EP0942995A1 - Novel calpaines, production and use thereof - Google Patents

Novel calpaines, production and use thereof

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Publication number
EP0942995A1
EP0942995A1 EP97952002A EP97952002A EP0942995A1 EP 0942995 A1 EP0942995 A1 EP 0942995A1 EP 97952002 A EP97952002 A EP 97952002A EP 97952002 A EP97952002 A EP 97952002A EP 0942995 A1 EP0942995 A1 EP 0942995A1
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Prior art keywords
leu
arg
ncl
val
glu
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German (de)
French (fr)
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Thomas Boehm
Neil T. Dear
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Abbott GmbH and Co KG
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BASF SE
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Priority claimed from DE1996150142 external-priority patent/DE19650142A1/en
Priority claimed from DE1997118248 external-priority patent/DE19718248A1/en
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Publication of EP0942995A1 publication Critical patent/EP0942995A1/en
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
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    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96466—Cysteine endopeptidases (3.4.22)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • 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 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.
  • 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.
  • 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.
  • CalpA calpain-homologous protein
  • 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.
  • 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).
  • 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.
  • 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.
  • calpastatin which consists of approximately 700 amino acids
  • calpastatin which consists of approximately 700 amino acids
  • calpastatin which consists of approximately 700 amino acids
  • calpastatin which consists of approximately 700 amino acids
  • calpastatin which consists of approximately 700 amino acids
  • calpastatin which consists of approximately 700 amino acids
  • calpastatin which consists of approximately 700 amino acids
  • calpastatin which consists of approximately 700 amino acids
  • calpastatin which consists of approximately 700 amino acids
  • calpastatin which
  • 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.
  • Calpain-specific primers 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.
  • This clone codes for a gene whose gene product was named nCL-3 as the new calpain.
  • 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 ⁇ Calpain, 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.
  • the intron shown in sequence SEQ ID NO: 5 was determined by comparison with the cDNA.
  • 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 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.
  • nCL-3 The greatest homology exists between nCL-3 and the human partial sequence ⁇ ST01106.
  • the partial sequence EST01106 was obtained from a hippocampus library. None 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 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 ⁇ -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.
  • 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), ⁇ (P07384), p94 (P20807); Rats m (Q07009), nCL-2 (D14480), p94 (P16259); Mouse p94 (X92523); Chickens m (D38026), ⁇ (D38027), ⁇ / m (P00789), p94 (D38028); Nematodes tra-3 (U12921); Drosophila Calp A (Q11002) and Dm (X78555), Schistosoma (P27730).
  • 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.
  • 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 ⁇ / m-Calpaingen indicates that the published sequence around this interface is not in agreement with the original cDNA sequence.
  • nCL-3 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.
  • nCL-3 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.
  • 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.
  • mice orthologist nCL-3 gene was localized between 44 and 53 cM on mouse chromosome 7.
  • 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.
  • 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.
  • 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.
  • Cathepsin B inhibition was determined analogously to a method by S. Hasnain et al., J. Biol. Chem. 1993, 268, 235-240.
  • cathepsin B human liver cathepsin B from Calbiochem, diluted to 5 units in 500 ⁇ M buffer
  • an inhibitor solution prepared from the chemical substance to be tested
  • a microbial or vegetable extract and DSMO final concentration: 100 ⁇ M up to 0.01 ⁇ M
  • 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.
  • 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.
  • 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.
  • ABP actin binding protein
  • Tissue parts such as brain sections or cell cultures are also suitable for testing membrane permeability.
  • nCL-3 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 ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • nCL-3 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.
  • 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.
  • 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
  • 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 ⁇ .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • vectors which are derived from pBR or pUC or shuttle vectors are particularly suitable.
  • PBluescript is very particularly suitable.
  • 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.
  • nCL-3 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.
  • 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.
  • 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
  • Genomic DNA from mouse cells ES E14 was used to clone the nCL-3 gene with the sequence SEQ ID NO: 1.
  • 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.).
  • the pBluescript vector with the cloned-in nCL-3 gene, the Escherichia coli strain DH10B according to Maniatis et al.
  • 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 ⁇ g 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
  • mRNA was isolated from dE12 embryos and from mouse, kidney, heart, lung, brain, thymus and small intestine tissue.
  • 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.
  • 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 ⁇ l and again precipitated in the cold with Na acetate / ethanol and washed with 70% ethanol, centrifuged (19000 ⁇ g, 4 ° C.) and dissolved in 300 ⁇ l water.
  • the mRNA concentration at 250 nm was then determined in a photometer and in an agarose gel with a 5 ⁇ l mRNA sample against a reference.
  • the cDNA was produced according to a protocol from GIBCO as follows:
  • 2 ⁇ l oligo (dT) and 12 ⁇ l DEPC dH 0 were added to 5 ⁇ g mRNA (isolated according to the method described above). This mixture was incubated at 70 ° C. for 10 minutes and then placed on ice. 2 ⁇ l 10 ⁇ buffer, 2 ⁇ l 25 mM MgCl 2 , 1 ⁇ l 10 mM dNTPs, 2 ⁇ l 0.1 M DTT were added to the sample in the order given. After 5 minutes of incubation at 23 ° C., 1 ⁇ l 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.
  • RNAse H and 79 ⁇ l 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.
  • PCR reaction for the detection of the expression of nCL-3 was carried out as follows:
  • Human hippocampus marathon-ready cDNA (Clontech) was used for the human sequence of the 5 'and 3' end.
  • 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').
  • 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.
  • 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.
  • 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.
  • Cathepsin B inhibition was determined analogously to a method by S.Hasnain et al., J. Biol. Chem. 1993, 268, 235-40.
  • an inhibitor solution prepared from inhibitor and DMSO (final concentrations: 100 ⁇ M to 0.01 ⁇ M), are added to 88 ⁇ L cathepsin B (cathepsin B from human liver (Calbiochem), diluted to 5 units in 500 ⁇ M buffer). This mixture is preincubated for 60 minutes at room temperature (25 ° C.) and the reaction is then started by adding 10 ⁇ L 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.
  • 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.
  • Example Platelet test for determining the cellular activity of calpain inhibitors
  • Platelets (0.1 ml) are preincubated for 5 minutes with 1 ⁇ l of various concentrations of inhibitors (dissolved in DMSO). This was followed by the addition of calcium ionophore A 23187 (1 ⁇ M 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.
  • ABSP actin binding protein
  • ORGANISM Mus musculus
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • ORGANISM Mus musculus
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • ORGANISM Mus musculus
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO

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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

Neue Calpaine, ihre Herstellung und VerwendungNew calpaine, their production and use
Beschreibungdescription
Die Erfindung betrifft neue Calpaine und ihre Herstellung. Weiterhin betrifft die Erfindung Verfahren zum Screening nach neuen Calpaininhibitoren und deren Verwendung.The invention relates to new calpains and their production. The invention further relates to methods for screening for new calpain inhibitors and their use.
Calpaine gehören zu den intrazellulären, nicht-lysosomalenCalpains are intracellular, non-lysosomal
Enzymen aus der Gruppe der Cystein-Proteasen. Sie sind an der Ca2+ -abhängigen Signaltransduktion in eukaryontisehen Zellen beteiligt, d.h. sie regulieren abhängig von der Ca2+ Konzentration zelluläre Funktionen. Calpaine kommen ubiquitär in tierischen Geweben bzw. Zellen von beispielsweise Mensch, Hühnern, Kaninchen oder in der Ratte vor. Auch in niederen Tieren wie beispielsweise in Drosophila melanogaster oder Caenorhabditis elegans wurden Calpaine gefunden. In Hefen, Pilzen oder Bakterien konnten bisher keine Calpaine nachgewiesen werden.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.
Bisher sind drei Hauptisoformen dieser ubiquitären Calpaine bekannt, die sich in vitro durch ihre Kalzium-abhängige Aktivier- barkeit unterscheiden. Calpain I ( = μCalpain ) wird durch μ-mo- lare Kalziumion-Konzentrationen aktiviert, während Calpain II (= mCalpain ) erst durch millimolare Konzentrationen an Kalziumionen aktiviert wird. Beide Calpaine bestehen aus zwei Untereinheiten, einer großen Untereinheit mit ca. 80 kDa und einer kleinen Untereinheit von ca. 30 kDa. Beide Untereinheiten des aktiven Hetero- dimers besitzen Bindungsstellen für Kalzium. Die große Unterein- heit wird aus folgenden vier Proteindomänen (= I - IV) aufgebaut: einer Proteasedomäne (= Domäne II) , einer Kalzium-bindenden Domäne (= Domäne IV) und zwei weiterer Domänen (= Domäne I und III) , deren Funktion unklar ist. Die kleine 30 K-Untereinheit besteht aus einer Kalzium-bindenden Untereinheit (= IV ) und einer weiteren Untereinheit (= V), deren Funktion unklar ist. Zusätzlich zu diesen beiden Calpaintypen wurde ein dritter bezüglich der Kalziumaktivierung intermediärer Typ (= μ/m 80K) im Huhn gefunden (Wang K.K.W, et al., TiPS, Vol. 15, 1994: 412 - 419, Suzuki, K et al., Biol. Chem. Hoppe-Seyler, Vol. 376, 1995: 523 - 529 ) .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).
Neben diesen ubiquitär vorkommenden Calpainen wurden in letzter Zeit zwei neue gewebespezifisch exprimierte Calpaine identifiziert. nCL-1 (= p94) ist ein in Hühnern, Ratten und Menschen vor- kommendes, Muskel-spezifisches Calpain, das vermutlich als Monomer aktiv sein könnte und nur aus der 80 kd Untereinheit besteht. Neben nCL-1 gibt es ein Magen-spezifische Calpain, das in zwei Splicing-Varianten nCL-2 und nC -2' vorkommen kann. nC -2' unterscheidet sich gegenüber nCL-2 durch das Fehlen der Kalzium-bindenden Region (Sorimachi, H.S. et al . , J. Biol. Chem. Vol. 268, No. 26, 1993: 19476 - 19482, Sorimachi, H:S: et al . , FEBS Lett . 5 343, 1994: 1 - 5). Auch in Drosophila wurde ein Calpain-homologes Protein (= CalpA) , das mit Actin interagiert und vermutliche eine wichtige Rolle in der Embryonalentwicklung spielt, gefunden, daß zwei verschiedene Splicing-Varianten aufweist (Mol. Cell. Biol. Vol. 15, No. 2, 1995: 824 - 834). Auch hier fehlt der kürzeren 0 Variante die Kalziumbindestelle.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.
Man vermutet, daß Calpaine bei verschiedenen physiologischen Prozessen wichtige Rollen spielen. Eine Vielzahl von cytoskele- talen, membrangebundenden oder regulatorischen Proteinen wie Pro- 5 teinkinase C, Phospholipase C, Spectrin, Cytoskelett-Proteine wie MAP2 , Muskelproteine, Neurofilamente und Neuropeptide, Plättchenproteine, -"Epidermal Growth Factor"-, NMDA-Rezeptor und Proteine, die an der Mitose beteiligt sind, sowie weitere Proteine sind Calpainsubstrate (Barrett M.J. et al . , Life Sei. 48, 1991: 0 1659 - 69, Wang K.K. et al . , Trends in Pharmacol. Sei., 15, 1994: 412 - 419). Die normale physiologische Funktion der Calpaine ist bis heute noch nicht klar verstanden.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.
Bei verschiedenen pathophysiologischen Prozessen und Krankheiten 5 wurden erhöhte Calpain-Spiegel gemessen, zum Beispiel bei: Ischämien des Herzen (z.B. Herzinfarkt), der Niere oder des Zentralnervensystems (z.B. Hirnschlag), Entzündungen, Muskel - dystrophien, Katarakten der Augen (Grauer Star) , Verletzungen des Zentralnervensystems (z.B. Trauma), Alzheimer Krankheit, HlV-in- 0 duzierte Neuropathy, Parkinsonsche- und Huntigtonsche Krankheit usw. (siehe Wang K.K. oben) . Man vermutet einen Zusammenhang dieser Krankheiten mit einem erhöhten und anhaltenden intrazellulären Kalziumspiegel. Dadurch werden Kalzium-abhängige Prozesse überaktiviert und unterliegen nicht mehr der physiologischen Regelung. Dementsprechend kann eine Überaktivierung von Calpainen auch pathophysiologische Prozesse auslösen.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.
