTCCAGCTGC GGCGACGCGG CAGGTGCCTC CCCTTCTTGG 180

65 70 75 CAG GTG GGC AAC TGC TGG TTT GTG GCT GCC TGC TCA TCA CTG GCC TCC 469 Gin Val Gly Asn Cys Trp Phe Val Ala Ala Cys Ser Ser Leu Ala Ser 80 85 90

CGA GAG TCA CTC TGG CAG AAG GTC ATC CCA GAC TGG AAG GAG CAG GAA 517 Arg Glu Ser Leu Trp Gin Lys Val Ile Pro Asp Trp Lys Glu Gin Glu 95 100 105

TGT TAC CAG GCC CTG GAC GGA GGC AAC ACG GCC GAT GCA TTG GTG GAT 757 Cys Tyr Gin Ala Leu Asp Gly Gly Asn Thr Ala Asp Ala Leu Val Asp 175 180 185

TTC ACA GGT GGT GTT TCT GAA CCC ATT GAC CTG ACC GAG GGG GAC TTG 805 Phe Thr Gly Gly Val Ser Glu Pro Ile Asp Leu Thr Glu Gly Asp Leu 190 195 200

GCA GCT GAC ATG GAG GCC CGC CTG GCA TGT GGC CTG GTG AAG GGC CAT 49 Ala Ala Asp Met Glu Ala Arg Leu Ala Cys Gly Leu Val Lys Gly His 240 245 250

CTG GCC TTC TTC AAG TCA GAG AAG CTT GAT ATG ATC CGT CTG AGG AAC 1045 Leu Ala Phe Phe Lys Ser Glu Lys Leu Asp Met Ile Arg Leu Arg Asn 270 275 280 CCC TGG GGC GAG CGG GAG TGG ACG GGG CCC TGG AGT GAC ACG TCA GAG 1O93

Pro Trp Gly Glu Arg Glu Trp Thr Gly Pro Trp Ser Asp Thr Ser Glu 285 290 295 300

AAG GAC ACT TTC TTC CAG AAC CCA CAG TAC GTA TTT GAA GTC AAG AAG 1381 Lys Asp Thr Phe Phe Gin Asn Pro Gin Tyr Val Phe Glu Val Lys Lys 385 390 395

CCA GAA GAT GAA GTG TTG ATC AGT ATC CAG CAG CGG CCG AAG CGC TCA 1429 Pro Glu Asp Glu Val Leu Ile Ser Ile Gin Gin Arg Pro Lys Arg Ser 400 405 410

CCA GGC CAC ACT GGC GAG TTC CTG CTC CGA GTC TTC ACA GAT GTC CCC 1669 Pro Gly His Thr Gly Glu Phe Leu Leu Arg Val Phe Thr Asp Val Pro 480 485 490 TCC AAC TGC CGG TGT GTG GGG GCT AGG GCT AGT GAC CGC ATG CAT ATA 1717 Ser Asn Cys Arg Cys Val Gly Ala Arg Ala Ser Asp Arg Met His Ile 495 500 505

TAC CCC ATG CTG GGC TAGATTTTAA C 1743

Tyr Pro Met Leu Gly 510

(2) INFORMATION ABOUT SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 513 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Phe Ser Cys Ala Lys Ala Tyr Glu Asp Gin Asn Tyr Ser Ala Leu 1 5 10 15

Lys Arg Ala Cys Leu Arg Lys Lys Val Leu Phe Glu Asp Pro Leu Phe 20 25 30

Pro Ala Thr Asp Asp Ser Leu Tyr Tyr Lys Gly Thr Pro Gly Pro Thr 35 40 45

Val Arg Trp Lys Arg Pro Lys Asp Ile Cys Asp Asp Pro Arg Leu Phe 50 55 60

Val Asp Gly Ile Ser Ser His Asp Leu His Gin Gly Gin Val Gly Asn 65 70 75 80

Cys Trp Phe Val Ala Ala Cys Ser Ser Leu Ala Ser Arg Glu Ser Leu

85 90 95

Trp Gin Lys Val Ile Pro Asp Trp Lys Glu Gin Glu Trp Asn Pro Glu 100 105 110

Lys Pro Asp Ser Tyr Ala Gly Ile Phe His Phe Asn Phe Trp Arg Phe 115 120 125

Gly Glu Trp Val Asp Val Ile Val Asp Asp Arg Leu Pro Thr Val Asn 130 135 140

Asn Gin Leu Ile Tyr Cys His Ser Asn Ser Lys Asn Glu Phe Trp Cys 145 150 155 160

Ala Leu Val Glu Lys Ala Tyr Ala Lys Leu Ala Gly Cys Tyr Gin Ala 165 170 175

Leu Asp Gly Gly Asn Thr Ala Asp Ala Leu Val Asp Phe Thr Gly Gly 180 185 190 Val Ser Glu Pro Ile Asp Leu Thr Glu Gly Asp Leu Ala Thr Asp Glu 195 200 205

Ala Lys Arg Asn Gin Leu Phe Glu Arg Val Leu Lys Val His Ser Arg 210 215 220

Gly Gly Leu Ile Ser Ala Ser Ile Lys Ala Val Thr Ala Ala Asp Met 225 230 235 240

Glu Ala Arg Leu Ala Cys Gly Leu Val Lys Gly His Ala Tyr Ala Val 245 250 255

Thr Asp Val Arg Lys Val Arg Leu Gly His Gly Leu Leu Ala Phe Phe 260 265 270

Lys Ser Glu Lys Leu Asp Met Ile Arg Leu Arg Asn Pro Trp Gly Glu 275 280 285

Arg Glu Trp Thr Gly Pro Trp Ser Asp Thr Ser Glu Glu Trp Gin Lys 290 295 300

Val Ser Lys Ser Glu Arg Glu Lys Met Gly Val Thr Val Gin Asp Asp 305 310 315 320

Gly Glu Phe Trp Met Thr Phe Glu Asp Met Cys Arg Tyr Phe Thr Asp 325 330 335

Ile Ile Lys Cys Arg Leu Ile Asn Thr Ser Tyr Leu Ser Ile His Lys 340 345 350

Thr Trp Glu Glu Ala Arg Leu His Gly Ala Trp Thr Arg His Glu Asp 355 360 365

Pro Gin Gin Asn Arg Ser Gly Gly Cys Ile Asn His Lys Asp Thr Phe 370 375 380

Phe Gin Asn Pro Gin Tyr Val Phe Glu Val Lys Lys Pro Glu Asp Glu 385 390 395 400

Val Leu Ile Ser Ile Gin Gin Arg Pro Lys Arg Ser Thr Arg Arg Glu 405 410 415

Gly Lys Gly Glu Asn Leu Ala Ile Gly Phe Asp Ile Tyr Lys Val Glu 420 425 430

Glu Asn Arg Gin Tyr Arg Met His Ser Leu Gin His Lys Ala Ala Ser 435 440 445

Ser Ile Tyr Ile Asn Ser Arg Ser Val Phe Leu Arg Thr Glu Leu Pro 450 455 460

Glu Gly Arg Tyr Val Ile Ile Pro Thr Thr Phe Glu Pro Gly His Thr 465 470 475 480 Gly Glu Phe Leu Leu Arg Val Phe Thr Asp Val Pro Ser Asn Cys Arg

485 490 495

Cys Val Gly Ala Arg Ala Ser Asp Arg Met His Ile Tyr Pro Met Leu 500 505 510

Gly

(2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 504 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO

(vi) ORIGINAL ORIGIN:

(A) ORGANISM: Mus musculus

(B) STEM: ES E14

(vii) DIRECT ORIGIN: (B) CLON: 29/30

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..33

(ix) FEATURES:

(A) NAME / KEY: intron

(B) LOCATION: 34..440

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 441..504

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

CGAGCGGGAG TGGACGGGCC CCTGGAGTGA CACGTGAGGC TCACCAGGGT TGGGGCTGGG 60

TATGGGCACA GAGGCAAGGA CAAGCGGTGA CACTGGACTG GGCCTTGCAG GGTCTGGGAG 120

AGATGCTCTG AGGAAAAAAT GGGAGACTTA CTTTCCAGTG TAAGTGTGGT GCTTGGGGGG 180

TAGGTTCATC AAGGACAGTG GCCAGAAGTG TGGCATGCTT TGTACGTGGA CAATGGCGCC 240

TCACCAGCTT TATTCCCTGA CTTCATAGCC TTAGCATAAA GGAAGATCAC AGTTCCTAGT 300 GGGAGAGAAC AGAGGCTTCT TAGCAGGGCT GGGCATGGCC TCCAGGTCTC TACCCACAGT J60

GCTCTGCAGG CGGCTTGGTC CAGAGCTCTC CCTTGGGCCA CTCCTCTTAT CCCGTTCCCT 420

CCCTGATACT CACTCCCCAG GTCAGAGGAA TGGCAGAAAG TGAGCAAGAG TGAGAGGGAG 480

AAGATGGGCG TGACCGTGCA GGAT 504 (2) INFORMATION ON SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1975 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO

(vi) ORIGINAL ORIGIN:

(A) ORGANISM: Homo sapiens

(vii) DIRECT ORIGIN:

(B) CLON: nCL-3

(ix) FEATURES:

(A) NAME / KEY: 5 'UTR

(B) LOCATION: 1..43

(ix) FEATURES:

(A) NAME / KEY: 3 'UTR

(B) LOCATION: 1964-1975

(ix) FEATURES:

(A) NAME / KEY: CDS

(B) LOCATION: 44..1963

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

ACTCACTATA GGGCTCGAGC GGCCGCCCGG GCAGGTAGCC ACC ATG TTC TCG TGT 55

Met Phe Ser Cys 1

GTG AAG CCC TAT GAG GAC CAG AAC TAC TCA GCC CTG AGG CGG GAC TGC 103 Val Lys Pro Tyr Glu Asp Gin Asn Tyr Ser Ala Leu Arg Arg Asp Cys 5 10 15 20

CGG CGC AGG AAG GTG CTC TTC GAG GAC CCC CTC TTC CCC GCC ACT GAC 151 Arg Arg Arg Lys Val Leu Phe Glu Asp Pro Leu Phe Pro Ala Thr Asp

25 30 35 GAC TCA CTC TAC TAT AAG GGC ACG CCG GGG CCC GCC GTC AGG CGG AAG r99 Asp Ser Leu Tyr Tyr Lys Gly Thr Pro Gly Pro Ala Val Arg Arg Lys 40 45 50

CGA CCC AAG GGC ATC TGC GAG GAC CCC CGC CTC TTT GTG GAT GGC ATC 247 Arg Pro Lys Gly Ile Cys Glu Asp Pro Arg Leu Phe Val Asp Gly Ile 55 60 65

AGC TCC CAC GAC CTG CAC CAG GGC CAG GTG GGC AAC TGC TGG TTT GTG 295 Ser Ser His Asp Leu His Gin Gly Gin Val Gly Asn Cys Trp Phe Val 70 75 80

GCA GCC TGC TCG TCA CTT GCC TCC CGG GAG TCG CTG TGG CAA AAG GTC 343 Ala Ala Cys Ser Ser Leu Ala Ser Arg Glu Ser Leu Trp Gin Lys Val 85 90 95 100

ATC CCA GAC TGG AAG GAG CAG GAA TGG GAC CCC GAA AAG CCC AAC GCC 391 Ile Pro Asp Trp Lys Glu Gin Glu Trp Asp Pro Glu Lys Pro Asn Ala 105 110 115

TAC GCG GGC ATC TTC CAC TTC CAC TTC TGG CGC TTC GGG GAA TGG GTG 439 Tyr Ala Gly Ile Phe His Phe His Phe Trp Arg Phe Gly Glu Trp Val 120 125 130

GAC GTG GTC ATC GAT GAC CGG CTG CCC ACA GTC AAC AAC CAG CTC ATC 487 Asp Val Val Ile Asp Asp Arg Leu Pro Thr Val Asn Asn Gin Leu Ile 135 140 145