Daher wurde postuliert, daß Inhibitoren der Calpain-Enzyme für die Behandlung dieser Krankheiten nützlich sein können. Ver- schiedene Untersuchungen bestätigten dies. So haben Seung-Chyul Hong et al . (Stroke 1994, 25 (3), 663 - 669) und Bartus R.T. et al. (Neurological Res. 1995, 17, 249 - 258) eine neuroprotektive Wirkung von Calpaininhibitoren in akuten neurodegenerativen Störungen, wie sie nach Hirnschlag auftreten, gezeigt. Ebenso verbesserten nach experimentellen Gehirntraumata Calpaininhibitoren die Erholung der auftretenden Gedächtnisleistungsdefizite und neuromotorischen Störungen (Saatman K.E. et al., Proc. Natl. Acad. Sei. USA, 93, 1996: 3428 - 3433). Edelstein C.L. et al . (Proc. Natl. Acad. Sei. USA, 92, 1995, 7662 - 7666) fand eine protektive Wirkung von Calpaininhibitoren auf durch Hypoxie geschädigten Nieren. Yoshida K.I. et al. (Jap. Circ. J. 59 (1), 5 1995, 40 - 48) konnten günstige Effekte von Calpaininhibitoren nach cardialen Schädigungen aufzeigen, die durch Ischämie oder Reperfusion erzeugt wurden. Da Calpaininhibitoren die Freisetzung von ß-AP4-Protein hemmen, wurde eine potentielle Anwendung als Therapeutikum der Alzheimer Krankheit vorgeschlagen (Higaki J. 0 et al., Neuron, 14, 1995: 651 - 659). Die Freisetzung von Inter- leukin-lα wird ebenfalls durch Calpaininhibitoren gehemmt (Watanabe N. et al., Cytokine, 6 (6), 1994: 597 - 601). Weiterhin wurde gefunden, daß Calpaininhibitoren cytotoxische Effekte an Tumorzellen zeigen (Shiba E. et al . , 20th Meeting Int. Ass. 5 Breast Cancer Res., Sendai Jp, 1994, 25. - 28. Sept., Int. J. Onco. 5 (Suppl.), 1994, 381). Auch bei der Restenose und bei Arthritis spielt Calpain eine wichtige Rolle und Calpaininhibitoren können das Krankheitsbild positiv beeinflussen (March K:L: et al. Circ. Res. 72, 1993: 413 - 423, Suzuki K. et al . , Biochem 0 J. , 285, 1992: 857 - 862) .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).
Weitere mögliche Anwendungen von Calpaininhibitoren sind Wang K.K (Trends in Pharmacol . Sei., 15, 1994: 412 - 419) zu entnehmen.Further possible uses of calpain inhibitors can be found in Wang K.K (Trends in Pharmacol. Sci., 15, 1994: 412-419).
5 Der potenteste und selektivste Calpaininhibitor ist das natürlich vorkommende intrazelluläre Protein Calpastatin. Es hemmt sowohl Calpain I als auch Calpain II, nicht jedoch andere Cystein- bzw. Thiolproteasen wie Cathepsin B, L oder Papain. Das aus ca. 700 Aminosäuren bestehende Calpastatin hat jedoch den Nachteil, 0 daß es für therapeutische Möglichkeiten aufgrund der Größe und der Unpassierbarkeit der Zellmembran nicht in Frage kommt. Neben niedermolekularen peptidischen Calpaininhibitoren wurden eine Reihe nicht-peptidischer Inhibitoren identifiziert. Nachteil dieser Inhibitoren ist, daß sie instabil sind, rasch metaboli- siert werden und zum Teil toxisch sind. Viele Calpaininhibitoren zeichnen sich außerdem durch eine mangelnde Selektivität aus, d.h. sie hemmen nicht nur Calpain I und II sondern auch andere Cysteinproteasen wie Papain, Chymotrypsin, Elastase oder Cathepsin B und L.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.
Es besteht daher nach wie vor ein Bedarf nach selektiven, hoch wirksamen Calpaininhibitoren. Für das Screening nach diesen selektiven, gut wirksamen Calpaininhibitoren sind hochspezifische Testsysteme erforderlich, die es ermöglichen selektive Inhibito- ren zu identifizieren. Üblicherweise werden die Screeningtests mit den ubiquitär vorkommenden Calpainen Calpain I und Calpain II durchgeführt. Für das Auffinden selektiver Inhibitoren ist es notwendig und wünschenswert weitere Calpaine zur Testung zur Verfügung zu stellen, die möglichst gewebespezifisch exprimiert werden, so daß die Inhibitoren auf ihre Selektivität zwischen den einzelnen Calpainen geprüft werden können.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.
Darüber hinaus sind weitere neue Calpaine gesuchte Proteine, da sie mit hoher Wahrscheinlichkeit bei den verschiedenen Krankheitsbildern bzw. Krankheiten unterschiedlich exprimiert werden und eine wichtige Rolle bei diesen Krankheiten spielen.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.
Aufgabe der vorliegenden Erfindung war es, Mittel zur Profilierung und Identifizierung von Calpaininhibitoren zur Verfügung zu stellen, die es ermöglichen Calpaininhibitoren zu identifizieren, die einerseits nur gegenüber einem Calpain inhibierende Wirkung aufweisen, und anderseits gegenüber mehreren Calpainen inhibierende Wirkung aufweisen und diese als therapeutisches Target zur Verfügung zu stellen.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.
Gegenstand der Erfindung ist ein neues Calpain sowie seine allelischen Varianten, Analoge oder Derivate.The invention relates to a new calpain and its allelic variants, analogs or derivatives.
Gegenstand der Erfindung ist auch ein Verfahren zur Identifizierung von Calpaininhibitoren, wobei man das Calpain nCL-3 codiert durch die Sequenz SEQ ID NO: 1 oder SEQ ID NO: 6 aus Geweben oder Zellen, in denen das Enzym nCL-3 exprimiert wird, isoliert und die Inhibierung der Spaltung eines Substrats des Enzyms nCL-3 und in mindestens einem weiteren Test die Inhibierung der Spaltung eines Substrats der Enzyme Calpain I und/oder II durch Test- Substanzen mißt und die Testsubstanzen auswählt, die mindestens gegen eines der Calpaine eine inhibierende Wirkung zeigen oder die Testsubstanzen auswählt, die das Enzym nCL-3 nicht hemmen, jedoch die Enzyme Calpain I und/oder II oder die das Enzym nCL-3 hemmen, nicht jedoch die Enzyme Calpain I und/oder II oder die nCL-3 und die Enzyme Calpain I und/oder II hemmen.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.
Weiterhin ist Gegenstand der Erfindung ein Verfahren zur Identifizierung von Calpaininhibitoren, dadurch gekennzeichnet, daß man die Inhibierung der Spaltung eines Substrates des Enzyms nCL-3 bzw. der Calpaine I und/oder II durch Testsubstanzen in zellulären Systemen bestimmt und solche Testsubstanzen auswählt, die die Zellmembran passieren und die intrazelluläre Aktivität des Enzyms nCL-3 und/oder der Calpaine I und/oder II hemmen.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.
Mit Hilfe der Calpain-spezifischen Primer wurden über das sogenannte Domain-Fingerprinting (Boehm T., Oncogene 8, 1993: 1385 - 1390) unter Verwendung von genomischer DNA Calpain-spezifische Sequenzsignaturen mittels PCR-Technik hergestellt, die vorteilhafterweise auch zur besseren Differenzierung der Calpainse- quenzen Intronsequenzen enthalten.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.
Die in Tabelle 1 genannten redundanten PCR-Primer wurden bei der Klonierung des Gens für nCL-3 verwendet .The redundant PCR primers listed in Table 1 were used in cloning the gene for nCL-3.
Tabelle 1Table 1
Redundante PCR Primer, die zur Klonierung von nCL-3 (=2930) verwendet wurdenRedundant PCR primers used to clone nCL-3 (= 2930)
Name SequenzName sequence
CA 1 5«- tng gng att gtt ggc tnc t - 3« c c t tCA 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 cCA 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 cCA 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 eCAL4 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 cCA 5 5 «- gag tan gca tgn cet ttn ac - 3« ta g c
CAL6 5«- ttn cgn aat cen tgg gg - 3« c c aCAL6 5 «- ttn cgn aat cen tgg gg - 3« c c a
CAL7 5«- cec can gga ttn cgn aan cg - 3« g g tCAL7 5 «- cec can gga ttn cgn aan cg - 3« g g t
CAL8 5«- gat ggn gaa ttt tgg atg - 3« c g cCAL8 5 «- gat ggn gaa ttt tgg atg - 3« c g c
CAL9 5«- gac atc caa aat ten cca tc - 3« et g c gCAL9 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 gCA 10 5 «- nag att aca tat ttc na - 3« a g g a c g
Mit dem Primerpaar Calδ und Cal9 (siehe Tab. 1) ließ sich ein Klon mit der Bezeichnung 29/30 (=2930) darstellen. Dieser Klon kodiert für ein Gen, dessen Genprodukt als neues Calpain die Bezeichnung nCL-3 erhielt. Die Nucleinsäuresequenz des Klons 29/30 (= nCL-3 bzw. 2930) ist Sequenz SEQ ID NO: 1 zu entnehmen. Die abgeleitete Aminosäuresequenz des Calpains nCL-3 ist der Sequenz SEQ ID NO: 2 zu entnehmen. Die unter Berücksichtigung eines vorhandenen Introns deduzierte Aminosäuresequenz weist eine typische Calpainsignatur auf, wobei eine Zuordnung zu den bekannten Calpain-Subfamilien μCalpain, mCalpain, nCL-1 oder nCL-2 aufgrund der geringen Homologie nicht möglich ist (siehe Tab. 2). Figur 3 ist die Homologie zu bekannten Calpain-Subfamilien zu entnehmen. Es handelt sich bei dem Calpain nCL-3 um ein neues bisher unbekanntes Calpain.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.
Sequenzanalysen zeigten die typischen drei Aminosäurereste (Cys81, His252 und Asn284) des katalytischen Zentrums von Cysteinproteasen. Die aus der Gensequenz abgeleiteten Aminosäure - reste 75 - 86 (QGQVGNCWFVAA) stimmen mit dem konservierten Muster typischer Thiolproteasen überein.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.
Tabelle 2Table 2
Homologie (%) auf Aminosäureebene zwischen Maus nCL-3 =2930) und anderen CalpainenHomology (%) at the amino acid level between mouse nCL-3 = 2930) and other calpains
Name % Homologie Nematode tra-3 34,5Name% homology nematode tra-3 34.5
Drosophila CalpA 31,5Drosophila CalpA 31.5
Huhn p94 31,2Chicken p94 31.2
Mensch p94 30,9Human p94 30.9
Maus p94 30,5 Ratte p94 30,0Mouse p94 30.5 rat p94 30.0
Huhn μ/m 28,8Chicken µ / m 28.8
Huhn m 27,8Chicken m 27.8
Mensch m 27,3Human m 27.3
Huhn μ 25,4 Schwein p94 25,4Chicken µ 25.4 Pig p94 25.4
Ratte m 24,4Rat m 24.4
Ratte nCL-2 23,9Rat nCL-2 23.9
Nematode CPL1 23,6Nematode CPL1 23.6
Mensch μ 23,1 Schistosoma 21,7Human μ 23.1 Schistosoma 21.7
Kaninchen m 16,1Rabbit m 16.1
Schwein m 15,8Pig m 15.8
Schwein μ 15,6Pig μ 15.6
Maus CAP4 13,7 Kaninchen μ 12,9Mouse CAP4 13.7 rabbit μ 12.9
Das in der Sequenz SEQ ID NO: 5 gezeigte Intron (von Nukleotid 109 bis 514) wurde durch Vergleich mit der cDNA festgelegt.The intron shown in sequence SEQ ID NO: 5 (from nucleotides 109 to 514) was determined by comparison with the cDNA.
Sequenzvergleiche zwischen der Maus 29/30-Sequenz (nCL-3) in verschiedensten Datenbanken wie Genbank.nr und Genbank. dbest ergaben Homologien zu CalpA, Tra-3 und einer humanen Sequenz mit der Be- Zeichnung EST01106 Homo sapiens cDNA clone HHCPE79 (siehe Figur 2). Die DNA- und Aminosäuresequenzen wurden auf Homologie mit nicht-redundanten Nucleinsäure-, Protein- und EST-Datenbanken am National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) untersucht. Die Aminosäuresequenz - vergleiche wurden mit Clustal W (Thompson et al . Nucl. Acids Res. 22, 1994, 4673 - 468=) durchgeführt.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 =).
nCL-3 weist im Vergleich zu den anderen Calpainen (Figur 2) neben einer verkürzten Domäne I ein verändertes C-terminales Ende auf, das keine ausgeprägte Homologie zur Domäne IV der anderen Calpaine hat. Im Bereich der Domäne IV liegt die Konsensussequenz der Ca2+~Bindungsstelle der Calpaine (sog. "EF-hand" ) . Diese Ca2+-Bindungsstelle fehlt im Falle von nCL-3, daß heißt möglicher- weise wird kein Ca2+ an der Domäne IV gebunden und das Protein auf anderem Wege aktiviert. Es ist damit das einzige Vertebraten- Calpain, dem die Calmodulin ähnliche Domäne IV fehlt.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.
Bei CalpA handelt es sich um ein gewebsspezifisches exprimiertes Calpainhomologes von Drosophila (Theopold V. et al., Mol. Cell. Biol., Vol. 15, No. 2, 1995: 824 - 834). Es wird in einigen Neuronen des Zentralen Nervensystems, in verstreuten Zellen des Mitteldarms und in Blutzellen von Drosophila exprimiert. Von CalpA wurden zwei verschiedene Splicing-Varianten gefunden. Der kürzeren Variante fehlt die Calpain-typische Kalziumbindungs- stelle.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.
Die Homologie auf Aminosäureebene zwischen CalpA und nCL-3 beträgt 31,5 % (siehe Tab. 2).The homology at the amino acid level between CalpA and nCL-3 is 31.5% (see Table 2).
Tra-3 ist an der Geschlechtsbestimmung von Caenorhabditis elegans beteiligt. In einer Kaskade von mehreren Genen und deren Genprodukten entscheidet tra-3 mit darüber, ob sich Caenorhabditis Männchen oder Hermaphroditen entwickeln (Kuwabara P.E. et al., TIG, Vol. 8, No.5, 1992: 164 - 168). Tra-3 scheint an der Sper- matogenese beteiligt zu sein.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.
Die Homologie auf Aminosäureebene zwischen tra-3 und nCL-3 beträgt 34,5 % (s. Tab 2). Möglicherweise ist auch nCL-3 an der Geschlechtsbestimmung beteiligt.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.
Weitere Homologien zwischen nCL-3 und anderen Calpainen sind Tabelle 2 zu entnehmen.Further homologies between nCL-3 and other calpaines can be found in Table 2.
Zwischen nCL-3 und der humanen Teilsequenz ΞST01106 besteht die größte Homologie. Die Teilsequenz EST01106 wurde aus einer Hippo- campusbibliothek gewonnen. Über die Funktion ist nichts bekannt (Nature 355, 6361, 1992: 632 - 634). Ebenfalls unbekannt ist die vollständige Gensequenz und ob es sich bei der Sequenz um ein Calpaingen handelt.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.
Die Sequenzvergleiche zwischen CalpA, Tra-3, EST01106 und nCL-3 werden in Figur 2 wiedergegeben.The sequence comparisons between CalpA, Tra-3, EST01106 and nCL-3 are shown in FIG. 2.
Ausgehend von humaner Hippocampus Marathon-Ready cDNA (Clontech) konnte mit Hilfe einer modifizierten RACE-Methode (= rapid ampli- fication of cDNA ends) nach Frohman et al. (Proc. Natl. Acad.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.
Sei. USA 85, 1988, 8998 - 9002) bzw. Edwards et al . (Nucl. Acids Res. 19, 1991, 5227 - 5232) unter Verwendung der oben genannten Primer (Cal6 und Cal9) die Gesamtsequenz des Klones EST01106 klo- niert werden. Wobei die 3 'Region der cDNA zunächst nicht mit Hilfe des Reversen-Primers des Clontech-Kit' s zu klonieren war. Erst unter Verwendung eines Primers, der komplementär zur humanen EST-Sequenz war, und eines Reversen Primers, der komplementär zur cDNA-Sequenz der letzten 6 Aminosäuren der Maus nCL-3-Sequenz (5 ' -tcagacagccgtgagagagg-3 ' ) war, konnte das 3 'Ende kloniert werden.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.
Die von der Gensequenz SEQ ID NO: 6 abgeleitete Aminosäuresequenz (SEQ ID NO: 7) zeigt 92.2 % Homologie zur Maus nCL-3 Sequenz (siehe Figur 4) . Diese Ähnlichkeit entspricht der Homologie auf Aminosäureebene zwischen dem Humanen und dem Mäuse m-Calpain (97 %) und dem Humanen und dem Mäuse p94 (93,5 %) wie unsere Sequenzierungen zeigten. EST01106 ist damit mit hoher Wahrscheinlichkeit das humane Ortholog der Maus nCL-3 Sequenz. Figur 4 zeigt außerdem die Sequenzen von Caenorhabditis tra-3, Drosophila CalpA, Maus p94. Maus m-Calpain, humanen μ-Calpain und Ratten nCL-2. Aminosäuren, die zwischen den verschiedenen Calpainen und nCL-3 übereinstimmen sind durch Punkte gekennzeichnet. Striche deuten Lücken an, die um eine maximale Übereinstimmung der Sequenzen zu erreichen, eingeführt wurden. Die C-terminalen Enden der alternativen Splicingprodukte der nCL-3- und CalpA-Tran- skripte sind über und unter der jeweiligen Gesamtsequenz angegeben, startend mit dem Beginn der unterschiedlichen Sequenz. Sterne geben die zwischen allen Calpainen konservierten Reste an. Die beiden Aminosäuresequenzen, die den Oligonucleotiden Cal6 und Cal9 entsprechen, wurden durch Kästchen gekennzeichnet. Durch Pfeile werden die "Splice sites" der korrespondierenden Maus nCL-3-DNA markiert.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.
Figur 5 gibt den phylogenetischen Stammbaum der verschiedenen Calpaine wieder. Die phylogenetischen Analysen zur Aufstellung dieses Stammbaums wurden unter Verwendung der nächsten Nachbar- Methode (Saitou et al . Mol. Biol. Evol . 4, 1987, 406 - 425) unter Ausschluß der Lücken durchgeführt. Mit Hilfe dieser phylogenetischen Analysen konnten die Vertebraten-Calpaine in sechs verschiedene Gruppen aufgeteilt werden (Figur 5, rechte Seite). Die Nicht-Vertebraten-Calpaine lassen sich der nCL-3 Gruppe als nächste Nachbarn zuordnen oder stehen in einer eigenen Gruppe. Die nCL-3 Gene bilden damit eine eigene Gruppe von Calpainen, die eine größere Ähnlichkeit zu Invertebraten Calpaine haben als zu Vertebraten Calpaine. Die Länge der horizontalen Linien ist proportional zur phylogenetischen Entfernung der verschiedenen Cal- paine. Die Länge der vertikalen Linien ist ohne Bedeutung. Die für die Aufstellung des phylogenetischen Stammbaums verwendeten Sequenzen haben die folgenden SWISSPROT- und EMBL-Nummern ("accession numbers") : Mensch m (P17655) , μ (P07384), p94 (P20807) ; Ratten m (Q07009) , nCL-2 (D14480), p94 (P16259); Maus p94 (X92523); Hühner m (D38026), μ (D38027), μ/m (P00789), p94 (D38028) ; Nematoden tra-3 (U12921); Drosophila Calp A (Q11002) und Dm (X78555) , Schistosoma (P27730) .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).
Die nCL-3 Genstruktur wird in Figur 6 wiedergegeben. Die Exon/In- tron-Splicing Übergänge innerhalb der kodierenden Sequenz des Genes wurden durch Vergleich der DNA-Sequenzen von genomischer DNA und cDNA ermittelt. Es wurden 11 Introns innerhalb der kodierenden Sequenz ermittelt. In Figur 4 wird die Position der "splicing sites" durch Pfeile markiert. Die Position des zuerst mit den Primern Cal6 und Cal9 amplifizierten genomischen Fragments wird durch Klammern markiert.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.
Figur 6b zeigt die Lage der "splicing sites" verschiedener Calpaine. Überraschenderweise besitzen nCL-3 und tra3 trotz der relativ großen Homologie keine gemeinsamen "splice sites". Die Übereinstimmung in der Lage einiger "splice sites" zwischen nCL-3 und den Vertebraten Calpainen deutet auf eine gemeinsame Entstehung der Gene hin. Das Fragezeichen über den letzten konservierten splice site im Hühner μ/m-Calpaingen deutet an, daß die publizierte Sequenz um diese Schnittstelle herum nicht in Übereinstimmung ist mit der ursprünglichen cDNA-Sequenz.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.
Neben dem in Sequenz SEQ ID NO: 1 dargestellten nCL-3-Gen wurde eine verkürzte Form, die in Sequenz SEQ ID NO: 3 dargestellt wird, identifiziert. Die abgeleitete Aminosäuresequenz des verkürzten nCL-3-Gens ist der Sequenz SEQ ID NO: 4 zu entnehmen. Dieses verkürzte nCL-3-Gen, das die Bezeichnung nCL-3' trägt, entsteht vermutlich durch alternatives Splicing. Semi-quanti- tative RT-PCR-Analysen mit mRNA, die aus dE17-Zellen isoliert wurden, unter Verwendung von Primern, die die flankierenden Regionen des Introns abdecken (siehe Figur 6) , zeigten, daß das nicht gesplicte Produkt etwa 0,5 % der n-CL3 mRNA ausmacht.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.
Das erfindungsgemäße neue Calpain nCL-3 wird in vielen Geweben mit unterschiedlicher Intensität exprimiert (Figur 1) . In der untersuchten mRNA Analysen wird nCL-3 klar ersichtlich in der Haut, der Niere, dem Herz, der Lunge, dem Thymus und in der Leber exprimiert.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.
Auch im Menschen wird das nCL-3 Gen unterschiedlich stark exprimiert. In allen untersuchten Geweben wurde eine geringe Expression nachgewiesen. Im Kolon, im Hoden, in der Niere, in der Leber und in der Luftröhre wurde eine starke Expression gefunden.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.
Das nCL-3 Gen wurde auf Chromosom 7 bei Mäusen und Chromosom 11 beim Menschen lokalisiert. Es liegt auf dem langen Arm des menschlichen Chromosoms bei etwa 84 cM (= centi Morgen) . Dies ist 12 - 14 cM von der kartierten Position des μ-Calpains (llql3) entfernt. nCL-3 konnte sehr nahe am Glycoprotein A-Gen katiert werden (llql3.5 - ql4) .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).
Das Maus Orthologe nCL-3 Gen wurde zwischen 44 und 53 cM auf dem Maus Chromosom 7 lokalisiert.The mouse orthologist nCL-3 gene was localized between 44 and 53 cM on mouse chromosome 7.
Für die Identifizierung von selektiven Calpaininhibitoren sind möglichst spezifische Verfahren zur Identifizierung der Inhibitoren erforderlich. Wichtig dabei ist, daß die selektierten Inhibitoren nur das gewünschte oder die gewünschten Calpaine hemmen, nicht jedoch andere Cystein-Proteasen und damit in physiologische Prozesse eingreifen.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.
Die auf ihre inhibitorische Aktivität hin zu prüfenden Test- substanzen können beispielsweise chemische Substanzen, mikro- bielle oder pflanzliche Extrakte sein. Sie werden üblicherweise neben den Test auf ihre Inhibitoraktivität gegenüber nCL-3, Calpain I und/oder II auf ihre Aktivität gegenüber Cathepsin B oder andere Thiolproteasen getestet.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.
Idealerweise sollten gute Inhibitoren keine oder nur geringe Aktivität gegenüber Cathepsin B, L, Elastase, Papain, Chymo- trypsin oder andere Cystein-Proteasen aufweisen, aber eine gute Aktivität gegenüber den Calpainen I und II aufweisen. Durch das erfindungsgemäße neue Calpain nCL-3 können mit den erfindungsgemäßen Verfahren Inhibitoren identifiziert werden, die zu ihrer inhibitorischen Wirkung zwischen den verschiedenen Calpainen Calpain I, II, nCL-1 nCL-2 und/oder nCL-3 diskrimi- nieren können.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.