TAC TGC CAC TCC AAC TCC CGC AAT GAG TTT TGG TGC GCC CTA GTG GAG 535 Tyr Cys His Ser Asn Ser Arg Asn Glu Phe Trp Cys Ala Leu Val Glu 150 155 160

AAG GCC TAT GCC AAA CTG GCA GGC TGT TAC CAG GCC CTG GAT GGA GGC 583 Lys Ala Tyr Ala Lys Leu Ala Gly Cys Tyr Gin Ala Leu Asp Gly Gly 165 170 175 180

AAC ACA GCA GAC GCA CTG GTG GAC TTC ACG GGT GGT GTT TCT GAG CCC 631 Asn Thr Ala Asp Ala Leu Val Asp Phe Thr Gly Gly Val Ser Glu Pro 185 190 195

ATC GAC CTG ACC GAG GGT GAC TTT GCC AAC GAT GAG ACT AAG AGG AAC 679 Ile Asp Leu Thr Glu Gly Asp Phe Ala Asn Asp Glu Thr Lys Arg Asn 200 205 210

CAG CTC TTT GAG CGC ATG TTA AAG GTG CAC AGC CGG GGC GGC CTC ATC 727 Gin Leu Phe Glu Arg Met Leu Lys Val His Ser Arg Gly Gly Leu Ile 215 220 225

AGT GCC TCC ATC AAG GCA GTG ACA GCA GCT GAC ATG GAG GCC CGC CTG 775 Ser Ala Ser Ile Lys Ala Val Thr Ala Ala Asp Met Glu Ala Arg Leu 230 235 240 GCG TGC GGC CTG GTA AAG GGC CAC GCA TAC GCC GTC ACT GAT GTG CGC 823 Ala Cys Gly Leu Val Lys Gly His Ala Tyr Ala Val Thr Asp Val Arg 245 250 255 260

AAG GTG CGC CTG GGC CAC GGC CTA CTG GCC TTC TTC AAG TCA GAG AAG 871 Lys Val Arg Leu Gly His Gly Leu Leu Ala Phe Phe Lys Ser Glu Lys 265 270 275

TTG GAC ATG ATC CGC CTG CGC AAC CCC TGG GGC GAG CGG GAG TGG AAC 919 Leu Asp Met Ile Arg Leu Arg Asn Pro Trp Gly Glu Arg Glu Trp Asn 280 285 290

GGG CCC TGG AGT GAC ACC TCG GAG GAG TGG CAG AAA GTG AGC AAG AGT 967 Gly Pro Trp Ser Asp Thr Ser Glu Glu Trp Gin Lys Val Ser Lys Ser 295 300 305

GAG CGG GAG AAG ATG GGT GTG ACC GTG CAG GAC GAC GGT GAG TTC TGG 1015 Glu Arg Glu Lys Met Gly Val Thr Val Gin Asp Asp Gly Glu Phe Trp 310 315 320

ATG ACC TTC GAG GAC GTG TGC CGG TAC TTC ACG GAC ATC ATC AAG TGC 1063 Met Thr Phe Glu Asp Val Cys Arg Tyr Phe Thr Asp Ile Ile Lys Cys 325 330 335 340

CGC GTG ATC AAC ACA TCC CAC CTG AGC ATC CAC AAG ACG TGG GAG GAG 1111 Arg Val Ile Asn Thr Ser His Leu Ser Ile His Lys Thr Trp Glu Glu 345 350 355

GCC CGG CTG CAT GGC GCC TGG ACG CTG CAT GAG GAC CCG CGA CAG AAC 1159 Ala Arg Leu His Gly Ala Trp Thr Leu His Glu Asp Pro Arg Gin Asn 360 365 370

CGC GGT GGC GGC TGC ATC AAC CAC AAG GAC ACC TTC TTC CAG AAC CCA 1207 Arg Gly Gly Gly Cys Ile Asn His Lys Asp Thr Phe Phe Gin Asn Pro 375 380 385

CAG TAC ATC TTC GAA GTC AAG AAG CCA GAA GAT GAA GTC CTG ATC TGT 1255 Gin Tyr Ile Phe Glu Val Lys Lys Pro Glu Asp Glu Val Leu Ile Cys 390 395 400