Die verschiedenen Inhibitortests wurden dabei wie folgt durchgeführt:The various inhibitor tests were carried out as follows:
Cathepsin B-TestCathepsin B test
Die Cathepsin B-Hemmung wurde analog einer Methode von S. Hasnain et al., J. Biol. Chem. 1993, 268, 235 - 240 bestimmt.Cathepsin B inhibition was determined analogously to a method by S. Hasnain et al., J. Biol. Chem. 1993, 268, 235-240.
Zu 88 μl Cathepsin B (Cathepsin B aus menschlicher Leber von der Firma Calbiochem, verdünnt auf 5 Units in 500μM Puffer) werden 2 μl einer Inhibitorlösung, hergestellt aus der zu testenden chemischen Substanz, einem mikrobiellen oder pflanzlichen Extrakt und DSMO (Endkonzentration: 100 μM bis 0,01 μM) zugegeben. Dieser Ansatz wird für 60 Minuten bei Raumtemperatur (= 25 °C) vorinku- biert und anschließend die Reaktion durch Zugabe von 10 μl 10 mM Z-Arg-Arg-pNA (in Puffer mit 10 % DMSO) gestartet. Die Reaktion wird 30 Minuten bei 405 nm im Mikrotiterplattenreader verfolgt. Aus den maximalen Steigungen werden anschließend die ICso's be- stimmt.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 und II-TestCalpain I and II test
Die Aktivität der Calpaininhibitoren wurde in einem colorimetri- sehen Test mit Casein nach Hammarsten (Merck, Darmstadt) als Substrat untersucht. Der Test wurde in Mikrotiterplatten, entsprechend der Veröffentlichung von Buroker-Kilgore und Wang in Anal. Biochem. 208, 1993, 387 - 392, durchgeführt. Als Enzyme wurde Calpain I (0,04 U/Test) aus Erythrozyten und Calpain II (0,2 U/Test) aus Nieren, beide vom Schwein, der Firma Calbiochem, benutzt. Die zu testenden Substanzen wurden mit dem Enzym für 60 Minuten bei Raumtemperatur inkubiert, wobei eine Konzentration von 1 % des Lösungsmittels DMSO nicht überschritten wurde. Nach Zugabe des Bio-Rad Farbreagenz erfolgte die Messung der optischen Dichte bei 595 nm in dem Easy Reader EAR 400 der Firma SLT. Die 50 %ige Aktivität des Enzyms ergibt sich aus den optischen Dichten, die bei der maximalen Aktivität des Enzyms ohne Inhibitoren und der Aktivität des Enzyms ohne Zugabe von Kalzium bestimmt wurden. Die Aktivität von Calpaininhibitoren kann ferner mit dem Substrat Suc-Leu Tyr-AMC bestimmt werden. Diese fluorimetrische Methode ist bei Zhaozhao Li et al , J. Med. Chem. 1993, 36, 3472-3480 beschrieben.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.
Da Calpaine intrazelluläre Cysteinproteasen sind, müssen Calpaininhibitoren die Zellmembran passieren, um den Abbau von intrazellulären Proteinen durch Calpain zu verhindern. Einige bekannte Calpaininhibitoren, wie zum Beispiel E 64 und Leupeptin, überwinden die Zellmembranen nur schlecht und zeigen dementsprechend, obwohl sie gute Calpaininhibitoren darstellen nur schlechte Wirkung an Zellen. Es ist deshalb vorteilhaft einen zusätzlichen Test für die Membrangängigkeit von potentiellen Calpaininhibitoren wie den humanen Plättchentest durchzuführen.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.
Plättchen-Test zur Bestimmung der zellulären Aktivität von CalpaininhibitorenPlatelet test to determine the cellular activity of calpain inhibitors
Der Calpainvermittelte Abbau von Proteinen in Plättchen wurde, wie von ZhaozhaoLi et al . , J. Med. Chem., 36, 1993, 3472- 3480 beschrieben, durchgeführt. Humane Plättchen wurden aus frischem Natrium-Citrat-Blut von Spendern isoliert und in Puffer ( 5 mM Hepes, 140 mM NaCl und 1 mg/ml BSA, pH 7,3) auf 107 Zellen/ml eingestellt .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).
Plättchen (0,1 ml) werden für 5 Minuten in 1 μl an verschiedenen Konzentrationen an potentiellen Inhibitoren (gelöst in DMSO) vor- inkubiert. Danach erfolgte die Zugabe von Kalziumionophor A 23187 (1 μM im Test) und Kalzium (5 mM im Test) und eine weitere Inku- bation von 5 Minuten bei 37 °C . Nach einem Zentrifugationsschritt wurden die Plättchen in SDS-Page Probenpuffer aufgenommen, 5 Minuten bei 95 °C gekocht und die Proteine in einem 8%igen Gel aufgetrennt. Der Abbau der beiden Proteine Actin bindendes Protein (= ABP) und Talin wurde durch quantitative Densitometrie verfolgt. Nach der Zugabe von Kalzium und lonophor verschwanden diese Proteine und es entstanden neue Banden von kleiner 200 Kd Molekulargewicht. Daraus wird die halb maximale Enzymaktivität mit oder als Kontrolle ohne Inhibitor bestimmt.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.
Ebenfalls geeignet für die Testung der Membrangängigkeit sind Gewebsteile wie Gehirnschnitte oder Zellkulturen.Tissue parts such as brain sections or cell cultures are also suitable for testing membrane permeability.
Test auf Hemmung gegenüber nCL-3 wird in Zellen durchgeführt, die dieses Protein exprimieren und sich dieses mit einem spezifischen Antikörper nachweisen läßt. Werden Zellen mit z. B: Kalzium und dem entsprechenden lonophor stimuliert, führt dies zu einer Aktivierung von nCL-3. Takaomi Saido beschrieb 1992 im J. Bio- chem. Vol. 11, 81-86 die autolytische Transition von μ-Calpain nach Aktivierung und der Nachweis mit Antikörpern. Entsprechende Antikörper werden für den Nachweis von nCL-3 erzeugt. Calpaininhibitoren verhindern die autolytische Transition und eine ent- sprechende Quantifizierung ist mit Antikörpern möglich.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.
Neben den beschriebenen in vitro Tests so wie dem zellulären Plättchentest eignen sich alle weiteren dem Fachmann bekannten Calpaintests wie der Test auf Hemmung des Glutamat induzierten Zelltods an corticalen Neuronen (Maulucci-Gedde M.A. et al.,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), der Kalzium-vermittelte Zelltod in NT2-Zellen (Squier M.K.T. et al . , J. Cell. Physiol., 159, 1994: 229 - 237, Patel T. et al., Faseb Journal 590, 1996: 587 - 597) oder die Analyse in Gewebsproben nach Abbauprodukten von Proteinen wie Spectrin, MAP2 oder Tau (Ami Arai et al . , Brain Research, 1991, 555, 276 - 280, James Brorson et al . , Stroke, 1995, 26, 1259 - 1267) .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).
Für die in vitro-Tests von nCL-3 wird das Calpain nCL-3 oder seine tierischen oder sein humanes Homologes aus Geweben oder Zellen in denen das Enzym exprimiert wird wie beispielsweise der Niere, dem Thymus, der Leber, der Lunge, oder aus Zellen oder Mikroorganismen, die mindestens eine Genkopie und/oder einen Vektor mit mindestens einer Genkopie des nCL-3-Gens enthalten, aufgereinigt und als Rohextrakt oder als reines Enzym verwendet.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.
Für die erfindungsgemäßen Verfahren werden die verschiedenen Calpaininhibitortests vorteilhafterweise in Kombination mit dem Test auf Hemmung der nCL-3-Enzymaktivität durch potentielle In- hibitoren durchgeführt. Dabei werden Inhibitoren so ausgewählt, daß sie entweder nur das Enzym nCL-3 hemmen und nicht die anderen Calpaine oder umgedreht nur die anderen Calpaine und nicht das Enzym nCL-3 oder das Enzym nCL-3 und mindestens ein weiteres Calpain.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.
Die verschiedenen Inhibitortests werden dabei so ausgeführt, das neben dem Test auf die inhibierende Wirkung der Testsubstanz gegenüber nCL-3, Calpain I und/oder II als Kontrolle die Tests ohne die Testsubstanz durchgeführt wird. Durch diese Testanord- nung lassen sich einfach die inhibitorischen Wirkungen der Test- substanzen erkennen.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.
Ein weiteres erfindungsgemäßes Verfahren verwendet das Enzym nCL-3 zum Screening nach neuen Calpaininhibitoren, wobei diese Inhibitoren vorteilhafterweise generell alle Calpaine oder einzelne Calpaine wie Calpain I, II, nCL-1, nCl-2 oder nCL-3 hemmen können. Die verschiedenen Testsubstanzen können dabei einzeln oder parallel in Testsystemen getestet werden. Vorteilhafterweise werden die Testsubstanzen in parallelen, automatisierten Testsystemen auf ihre inhibitorische Wirkung hin gescreent.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.
Für die Inhibitortests sind generell alle Substanzen geeignet. So stammen die Substanzen beispielsweise aus der klassischen chemischen Synthese, aus der Kombinatorik, aus mikrobiellen, tierischen oder pflanzlichen Extrakten. Unter mikrobiellen Extrakten sind beispielsweise Fermentationsbrühen, Zellaufschlüsse von Mikroorganismen oder Substanzen nach Biotransformation zu verstehen. Auch Zellfraktionen sind für die Tests geeignet.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.
Für die Klonierung des nCL-3 Gens oder seiner tierischen Homologen oder seines humanen Homologen eignen sich alle prokaryon- tischen oder eukaryontischen Expressionssysteme, die zur Isolierung eines enzymatisch aktiven Genprodukts geeignet sind. Bevorzugt werden Expressionssysteme, die eine Expression der nCL-3-Gensequenzen in bakteriellen, pilzlichen oder tierischen Zellen ganz besonders bevorzugt in Insektenzellen ermöglicht. Unter enzymatisch aktivem Genprodukt sind nCL-3-Proteine zu verstehen, die direkt nach Isolierung aus dem Expressionsorganismus wie beispielsweise aus einer prokaryotischen oder eukaryotischen Zelle oder nach Renaturierung ein aktives Protein ergeben, das in der Lage ist mindestens ein bekanntes Calpainsubstrat wie die oben genannten oder über Autokatalyse sich selbst zu spalten.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.
Für die Bestimmung der enzymatischen Aktivität sind alle dem Fachmann bekannten Calpaintests wie in vitro-Tests wie die oben beschriebenen Tests für Calpain I und II oder zelluläre Tests wie der Plättchentest geeignet. Dabei können als Detektionsmöglich- keit Tests verwendet werden, die auf Basis eines colorimetrischen Assays (Buroker-Kilgore M. et al., Anal. Biochem. 208, 1993: 387 - 392) oder auf Basis eines Fluoreszenz-Assays beruhen.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.
Außerdem sind auch alle Teilsequenzen, die das katalytischeIn addition, all partial sequences that are the catalytic
Zentrum des nCL-3 Gens und/oder weitere Sequenzen des nCL-3 Gens und/oder andere Calpaingensequenzen und/oder andere Sequenzen enthalten und enzymatische Aktivität zeigen, unter enzymatische aktivem Genprodukt von nCL-3 zu verstehen.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.
Unter Wirtsorganismen sind alle prokaryotischen oder eukaryontischen Organismen, die als Wirtsorganismen geeignet sind, sind beispielsweise Bakterien wie Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptococcus carnosus, Hefen wie Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pilze wie Aspergillus niger, Insektenzellen wie Spodoptera frugiperda, Trichoplusia-Zellen oder alle anderen Insektenzellen, die für eine virale Expression geeignet sind, oder tierische Zellen wie CV1, COS, C127, 3T3 oder CHO oder humane Zellen zu verstehen.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.
Unter Expressionssysteme sind die Kombination aus den oben bei- spielhaft genannten Expressionsorganismen und den zu den Organismen passenden Vektoren wie Plasmide, Viren oder Phagen wie das T7 RNA Polymerase/Promoter System oder Vektoren mit regulatorischen Sequenzen für den Phagen λ zu verstehen.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.
Bevorzugt sind unter dem Begriff Ξxpressionssysteme die Kombination aus Escherichia coli und seinen Plasmiden und Phagen oder das Baculovirus-System und die entsprechenden Insektenzellen wie Spodoptera frugiperda zu verstehen.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.
Für die vorteilhafte erfindungsgemäße Expression des nCL-3-Gens sind außerdem weitere 3' und/oder 5' Terminale regulatorische Sequenzen geeignet.Additional 3 'and / or 5' terminal regulatory sequences are also suitable for the advantageous expression of the nCL-3 gene according to the invention.
Diese regulatorischen Sequenzen sollen die gezielte Expression des nCL-3 Gens ermöglichen. Dies kann beispielsweise je nach Wirtsorganismus bedeuten, daß das Gen erst nach Induktion exprimiert oder überexprimiert wird, oder daß es sofort exprimiert und/oder überexprimiert wird.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.
Die regulatorischen Sequenzen bzw. Faktoren können dabei vorzugsweise die nCL-3 Genexpression positiv beeinflussen und dadurch erhöhen. So kann eine Verstärkung der regulatorischen Elemente vorteilhafterweise auf der Transkriptionsebene erfolgen, indem starke Transkriptionssignale wie Promotoren und/oder "Enhancer" verwendet werden. Daneben ist aber auch eine Verstärkung derThe 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 möglich, indem beispielsweise die Stabilität der mRNA verbessert wird.Translation possible, for example by improving the stability of the mRNA.
Unter "Enhancer" sind beispielsweise DNA-Sequenzen zu verstehen, die über eine verbesserte Wechselwirkung zwischen RNA-Polymerase und DNA eine erhöhte nCL-3-Genexpression bewirken.“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.
Dem nCL-3-Gen mit oder ohne vorgeschaltetem Promotor bzw. mit oder ohne Regulatorgen können ein oder mehrere DNA-Sequenzen vor- und/oder nachgeschaltet sein, so daß das Gen in einer Genstruktur enthalten ist.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.
Die Genexpression des nCL-3-Gens läßt sich darüber hinaus auch durch Erhöhen der nCL-3-Genkopienzahl erhöhen. Zur Erhöhung der Genkopienzahl wird das nCL-3-Gen beispielsweise in einem CHO- Expressionsvektor amplifiziert. Als Vektoren eignen sich auch Vektoren der pED-Reihe - dicistronische Vektoren -, die auch das amplifizierbare Markergen Dihydrofolat Reduktase enthalten. Details können den Current Protocols in Molecular Biology Vol 2, 1994 entnommen werden.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.
Eine Steigerung der nCL-3-Enzymaktivität läßt sich zum Beispiel gegenüber dem Ausgangsenzym durch Veränderung des nCL-3-Gens oder seiner tierischen Homologen durch klassische Mutagenese wie UV- Bestrahlung oder Behandlung mit chemischen Mutagentien und/oder durch gezielte Mutagenese wie site directed mutagenesis, Deletion (en) , Insertion (en) und/oder Substitution (en) erzielen. Eine Erhöhung der Enzymaktivität kann beispielsweise erreicht werden, indem das katalytische Zentrum so verändert wird, daß das zu spaltende Substrat rascher umgesetzt wird. Auch kann eine erhöhte Enzymaktivität neben der beschriebenen Genamplifikation durch Ausschaltung von Faktoren, die die Enzymbiosynthese re- primieren und/oder durch Synthese aktiver statt inaktiver nCL-3-Proteine erreicht werden. Auf diesem Weg können erhöhte Enzymmengen für die in vitro-Tests zur Verfügung gestellt werden.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 oder seine tierischen Homologen lassen sich vorteilhafter- weise ausgehend von genomischer DNA oder cDNA unter Verwendung beispielsweise der PCR-Technik (siehe Molekular Cloning, Sambrok, Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press, Second Edition 1989, Kapitel 14, 1 - 35, ISBN 0-87969-309-6 und Saiki et al . , Science, 1988, Vol. 239, 487ff) klonieren, bevorzugt läßt sich nCL-3 unter Verwendung von genomischer DNA und besonders bevorzugt unter Verwendung von genomischer DNA aus Mauszellen oder humanen Zellen klonieren.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.
Als Wirtsorganismus für die Klonierung eignen sich beispielsweise alle Escherichia coli-Stämme, bevorzugt der Escherichia coli Stamm DH10B. Als Vektoren für die Klonierung sind alle Vektoren geeignet, die für die Expression in Escherichia coli geeignet sind (siehe Molecular Cloning, Sambrok, Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press, Second Edition 1989, ISBN 0-87969-309-6) . Besonders geeignet sind beispielsweise Vektoren, .die sich von pBR oder pUC ableiten oder shuttle-Vektoren, ganz besonders geeignet ist pBluescript.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.
Nach Isolierung und Sequenzierung sind nCL-3-Gene mit Nukleotid- sequenzen erhältlich, die für die in SEQ ID NO: 2 angegebene Aminosäuresequenz oder deren Allelvariantionen kodieren. Unter Allelvarianten sind nCL-3-Varianten zu verstehen, die 60 bis 100 % Homologie auf Aminosäureebene, bevorzugt 70 bis 100 %, ganz be- sonders bevorzugt 80 bis 100 % aufweisen. Allelvarianten umfassen insbesondere funktioneile Varianten, die durch Deletion, Insertion oder Substitution von Nukleotiden aus der in SEQ ID NO: 1 oder SEQ ID NO: 6 dargestellten Sequenz erhältlich sind, wobei die nCL-3-Aktivität aber erhalten bleibt.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.
Unter Analoge von nCL-3 sind beispielsweise seine tierischen Homologen, verkürzte Sequenzen wie nCL-3' (siehe SEQ ID NO: 3), Einzelstrang-DNA oder RNA der codierenden und nichtcodierenden DNA-Sequenz besonders antisense RNA zu verstehen.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.
Derivate von nCL-3 sind beispielsweise solche Derivate, die enzy- matisch nicht oder nur schwer spaltbar sind wie die Nucleinsäure - phosphonate oder -phosphothioate, bei denen die Phospatgruppe der Nucleinsäuren gegen eine Phosphonat- bzw Thioatgruppe ersetzt wurde.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.
Auch der Promotor, der der angegebenen Nukleotidsequenz vorgeschalten ist, kann durch ein oder mehrere Nukleotidaustausche, durch Insertion(en) und/oder Deletion (en) verändert sein, ohne daß aber die Funktionalität bzw. Wirksamkeit des Promotors beeinträchtigt ist. Des weiteren kann der Promotor auch durch Ver- änderung seiner Sequenz in seiner Wirksamkeit erhöht oder komplett durch wirksamere Promotoren auch artfremder Organismen ausgetauscht werden.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.
Die nach den erfindungsgemäßen Verfahren identifizierten Cal- paininhibitoren eignen sich zur Herstellung von Medikamenten zur Behandlung von Krankheiten ausgewählt aus der Gruppe der kardiovaskulären, immunologischen, entzündlichen, allergischen, neurologischen, neurodegenerativen, oder onkologischen Erkrankungen wie beispielsweise Restenose, Arthritis, Ischämien des Herzen, der Niere oder des Zentralnervensystems (z.B. Hirnschlag), Entzündungen, Muskeldystrophien, Katarakten der Augen (Grauer Star) , Verletzungen des Zentralnervensystems (z.B. Trauma), Alzheimer Krankheit, HlV-induzierte Neuropathy, Parkinsonsche- und Huntigtonsche Krankheit.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.
Die erfindungsgemäßen nCL-3-Gensequenzen eignen sich vorteilhafterweise auch zur Diagnose von Krankheiten beispielsweise zur Diagnose von Muskeldystrophie oder zur Gentherapie. BeispieleThe 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
Beispiel 1: Klonierung des nCL-3-GensExample 1: Cloning of the nCL-3 gene
Für die Klonierung des nCL-3-Gen mit der Sequenz SEQ ID NO: 1 wurde genomische DNA aus Mauszellen ES E14 verwendet. Es wurden die 5 '-3' (= Vorwärts) und 3 '-5' (= Rückwärts) -Sequenzen der Primer CAL6 und CAL9 (siehe Tabelle 1) sowie folgende PCR-Bedingungen (siehe Molekular Cloning, Sambrok, Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press, Second Edition 1989, Kapitel 14,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 und Saiki et al., Science, 1988, Vol. 239, 487ff) für die Klonierung verwendet:1-35, ISBN 0-87969-309-6 and Saiki et al., Science, 1988, Vol. 239, 487ff) are used for the cloning:
250 ng Vorwärts-Primer 250 ng Rückwärts-Primer250 ng forward primer 250 ng reverse primer
1, 5 mM MgC121.5 mM MgC12
0 , 2 mM dNTPs0.2mM dNTPs
50 mM KC150 mM KC1
10 mM Tris pH 9 , 0 1 μg genomische DNA10 mM Tris pH 9.0 1 ug genomic DNA
2 Units Taq Polymerase.2 units of Taq polymerase.
Es wurden 35 PCR-Zyklen durchgeführt, wobei 45 Sekunden 94 °C, 45 Sekunden 48 °C und 2 Minuten die Temperatur auf 72 °C gehalten wurde. Das nCL-3-Gen wurde in den Vektor pBluescript (SK+) mit dem Enzym EcoRV einkloniert (siehe Holton et al., Nucleic Acids Research, Vol. 19., No. 5, 1156ff.). Mit dem pBluescript-Vektor mit dem einklonierten nCL-3-Gen wurde der Escherichia coli Stamm DH10B entsprechend Maniatis et al . transformiert (siehe Molekular Cloning, Sambrok, Fritsch and Maniatis, Cold Spring Harbor, Laboratory Press, Second Edition 1989, Band 1, Kapitel 1, 74 - 84, ISBN 0-87969-309-6 und Saiki et al., Science, 1988, Vol. 239, 487ff ) .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).
Auf diesem Wege läßt sich auch die humane Sequenz des nCL-3 Gens mit der SEQ ID NO: 6 klonieren, wobei von 0,1 ng cDNA oder 0,5 μg genomischer DNA ausgegangen werden kann. Der Klonierungsansatz kann außerdem vorteilhafterweise 0,1 % Triton X-100 enthalten.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.
Beispiel 2: Expression des nCL-3-Gens in verschiedenen MausgewebenExample 2: Expression of the nCL-3 gene in different mouse tissues
Für die Expression wurde mRNA aus dE12 Embryonen und aus Haut-, Niere-, Herz-, Lunge-, Gehirn-, Thymus- und Dünndarmgewebe der Maus isoliert. Für die Extraktion der mRNA wurden die Gewebe in flüssigem Stickstoff dispergiert und in 10 ml einer Lösung aus 4 M Guanidinium-Isothiocyanat, 25 mM NaCitrat, 0,5 % Sarcosyl und 72 μl 2-Mercaptoethanol resuspendiert. Anschließend wurden 1 ml NaAcetat (pH 4,0), 10 ml wassergesättigtes Phenol und 2 ml Chloroform unter Mischen zugegeben. Die Proben wurden 20 Minuten in zentrifugiert (5000 x g, 4 °C) . Der Niederschlag wurde verworfen. Aus dem Überstand wurde die RNA mit einem Volumen Isopropanol bei - 20 °C (mindestens eine Stunde Fällung) ausgefällt und erneut für 30 Minuten (19000 x g, 4 °C) zentrifugiert. Der Niederschlag wurde in 300 μl resuspendiert und erneut mit NaAcetat/Ethanol in der Kälte gefällt und mit 70 % Ethanol, abzentrifugiert (19000 x g, 4 °C) , gewaschen und in 300 μl Wasser gelöst. Anschließend wurde die mRNA-Konzentration bei 250 nm im Photometer und in einem Aga- rosegel mit einer 5 μl mRNA-Probe gegen eine Referenz bestimmt.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.
Über RT-PCR, in dem ausgehend von der isolierten mRNA zunächst eine cDNA-Kopie mit Hilfe der Reversen Transcriptase erstellt wurde, wurde mit Hilfe der folgenden zwei Primer a) Vorwärtsprimer 5' -tagctcgagtggacgtaatcgtcgatgac-3 ' b) Rückwärtsprimer 5' -tagctcgagtgctgtaggctgtgcatacg-3 ' die Expression des nCL-3-Gens in den verschiedenen Mausgeweben bestimmt (siehe Figur 1) .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).