ATC CAG CAG CGG CCA AAG CGG TCT ACG CGC CGG GAG GGC AAG GGT GAG 1303 Ile Gin Gin Arg Pro Lys Arg Ser Thr Arg Arg Glu Gly Lys Gly Glu 405 410 415 420

AAC CTG GCC ATT GGC TTT GAC ATC TAC AAG GTG GAG GAG AAC CGC CAG 1351 Asn Leu Ala Ile Gly Phe Asp Ile Tyr Lys Val Glu Glu Asn Arg Gin 425 430 435

TAC CGC ATG CAC AGC CTG CAG CAC AAG GCC GCC AGC TCC ATC TAC ATC 1399 Tyr Arg Met His Ser Leu Gin His Lys Ala Ala Ser Ser Ile Tyr Ile 440 445 450 AAC TCA CGC AGC GTC TTC CTG CGC ACC GAC CAG CCC GAG GGC CGC TAT 1447 Asn Ser Arg Ser Val Phe Leu Arg Thr Asp Gin Pro Glu Gly Arg Tyr 455 460 465

GTC ATC ATC CCC ACA ACC TTC GAG CCA GGC CAC ACT GGC GAG TTC CTG 1495 Val Ile Ile Pro Thr Thr Phe Glu Pro Gly His Thr Gly Glu Phe Leu 470 475 480

CTC CGA GTC TTC ACT GAT GTG CCC TCC AAC TGC CGG GAG CTG CGC CTG 1543 Leu Arg Val Phe Thr Asp Val Pro Ser Asn Cys Arg Glu Leu Arg Leu 485 490 495 500

GAT GAG CCC CCA CAC ACC TGC TGG AGC TCC CTC TGT GGC TAC CCC CAG 1591 Asp Glu Pro Pro His Thr Cys Trp Ser Ser Leu Cys Gly Tyr Pro Gin 505 510 515

CTG GTG ACC CAG GTA CAT GTC CTG GGA GCT GCT GGC CTC AAG GAC TCC 1639 Leu Val Thr Gin Val His Val Leu Gly Ala Ala Gly Leu Lys Asp Ser 520 525 530

CCA ACA GGG GCT AAC TCT TAT GTG ATC ATC AAG TGT GAG GGA GAC AAA 1687 Pro Thr Gly Ala Asn Ser Tyr Val Ile Ile Lys Cys Glu Gly Asp Lys 535 540 545

GTC CGC TCG GCT GTG CAG AAG GGC ACC TCC ACA CCA GAG TAC AAT GTG 1735 Val Arg Ser Ala Val Gin Lys Gly Thr Ser Thr Pro Glu Tyr Asn Val 550 555 560

AAA GGC ATC TTC TAC CGC AAG AAG CTG AGC CAG CCC ATC ACT GTA CAG 1783 Lys Gly Ile Phe Tyr Arg Lys Lys Leu Ser Gin Pro Ile Thr Val Gin 565 570 575 580

GTC TGG AAC CAC CGA GTG CTG AAG GAT GAA TTT CTG GGC CAG GTG CAC 1831 Val Trp Asn His Arg Val Leu Lys Asp Glu Phe Leu Gly Gin Val His 585 590 595

CTA AAG GCT GAC CCG GAC AAC CTC CAG GCC CTG CAT ACC CTC CAC CTC 1879 Leu Lys Ala Asp Pro Asp Asn Leu Gin Ala Leu His Thr Leu His Leu 600 605 610

CGG GAC CGA AAT AGC CGG CAG CCC AGC AAC CTG CCA GGC ACT GTG GCC 1927 Arg Asp Arg Asn Ser Arg Gin Pro Ser Asn Leu Pro Gly Thr Val Ala 615 620 625

GTG CAC ATT CTC AGC AGC ACC TCT CTC ACG GCT GTC TGACTCGAGC 1973

Val His Ile Leu Ser Ser Thr Ser Leu Thr Ala Val 630 635 640

TA 1975

(2) INFORMATION ABOUT SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 640 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Met Phe Ser Cys Val Lys Pro Tyr Glu Asp Gin Asn Tyr Ser Ala Leu 1 5 10 15