Die cDNA wurde nach einem Protokoll von der Firma GIBCO wie folgt hergestellt:The cDNA was produced according to a protocol from GIBCO as follows:
Zu 5 μg mRNA (isoliert nach der oben beschriebenen Methode) wurden 2 μl oligo(dT) und 12 μl DEPC dH 0 gegeben. Dieser Ansatz wurde 10 Minuten bei 70 °C inkubiert und anschließend auf Eis gestellt. Zur Probe wurden in der angegebenen Reihenfolge 2 μl 10 x Puffer, 2 μl 25 mM MgCl2, 1 μl 10 mM dNTPs, 2 μl 0,1 M DTT gegeben. Nach 5 Minuten Inkubation bei 23 °C wurde 1 μl Super- Script Reverse Transcriptase hinzugegeben und das ganze 10 Min. bei 25 °C,50 Min. bei 42 °C und 15 Min. bei 70 °C weiter inkubiert. Danach wurde 1 μl RNAse H und 79 μl dH0 zugegeben und je 20 Min. bei 37 °C und 15 Min. bei 70 °C die Reaktion weiter durch- geführt. 1 μl dieser cDNA wurde für die RT-PCR-Reaktion verwendet . 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.
Die PCR-Reaktion für die Nachweis der Expression von nCL-3 wurde wie folgt durchgeführt:The PCR reaction for the detection of the expression of nCL-3 was carried out as follows:
250 ng Vorwärts-Primer 5 250 ng Rückwärts-Primer 1,5 mM MgC12 0,2 mM dNTPs 50 mM KC1 10 mM Tris pH 9 , 0 0 1 μg CDNA250 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 Taq Polymerase.2 units of Taq polymerase.
Es wurden 35 PCR-Zyklen durchgeführt, wobei 45 Sekunden 94 °C, 45 Sekunden 58 °C und 1 Minute die Temperatur auf 72 °C gehalten 5 wurde.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.
In den getesteten Geweben zeigte sich, daß nCL-3 in den dE12-Em- bryonen exprimiert wird. nCL-3 wird auch in der Haut, der Niere, dem Herz, der Lunge und im Thymus exprimiert. Im Gehirn und im 0 Dünndarm ist die Expression schwächer als in den vorgenannten Organen (in Abbildung 1 nicht zu sehen) . Als interner Standard wurde hprt (= Hypoxanthin-phosphor-ribosyl-transferase) verwendet .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. Beispiel: Klonierung der humanen Sequenz des nCL-33. Example: Cloning of the human sequence of nCL-3
Das 3' Ende der Maus bzw. Humanen nCL-3 cDNA wurde mit der sog. RACE-Methode (= rapid amplification of cDNA ends) , wie von Frohman et al. (Proc. Natl . Acad. Sei. USA 85, 1988, 8998 - 9002) bzw. Edwards et al. (Nucl. Acids Res. 19, 1991, 5227 - 5232) beschrieben, ermittelt. Für die humane Sequenz des 5' und 3' Endes wurde humane Hippocampus Marathon-Ready cDNA (Clontech) verwendet. Für die Maus-Sequenz wurde wie unter Beispiel 2 beschrieben cDNA aus Tag 12 Mäuseembryonen verwendet. Das humane 3' Ende konnte nicht mit dem Reversen-Primer des Kit's isoliert werden. Die Klonierung gelang mit Hilfe eines zur humanen EST-Sequenz komplementären Vorwärts-Primers und eines Reversen-Primers, der den letzten 6 Aminosäuren der Maus nCL-3 Sequenz (5' -teagacage- cgtgagagagg-3' ) entsprach.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. Beispiel: Isolierung und Charakterisierung von Cosmid Klonen4. Example: Isolation and Characterization of Cosmid Clones
Cosmid Klone mit dem Maus nCl-3 Gen wurden aus einer Cosmidbi- bliothek, die aus genomischer ES-Mäusezellen-DNA durch Klonierung in den Vector pSuperCos (Stratagene) hergestellt wurde, isoliert. Die Bibliothek war in 348 "Pools" a 1000 Klone unterteilt worden. Positive "Pools" wurden mit Hilfe der PCR-Analyse unter Verwen- düng von nCL-3-spezifischen Primern identifiziert. Diese "Pools" wurden anschließend ausplattiert und und gescreent. Positive Klone wurden über Koloniehybridisierung mit 32P-markierten Maus nCL-3 cDNA-Fragmenten identifiziert.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. Beispiel: RNA Expressionsanalyse5. Example: RNA expression analysis
Die Expression des humanen nCL-3 wurde über Hybridisierung eines Humanen RNA "Master Blots" (Clontech) mit einem 32P-markierten humanen nCL-3 Fragment (Nucleotid 1 - 928 kodierend für die Aminosäuren 1 - 295) untersucht. Die Hybridisierung und die hoch- stringenten Waschbedingungen wurden entsprechend den Anweisungen des Hersteller durchgeführt.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. Beispiel: Lokalisierung des nCL-3 Gens auf dem Chromosom6. Example: Localization of the nCL-3 gene on the chromosome
Die Lokalisierung des Gens in der Maus erfolgte durch PCR-Analyse genomischer DNA, die von somatischen Maus x Hamster-Zellhybriden isoliert worden war, wie sie von Williamson et al. (Mamm. Genome 6, 1995, 429 - 432) beschrieben worden war, unter Verwendung eines Sets von DNAs wie sie Schupp et al. (Immunogenet . 45, 1997, 180 - 187) zu entnehmen ist. Als Primer-Sequenzen wurden 5'-tgca- cagcctacagcataag-3 ' und 5' -tcagacagccgtgagagagg-3' verwendet. Mit Hilfe dieser Primer konnte ein etwa 2.7 kb großes Fragment von Maus und keine Hamster DNA amplifiziert werden. Die PCR Reaktionen wurden mit dem "Expand Long Template PCR System (Boehrin- ger Mannheim) entsprechend den Anweisungen des Herstellers bei einer "Annealing" -Temperatur von 58 °C durchgeführt.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.
Die Lokalisierung des Gens im Mensch erfolgte mit Hilfe des NIGMS Mensch/Nager somatischen Zellhybrid "mapping panels" (Coriell Cell Repositories) . Als Primer-Sequenzen für die PCR-Reaktion wurden folgende Primer verwendet : 5' -acttcatcttc- tggcttcttgacttc-3' und 5' -gctgcatcaaccacaaggacac-3 ' . Die PCR- Amplification wurde mit einer "Annealing" -Temperatur von 58 °C durchgeführt und führte zu einem 600 bp Fragment. Die Ergebnisse wurden auf Übereinstimmung zwischen der Anwesenheit von menschlichen Chromosomen und dem PCR-Produkt untersucht. Die genaue Lokalisierung des Gens im menschlichen Chromosom erfolgte mit Hilfe des "Genebridge 4 RH Panels" (Research Genetics) und Übermittelung der PCR-Ergebnisse an den Lokalisierungsservice das MIF Center for Genome Research (http://www-genome.wi.mit.edu). 7. Beispiel: Cathepsin B-TestThe 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
Die Cathepsin B-Hemmung wurde analog einer Methode von S.Hasnain et al., J. Biol. Chem. 1993, 268, 235-40 bestimmt.Cathepsin B inhibition was determined analogously to a method by S.Hasnain et al., J. Biol. Chem. 1993, 268, 235-40.
Zu 88μL Cathepsin B ( Cathepsin B aus menschlicher Leber (Calbiochem) , verdünnt auf 5 Units in 500 μM Puffer) werden 2μL einer Inhibitor-Lösung, hergestellt aus Inhibitor und DMSO (Endkonzentrationen: 100 μM bis 0,01 μM) . Dieser Ansatz wird für 60 Minuten bei Raumtemperatur (25°C) vorinkubiert und anschließend die Reaktion durch Zugabe von 10 μL lOmM Z-Arg-Arg-pNA (in Puffer mit 10 % DMSO) gestartet. Die Reaktion wird 30 Minuten bei 405 nM im Mikrotiterplattenreader verfolgt. Aus den maximalen Steigungen werden anschließend die IC50 bestimmt.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. Beispiel: Calpain-Test8. Example: Calpain test
Die Aktivität der Calpain Inhibitoren wurde in einem colorimetri- schen Test mit Casein nach Hammarsten (Merck, Darmstadt) als Sub- strat untersucht. Der Test wurde in der Mikrotiterplatte, entsprechend der Veröffentlichung von Buroker-Kilgore und Wang in Anal. Biochemistry 208, 387-392 (1993), durchgeführt. Als Enzym wurde 29/30, welches in einem der oben beschriebenen Systemen exprimiert und anschließend gereinigt wurde, verwendet. Die Sub- stanzen wurden mit dem Enzym für 60 Minuten bei Raumtemperatur inkubiert, wobei eine Konzentration von 1% des Lösungsmittels DMSO nicht überschritten wurde. Nach Zugabe des Bio-Rad Farbreagenz erfolgte die Messung der optischen Dichte bei 595 nm in dem Easy Reader EAR 400 der Firma SLT. Die 50%ige Aktivität des En- zyms ergibt sich aus den optischen Dichten, die bei der maximalen Aktivität des Enzyms ohne Inhibitoren und der Aktivität des En¬ zyms ohne Zugabe von Kalzium bestimmt wurden.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. Beispiel: Plättchen-Test zur Bestimmung der zellulären Aktivi- tat von Calpain-Inhibitoren9. Example: Platelet test for determining the cellular activity of calpain inhibitors
Der Calpain-vermittelte Abbau von Proteinen in Plättchen wurde, wie von ZhaozhaoLi et al . , J. Med. Chem., 1993, 36, 3472-3480 beschrieben, durchgeführt. Humane Plättchen wurden aus frischem Natrium-Citrat-Blut von Spendern isoliert und in Puffer (5 mM Hepes, 140 mM NaCl und 1 mg/ml BSA, pH 7,3) auf 107 Zellen/ ml eingestellt.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).
Plättchen (0,1ml) werden für 5 Minuten mit 1 μl an verschiedenen Konzentrationen an Inhibitoren (gelöst in DMSO) vorinkubiert. Danach erfolgte die Zugabe von Kalziumionophor A 23187 (1 μM im Test) und Kalzium (5 mM im Test) und eine weitere Inkubation von 5 Minuten bei 37 C. Nach einem Zentrifugationsschritt wurden die Plättchen in SDS-Page Probenpuffer aufgenommen, 5 Minuten bei 95 °C gekocht und die Proteine in einem 8% igen Gel aufgetrennt. Der Abbau der beiden Proteine Actin bindendes Protein (ABP) und Talin wurde durch quantitative Densitometrie verfolgt, da nach der Zugabe von Kalzium und lonophor diese Proteine verschwanden und eine neue Bande im Bereich von 200 Kd Molekulargewicht entstand. Daraus wird die halb maximale Enzymaktivität bestimmt. 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.
SEQUENZPROTOKOLLSEQUENCE LOG
(1) ALGEMEINE INFORMATION:(1) GENERAL INFORMATION:
(i) ANMELDER:(i) APPLICANT:
(A) NAME: BASF Aktiengesellschaft(A) NAME: BASF Aktiengesellschaft
(B) STRASSE: Carl Bosch Strasse(B) STREET: Carl Bosch Strasse
(C) ORT: Ludwigshafen(C) LOCATION: Ludwigshafen
(D) BUNDESLAND: Rheinland-Pfalz(D) FEDERAL STATE: Rhineland-Palatinate
(E) LAND: Germany(E) COUNTRY: Germany
(F) POSTLEITZAHL: D-67056(F) POSTCODE: D-67056
(ii) ANMELDETITEL: Neue Calpaine, ihre Herstellung und Verwendung(ii) APPLICATION TITLE: New Calpaine, their production and use
(iii) ANZAHL DER SEQUENZEN: 7(iii) NUMBER OF SEQUENCES: 7
(iv) COMPUTER-LESBARE FORM:(iv) COMPUTER READABLE FORM:
(A) DATENTRÄGER: Floppy disk(A) DISK: Floppy disk
(B) COMPUTER: IBM PC compatible(B) COMPUTER: IBM PC compatible
(C) BETRIEBSSYSTEM: PC-DOS/MS-DOS(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPA)(D) SOFTWARE: Patentin Release # 1.0, Version # 1.25 (EPA)
(2) INFORMATION ZU SEQ ID NO: 1:(2) INFORMATION ABOUT SEQ ID NO: 1:
(i) SEQUENZ CHARAKTERISTIKA:(i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 2459 Basenpaare(A) LENGTH: 2459 base pairs
(B) ART: Nukleinsäure(B) TYPE: nucleic acid
(C) STRANGFORM: Einzel(C) STRAND FORM: Single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: CDNS(ii) MOLECULE TYPE: CDNS
(iii) HYPOTHETISCH: NEIN(iii) HYPOTHETICAL: NO
(iii) ANTISENSE: NEIN(iii) ANTISENSE: NO
(vi) URSPRÜNLICHE HERKUNFT:(vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Mus musculus(A) ORGANISM: Mus musculus
(ix) MERKMALE:(ix) FEATURES:
(A) NAME/SCHLÜSSEL: 5 ' UTR(A) NAME / KEY: 5 'UTR
(B) LAGE: 1..193(B) LOCATION: 1..193
(ix) MERKMALE:(ix) FEATURES:
(A) NAME/SCHLÜSSEL: 3 ' UTR(A) NAME / KEY: 3 'UTR
(B) LAGE: 2117..2459(B) LOCATION: 2117..2459
(ix) MERKMALE:(ix) FEATURES:
(A) NAME/SCHLÜSSEL: CDS(A) NAME / KEY: CDS
(B) LAGE: 194..2116(B) LOCATION: 194..2116
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 1: CTGAAGCCCG GGGGTCCAAG TTCCAACCCC CGCCTGCGGG CTGCCGGGGT ATCATCTCCC ^60(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CTGAAGCCCG GGGGTCCAAG TTCCAACCCC CGCCTGCGGG CTGCCGGGGT ATCATCTCCC ^ 60
CGCAGAGTCC CGGCCGTGGC GCGGGCTGGT CTAGCCTCCG CTCCAGTGCC CGCACTGTGC 120CGCAGAGTCC CGGCCGTGGC GCGGGCTGGT CTAGCCTCCG CTCCAGTGCC CGCACTGTGC 120
TCTGCATCCC GGGAGTCCAG CTCCAGCTGC GGCGACGCGG CAGGTGCCTC CCCTTCTTGG 180TCTGCATCCC 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 10GGACGTGGTC 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 25TAC 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 40GAT 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 60CCA 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 GlyCCC 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 7565 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 90CAG 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 105CGA 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 120TGG 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 140TTC 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 155CCC 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 170GAG 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 200TGT 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 220GCC 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 235GTG 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 250GCA 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 265GCA 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 280CTG 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 300CCC 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 315GAA 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 330GTG 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 345TAC 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 360AGC 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 380AGA 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 410AAG 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 425ACT 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 440TAT 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 460AAG 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 475ACA 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 490CCA 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 505TCC 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 520AGT 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 540GGG 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 555ATC 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 570ACC 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 585CTG 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 620GAT 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 635AGT 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 2153CTC ACG GCT GTC TGACCCCAGC CTGCCTGTCC TGCCCCACTA GTCCTCACCA 2153
Leu Thr Ala Val 640Leu Thr Ala Val 640
CTACTCGCAT GTCCCCACCT TGCCTGGGAC CAGCCTGGGA ACCAGACACT GGGGCCCTTT 2213CTACTCGCAT GTCCCCACCT TGCCTGGGAC CAGCCTGGGA ACCAGACACT GGGGCCCTTT 2213
CCTCACTCTT CCACTGACCC ACTGTGTGAC CTGAAGAGAG CCCTGCCCTC TCTGAGCCTC 2273CCTCACTCTT CCACTGACCC ACTGTGTGAC CTGAAGAGAG CCCTGCCCTC TCTGAGCCTC 2273
AGTGTTTGGA GGGCCCCAAA GAATTCCCGT CTTGTGGGGG AGTTTTCTTG CCTAAGATTT 2333AGTGTTTGGA GGGCCCCAAA GAATTCCCGT CTTGTGGGGG AGTTTTCTTG CCTAAGATTT 2333
AATGCAGTTC TCTCTACCCA GTGGGCGCTG CTGTTAAGGG GCCATCTGCT GAAAACGTTT 2393AATGCAGTTC TCTCTACCCA GTGGGCGCTG CTGTTAAGGG GCCATCTGCT GAAAACGTTT 2393
CCCCAGGCCC TGCTGTCTGC CAGGAGTGCC AAGTGTCAAC TGTTTACACA CAAACTGCCA 2453CCCCAGGCCC TGCTGTCTGC CAGGAGTGCC AAGTGTCAAC TGTTTACACA CAAACTGCCA 2453
TGTCCC 2459TGTCCC 2459
(2) INFORMATION ZU SEQ ID NO : 2:(2) INFORMATION ABOUT SEQ ID NO: 2:
(i) SEQUENZ CHARAKTERISTIK:(i) SEQUENCE CHARACTERISTICS:
(A) L0NGE: 640 Aminosäuren(A) L0NGE: 640 amino acids
(B) ART: Aminosäure (D) TOPOLOGIE: linear(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Protein(ii) MOLECULE TYPE: Protein
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 2:(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 15Met 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 30Lys 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 45Pro 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 60Val 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 80Val 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 LeuCys 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 11085 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 125Lys 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 140Gly 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 160Asn 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 175Ala 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 190Leu 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 205Val 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 220Ala 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 240Gly 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 255Glu 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 270Thr 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 285Lys 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 300Arg 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 320Val 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 335Gly 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 350Ile 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 365Thr 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 400Pro 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 415Val 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 430Gly 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 445Glu 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 460Ser 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 480Glu 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 495Gly 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 510Glu 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 525Gly 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 540Leu 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 560Glu 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 575Glu 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 590Ile 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 605Gly 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 620Thr 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 640Gly Ile Val Ala Val Arg Val Leu Cys Ser Ala Ser Leu Thr Ala Val 625 630 635 640
(2) INFORMATION ZU SEQ ID NO: 3:(2) INFORMATION ON SEQ ID NO: 3:
(i) SEQUENZ CHARAKTERISTIK:(i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 1743 Basenpaare(A) LENGTH: 1743 base pairs
(B) ART: Nukleinsäure (C) STRANGFORM: Einzel(B) TYPE: nucleic acid (C) STRAND FORM: Single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: cDNS (iii) HYPOTHETISCH: NEIN (iii) ANTISENSE: NEIN(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO
(vi) URSPRÜNLICHE HERKUNFT:(vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Mus musculus(A) ORGANISM: Mus musculus
(B) STAMM: balb/c(B) STEM: balb / c
(ix) MERKMALE:(ix) FEATURES:
(A) NAME/ SCHLÜSSEL: 5 ' UTR(A) NAME / KEY: 5 'UTR
(B) LAGE: 1..193(B) LOCATION: 1..193
(ix) MERKMALE:(ix) FEATURES:
(A) NAME/SCHLÜSSEL: 3 ' UTR(A) NAME / KEY: 3 'UTR
(B) LAGE: 1736..1743(B) LOCATION: 1736..1743
(ix) MERKMALE:(ix) FEATURES:
(A) NAME/SCHLÜSSEL: CDS(A) NAME / KEY: CDS
(B) LAGE: 194..1735(B) LOCATION: 194..1735
(xi) SEQUENZBΞSCHREIBUNG: SEQ ID NO: 3:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CTGAAGCCCG GGGGTCCAAG TTCCAACCCC CGCCTGCGGG CTGCCGGGGT ATCATCTCCC 60CTGAAGCCCG GGGGTCCAAG TTCCAACCCC CGCCTGCGGG CTGCCGGGGT ATCATCTCCC 60
CGCAGAGTCC CGGCCGTGGC GCGGGCTGGT CTAGCCTCCG CTCCAGTGCC CGCACTGTGC 120CGCAGAGTCC CGGCCGTGGC GCGGGCTGGT CTAGCCTCCG CTCCAGTGCC CGCACTGTGC 120
TCTGCATCCC GGGAGTCCAG CTCCAGCTGC GGCGACGCGG CAGGTGCCTC CCCTTCTTGG 180TCTGCATCCC 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 10GGACGTGGTC 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 25TAC 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 40GAT 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 60CCA 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 GlyCCC 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 9065 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 105CGA 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
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 120TGG 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 140TTC 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 155CCC 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 170GAG 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 185TGT 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 200TTC 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 220GCC 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 235GTG 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 49 Ala Ala Asp Met Glu Ala Arg Leu Ala Cys Gly Leu Val Lys Gly His 240 245 250GCA 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
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 265GCA 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 1O93CTG 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 300Pro 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 315GAA 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 330GTG 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 345TAC 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 360AGC 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 380AGA 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 395AAG 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 410CCA 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
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 425ACT 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 440TAT 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 460AAG 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 475ACA 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 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 505CCA 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 1743TAC CCC ATG CTG GGC TAGATTTTAA C 1743
Tyr Pro Met Leu Gly 510Tyr Pro Met Leu Gly 510
(2) INFORMATION ZU SEQ ID NO: 4:(2) INFORMATION ABOUT SEQ ID NO: 4:
(i) SEQUENZ CHARAKTERISTIKA:(i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 513 Aminosäuren(A) LENGTH: 513 amino acids
(B) ART: Aminosäure (D) TOPOLOGIE: linear(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Protein(ii) MOLECULE TYPE: Protein
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 4:(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 15Met 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 30Lys 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 45Pro 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 60Val 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 80Val 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 LeuCys Trp Phe Val Ala Ala Cys Ser Ser Leu Ala Ser Arg Glu Ser Leu
85 90 9585 90 95
Trp Gin Lys Val Ile Pro Asp Trp Lys Glu Gin Glu Trp Asn Pro Glu 100 105 110Trp 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 125Lys 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 140Gly 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 160Asn 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 175Ala 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 205Leu 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 220Ala 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 240Gly 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 255Glu 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 270Thr 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 285Lys 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 300Arg 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 320Val 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 335Gly 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 350Ile 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 365Thr 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 380Pro 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 400Phe 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 415Val 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 430Gly 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 445Glu 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 460Ser 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 ArgGlu 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 495485 490 495
Cys Val Gly Ala Arg Ala Ser Asp Arg Met His Ile Tyr Pro Met Leu 500 505 510Cys Val Gly Ala Arg Ala Ser Asp Arg Met His Ile Tyr Pro Met Leu 500 505 510
GlyGly
(2) INFORMATION ZU SEQ ID NO: 5:(2) INFORMATION ON SEQ ID NO: 5:
(i) SEQUENZ CHARAKTERISTIKA:(i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 504 Basenpaare(A) LENGTH: 504 base pairs
(B) ART: Nukleinsäure(B) TYPE: nucleic acid