Arg Arg Asp Cys Arg Arg Arg Lys Val Leu Phe Glu Asp Pro Leu Phe 20 25 30

Pro Ala Thr Asp Asp Ser Leu Tyr Tyr Lys Gly Thr Pro Gly Pro Ala 35 40 45

Val Arg Arg Lys Arg Pro Lys Gly Ile Cys Glu Asp Pro Arg Leu Phe 50 55 60

Val Asp Gly Ile Ser Ser His Asp Leu His Gin Gly Gin Val Gly Asn 65 70 75 80

Cys Trp Phe Val Ala Ala Cys Ser Ser Leu Ala Ser Arg Glu Ser Leu

85 90 95

Trp Gin Lys Val Ile Pro Asp Trp Lys Glu Gin Glu Trp Asp Pro Glu 100 105 110

Lys Pro Asn Ala Tyr Ala Gly Ile Phe His Phe His Phe Trp Arg Phe 115 120 125

Gly Glu Trp Val Asp Val Val Ile Asp Asp Arg Leu Pro Thr Val Asn 130 135 140

Asn Gin Leu Ile Tyr Cys His Ser Asn Ser Arg Asn Glu Phe Trp Cys 145 150 155 160

Ala Leu Val Glu Lys Ala Tyr Ala Lys Leu Ala Gly Cys Tyr Gin Ala 165 170 175

Leu Asp Gly Gly Asn Thr Ala Asp Ala Leu Val Asp Phe Thr Gly Gly 180 185 190

Val Ser Glu Pro Ile Asp Leu Thr Glu Gly Asp Phe Ala Asn Asp Glu 195 200 205

Thr Lys Arg Asn Gin Leu Phe Glu Arg Met Leu Lys Val His Ser Arg 210 215 220

Gly Gly Leu Ile Ser Ala Ser Ile Lys Ala Val Thr Ala Ala Asp Met 225 230 235 240 Glu Ala Arg Leu Ala Cys Gly Leu Val Lys Gly His Ala Tyr Ala Val 245 250 255

Thr Asp Val Arg Lys Val Arg Leu Gly His Gly Leu Leu Ala Phe Phe 260 265 270

Lys Ser Glu Lys Leu Asp Met Ile Arg Leu Arg Asn Pro Trp Gly Glu 275 280 285

Arg Glu Trp Asn Gly Pro Trp Ser Asp Thr Ser Glu Glu Trp Gin Lys 290 295 300

Val Ser Lys Ser Glu Arg Glu Lys Met Gly Val Thr Val Gin Asp Asp 305 310 315 320

Gly Glu Phe Trp Met Thr Phe Glu Asp Val Cys Arg Tyr Phe Thr Asp 325 330 335

Ile Ile Lys Cys Arg Val Ile Asn Thr Ser His Leu Ser Ile His Lys 340 345 350

Thr Trp Glu Glu Ala Arg Leu His Gly Ala Trp Thr Leu His Glu Asp 355 360 365

Pro Arg Gin Asn Arg Gly Gly Gly Cys Ile Asn His Lys Asp Thr Phe 370 375 380

Phe Gin Asn Pro Gin Tyr Ile Phe Glu Val Lys Lys Pro Glu Asp Glu 385 390 395 400

Val Leu Ile Cys Ile Gin Gin Arg Pro Lys Arg Ser Thr Arg Arg Glu 405 410 415

Gly Lys Gly Glu Asn Leu Ala Ile Gly Phe Asp Ile Tyr Lys Val Glu 420 425 430

Glu Asn Arg Gin Tyr Arg Met His Ser Leu Gin His Lys Ala Ala Ser 435 440 445

Ser Ile Tyr Ile Asn Ser Arg Ser Val Phe Leu Arg Thr Asp Gin Pro 450 455 460

Glu Gly Arg Tyr Val Ile Ile Pro Thr Thr Phe Glu Pro Gly His Thr 465 470 475 480

Gly Glu Phe Leu Leu Arg Val Phe Thr Asp Val Pro Ser Asn Cys Arg 485 490 495

Glu Leu Arg Leu Asp Glu Pro Pro His Thr Cys Trp Ser Ser Leu Cys 500 505 510

Gly Tyr Pro Gin Leu Val Thr Gin Val His Val Leu Gly Ala Ala Gly 515 520 525 Leu Lys Asp Ser Pro Thr Gly Ala Asn Ser Tyr Val Ile Ile Lys Cys 530 535 540

Glu Gly Asp Lys Val Arg Ser Ala Val Gin Lys Gly Thr Ser Thr Pro 545 550 555 560

Glu Tyr Asn Val Lys Gly Ile Phe Tyr Arg Lys Lys Leu Ser Gin Pro 565 570 575

Ile Thr Val Gin Val Trp Asn His Arg Val Leu Lys Asp Glu Phe Leu 580 585 590

Gly Gin Val His Leu Lys Ala Asp Pro Asp Asn Leu Gin Ala Leu His 595 600 605

Thr Leu His Leu Arg Asp Arg Asn Ser Arg Gin Pro Ser Asn Leu Pro 610 615 620

Gly Thr Val Ala Val His Ile Leu Ser Ser Thr Ser Leu Thr Ala Val 625 630 635 640

Claims

claims

1. nCL-3-Calpaingen with the sequence SEQ ID NO: 1 and its allelic variants, which have a homology of 60 to 100% at the derived amino acid level, analogs or derivatives.

2. nCL-3-calpaingen according to claim 1, characterized in that it is a shortened gene sequence in the analog.

3. nCL-3 calpaingen according to claim 1 encoded by the sequence SEQ ID NO: 6.

4. gene construct containing an nCL-3 calpaingen according to claims 1 to 3, which is functionally linked to one or more regulatory signals to increase gene expression.

5. Amino acid sequences encoded by nCL-3 genes according to claim 1.

6. A method for identifying calpain inhibitors, wherein a calpain coded by a sequence according to claim 1 is isolated from tissues or cells in which the enzyme nCL-3 is expressed, and the inhibition of the cleavage of a substrate of the enzyme nCL-3 and in at least a further test measures the inhibition of the cleavage of a substrate of the enzymes calpain I and / or II by test substances and selects the test substances which show an inhibitory effect against at least one of the calpains.

7. The method according to claim 6, characterized in that one selects the test substances that do not inhibit the enzyme nCL-3, but the enzymes Calpain I and / or II.

8. The method according to claim 6, characterized in that one selects the test substances which inhibit the enzyme nCL-3, but not the enzymes Calpain I and / or II.

9. The method according to claims 6 to 8, characterized in that one selects the test substances that pass through the cell membrane in cellular systems.

10. A process for the preparation of the enzyme nCL-3, αaαurcn indicates that a copy of the gene sequences for nCL-3 according to claim 1 is cloned into a vector and the gene for the enzyme nCL-3 in a host organism corresponding to the vector

5 expressed and then the enzyme isolated from the host organism.

11. The method according to claim 10, characterized in that one uses a vector in prokaryotic or eukaryonic

10 cells allow expression of the gene for nCL-3.

12. The method according to claim 10, characterized in that bacteria, fungal or animal cells are used as the host organism.

15

13. The method according to claim 10, characterized in that one uses baculoviruses as vector and insect cells as host organism.

14. Use of a calpain inhibitor identifiable according to claims 6 to 9 for the manufacture of medicaments for the treatment of diseases in which a calpain malfunction is present.

15. Use of a calpain inhibitor according to claim 14 for the manufacture of medicaments for the treatment of diseases selected from the group of cardiovascular, immunological, inflammatory, allergic, neurological, neurodegenerative or oncological diseases.

30

16. Use of the Calpain nCL-3 according to claim 5 in test systems.

17. Use of the calpain nCL-3 according to claim 5 for the production of antibodies.

18. Use of a gene sequence according to claim 1 for the production of antisense mRNA.

40 19. Use of the antisense mRNA according to claim 18 for the manufacture of medicaments for the treatment of diseases in which a calpain malfunction is present.

20. Use of a calpaing gene according to claim 1 for the diagnosis 45 of diseases or in gene therapy.