(C) STRANGFORM: Einzel(C) STRAND FORM: Single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: DNS (genomisch) (iii) HYPOTHETISCH: NEIN (iii) ANTISENSE: NEIN(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO
(vi) URSPRÜNLICHE HERKUNFT:(vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Mus musculus(A) ORGANISM: Mus musculus
(B) STAMM: ES E14(B) STEM: ES E14
(vii) UNMITTELBARE HERKUNFT: (B) CLON: 29/30(vii) DIRECT ORIGIN: (B) CLON: 29/30
(ix) MERKMALE:(ix) FEATURES:
(A) NAME/SCHLÜSSEL: exon(A) NAME / KEY: exon
(B) LAGE: 1..33(B) LOCATION: 1..33
(ix) MERKMALE:(ix) FEATURES:
(A) NAME/SCHLÜSSEL: intron(A) NAME / KEY: intron
(B) LAGE: 34..440(B) LOCATION: 34..440
(ix) MERKMALE:(ix) FEATURES:
(A) NAME/SCHLÜSSEL: exon(A) NAME / KEY: exon
(B) LAGE: 441..504(B) LOCATION: 441..504
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 5:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CGAGCGGGAG TGGACGGGCC CCTGGAGTGA CACGTGAGGC TCACCAGGGT TGGGGCTGGG 60CGAGCGGGAG TGGACGGGCC CCTGGAGTGA CACGTGAGGC TCACCAGGGT TGGGGCTGGG 60
TATGGGCACA GAGGCAAGGA CAAGCGGTGA CACTGGACTG GGCCTTGCAG GGTCTGGGAG 120TATGGGCACA GAGGCAAGGA CAAGCGGTGA CACTGGACTG GGCCTTGCAG GGTCTGGGAG 120
AGATGCTCTG AGGAAAAAAT GGGAGACTTA CTTTCCAGTG TAAGTGTGGT GCTTGGGGGG 180AGATGCTCTG AGGAAAAAAT GGGAGACTTA CTTTCCAGTG TAAGTGTGGT GCTTGGGGGG 180
TAGGTTCATC AAGGACAGTG GCCAGAAGTG TGGCATGCTT TGTACGTGGA CAATGGCGCC 240TAGGTTCATC AAGGACAGTG GCCAGAAGTG TGGCATGCTT TGTACGTGGA CAATGGCGCC 240
TCACCAGCTT TATTCCCTGA CTTCATAGCC TTAGCATAAA GGAAGATCAC AGTTCCTAGT 300 GGGAGAGAAC AGAGGCTTCT TAGCAGGGCT GGGCATGGCC TCCAGGTCTC TACCCACAGT J60TCACCAGCTT TATTCCCTGA CTTCATAGCC TTAGCATAAA GGAAGATCAC AGTTCCTAGT 300 GGGAGAGAAC AGAGGCTTCT TAGCAGGGCT GGGCATGGCC TCCAGGTCTC TACCCACAGT J60
GCTCTGCAGG CGGCTTGGTC CAGAGCTCTC CCTTGGGCCA CTCCTCTTAT CCCGTTCCCT 420GCTCTGCAGG CGGCTTGGTC CAGAGCTCTC CCTTGGGCCA CTCCTCTTAT CCCGTTCCCT 420
CCCTGATACT CACTCCCCAG GTCAGAGGAA TGGCAGAAAG TGAGCAAGAG TGAGAGGGAG 480CCCTGATACT CACTCCCCAG GTCAGAGGAA TGGCAGAAAG TGAGCAAGAG TGAGAGGGAG 480
AAGATGGGCG TGACCGTGCA GGAT 504 (2) INFORMATION ZU SEQ ID NO: 6:AAGATGGGCG TGACCGTGCA GGAT 504 (2) INFORMATION ON SEQ ID NO: 6:
(i) SEQUENZ CHARAKTERISTIKA:(i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 1975 Basenpaare(A) LENGTH: 1975 base pairs
(B) ART: Nukleinsäure(B) TYPE: nucleic acid
(C) STRANGFORM: Einzel(C) STRAND FORM: Single
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: cDNS (iii) HYPOTHETISCH: NEIN (iii) ANTISENSE: NEIN(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTISENSE: NO
(vi) URSPRÜNLICHE HERKUNFT:(vi) ORIGINAL ORIGIN:
(A) ORGANISMUS: Homo sapiens(A) ORGANISM: Homo sapiens
(vii) UNMITTELBARE HERKUNFT:(vii) DIRECT ORIGIN:
(B) CLON: nCL-3(B) CLON: nCL-3
(ix) MERKMALE:(ix) FEATURES:
(A) NAME/SCHLÜSSEL: 5 ' UTR(A) NAME / KEY: 5 'UTR
(B) LAGE: 1..43(B) LOCATION: 1..43
(ix) MERKMALE:(ix) FEATURES:
(A) NAME/SCHLÜSSEL: 3 ' UTR(A) NAME / KEY: 3 'UTR
(B) LAGE: 1964..1975(B) LOCATION: 1964-1975
(ix) MERKMALE:(ix) FEATURES:
(A) NAME/SCHLÜSSEL: CDS(A) NAME / KEY: CDS
(B) LAGE: 44..1963(B) LOCATION: 44..1963
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 6:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
ACTCACTATA GGGCTCGAGC GGCCGCCCGG GCAGGTAGCC ACC ATG TTC TCG TGT 55ACTCACTATA GGGCTCGAGC GGCCGCCCGG GCAGGTAGCC ACC ATG TTC TCG TGT 55
Met Phe Ser Cys 1Met 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 20GTG 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 AspCGG 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 5025 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 65CGA 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 80AGC 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 100GCA 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 115ATC 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 130TAC 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 145GAC 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 160TAC 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 180AAG 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 195AAC 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 210ATC 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 225CAG 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 260AGT 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 275AAG 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 290TTG 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 305GGG 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 320GAG 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 340ATG 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 355CGC 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 370GCC 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 385CGC 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 400CAG 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 420ATC 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 435AAC 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 465TAC 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 480GTC 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 500CTC 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 515GAT 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 530CTG 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 545CCA 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 560GTC 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 580AAA 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 595GTC 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 610CTA 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 625CGG 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 1973GTG 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 640Val His Ile Leu Ser Ser Thr Ser Leu Thr Ala Val 630 635 640
TA 1975TA 1975
(2) INFORMATION ZU SEQ ID NO: 7: (i) SEQUENZ CHARAKTERISTIKA:(2) INFORMATION ABOUT SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS:
(A) LÄNGE: 640 Aminosäuren(A) LENGTH: 640 amino acids
(B) ART: Aminosäure (D) TOPOLOGIE: linear(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Protein(ii) MOLECULE TYPE: Protein
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 7:(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 15Met 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 30Arg 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 45Pro 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 60Val 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 80Val 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 LeuCys Trp Phe Val Ala Ala Cys Ser Ser Leu Ala Ser Arg Glu Ser Leu
85 90 9585 90 95
Trp Gin Lys Val Ile Pro Asp Trp Lys Glu Gin Glu Trp Asp Pro Glu 100 105 110Trp 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 125Lys 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 140Gly 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 160Asn 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 175Ala 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 190Leu 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 205Val 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 220Thr 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 255Gly 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 270Thr 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 285Lys 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 300Arg 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 320Val 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 335Gly 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 350Ile 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 365Thr 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 380Pro 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 400Phe 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 415Val 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 430Gly 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 445Glu 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 460Ser 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 480Glu 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 495Gly 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 510Glu 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 540Gly 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 560Glu 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 575Glu 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 590Ile 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 605Gly 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 620Thr 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 Gly Thr Val Ala Val His Ile Leu Ser Ser Thr Ser Leu Thr Ala Val 625 630 635 640

Claims

Patentansprüche claims
1. nCL-3-Calpaingen mit der Sequenz SEQ ID NO: 1 sowie seine allelischen Varianten, die auf der abgeleiteten Aminosäureebene eine Homologie von 60 bis 100 % aufweisen, Analoge oder Derivate.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 nach Anspruch 1, dadurch gekennzeichnet, daß es sich bei dem Analogen um eine verkürzte Gensequenz handelt.2. nCL-3-calpaingen according to claim 1, characterized in that it is a shortened gene sequence in the analog.
3. nCL-3-Calpaingen nach Anspruch 1 codiert durch die Sequenz SEQ ID NO: 6.3. nCL-3 calpaingen according to claim 1 encoded by the sequence SEQ ID NO: 6.
4. Genkonstrukt enthaltend ein nCL-3-Calpaingen gemäß den Ansprüchen 1 bis 3, das funktioneil mit einem oder mehreren Regulationssignalen zur Erhöhung der Genexpression funktioneil verknüpft ist.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. Aminosäuresequenzen codiert durch nCL-3-Gene gemäß Anspruch 1.5. Amino acid sequences encoded by nCL-3 genes according to claim 1.
6. Verfahren zur Identifizierung von Calpaininhibitoren, wobei man ein Calpain codiert durch eine Sequenz gemäß Anspruch 1 aus Geweben oder Zellen, in denen das Enzym nCL-3 exprimiert wird, isoliert und die Inhibierung der Spaltung eines Substrats des Enzyms nCL-3 und in mindestens einem weiteren Test die Inhibierung der Spaltung eines Substrats der Enzyme Calpain I und/oder II durch TestSubstanzen mißt und die Test- substanzen auswählt, die mindestens gegen eines der Calpaine eine inhibierende Wirkung zeigen.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. Verfahren nach Anspruch 6, dadurch gekennzeichnet, daß man die Testsubstanzen auswählt, die das Enzym nCL-3 nicht hemmen, jedoch die Enzyme Calpain I und/oder II.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. Verfahren nach Anspruch 6, dadurch gekennzeichnet, daß man die Testsubstanzen auswählt, die das Enzym nCL-3 hemmen, nicht jedoch die Enzyme Calpain I und/oder 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. Verfahren nach den Ansprüchen 6 bis 8, dadurch gekenn- zeichnet, daß man die Testsubstanzen auswählt, die in zellulären Systemen die Zellmembran passieren. 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. Verfahren zur Herstellung des Enzyms nCL-3, αaαurcn ge enn- zeichnet, daß man eine Kopie der Gensequenzen für nCL-3 gemäß Anspruch 1 in einen Vektor kloniert und in einem dem Vektor entsprechenden Wirtsorganismus das Gen für das Enzym nCL-310. 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 exprimiert und anschließend das Enzym aus dem Wirtsorganismus isoliert.5 expressed and then the enzyme isolated from the host organism.
11. Verfahren nach Anspruch 10, dadurch gekennzeichnet, daß man einen Vektor verwendet der in prokaryontischen oder eukaryon-11. The method according to claim 10, characterized in that one uses a vector in prokaryotic or eukaryonic
10 tischen Zellen die Expression des Gens für nCL-3 ermöglicht.10 cells allow expression of the gene for nCL-3.
12. Verfahren nach Anspruch 10, dadurch gekennzeichnet, daß man als Wirtsorganismus Bakterien, pilzliche oder tierische Zellen verwendet.12. The method according to claim 10, characterized in that bacteria, fungal or animal cells are used as the host organism.
1515
13. Verfahren nach Anspruch 10, dadurch gekennzeichnet, daß man als Vektor Baculoviren und als Wirtsorganismus Insektenzellen verwendet.13. The method according to claim 10, characterized in that one uses baculoviruses as vector and insect cells as host organism.
20 14. Verwendung eines Calpaininhibitors identifizierbar gemäß den Ansprüchen 6 bis 9 zur Herstellung von Medikamenten zur Behandlung von Krankheiten bei denen eine Calpainfehlfunktion vorliegt.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.
25 15. Verwendung eines Calpaininhibitors nach Anspruch 14 zur Herstellung von Medikamenten zur Behandlung von Krankheiten ausgewählt aus der Gruppe der kardiovaskulären, immunologischen, entzündlichen, allergischen, neurologischen, neurodegene- rativen, oder onkologischen Erkrankungen.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.
3030
16. Verwendung des Calpains nCL-3 gemäß Anspruch 5 in Test- systemen.16. Use of the Calpain nCL-3 according to claim 5 in test systems.
17. Verwendung des Calpains nCL-3 gemäß Anspruch 5 zur Her- 35 Stellung von Antikörpern.17. Use of the calpain nCL-3 according to claim 5 for the production of antibodies.
18. Verwendung einer Gensequenz gemäß Anspruch 1 zur Herstellung von antisense mRNA.18. Use of a gene sequence according to claim 1 for the production of antisense mRNA.
40 19. Verwendung der antisense mRNA nach Anspruch 18 zur Herstellung von Medikamenten zur Behandlung von Krankheiten bei denen eine Calpainfehlfunktion vorliegt.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. Verwendung eines Calpaingens gemäß Anspruch 1 zur Diagnose 45 von Krankheiten oder in der Gentherapie. 20. Use of a calpaing gene according to claim 1 for the diagnosis 45 of diseases or in gene therapy.
EP97952002A 1996-12-04 1997-11-28 Novel calpaines, production and use thereof Withdrawn EP0942995A1 (en)

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DE1996150142 DE19650142A1 (en) 1996-12-04 1996-12-04 nCL-3, a truncated calpain
DE19650142 1996-12-04
DE19718248 1997-04-30
DE1997118248 DE19718248A1 (en) 1997-04-30 1997-04-30 New nucleic acid encoding nCL-3 calpain
PCT/EP1997/006644 WO1998024916A1 (en) 1996-12-04 1997-11-28 Novel calpaines, production and use thereof

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AUPQ656500A0 (en) * 2000-03-28 2000-04-20 Autogen Pty Ltd A method of treatment and agents for same
WO2005082860A1 (en) * 2004-02-27 2005-09-09 National Research Council Of Canada Tetracyclines and their use as calpain inhibitors
CA2631071A1 (en) * 2008-05-09 2009-11-09 Tong-Jun Lin Inhibition of calpain reduces allergic inflammation
CN104458709A (en) * 2013-09-12 2015-03-25 中国药科大学 High flux screening method for screening calcium activated neutral protease-1 inhibitor
CN105334329B (en) * 2015-12-10 2017-07-28 中国农业科学院农产品加工研究所 The assay method of calpain phosphorylation level
US11981943B2 (en) * 2016-10-11 2024-05-14 University Of Iowa Research Foundation Methods and composition for detecting CAPN5
US20220364142A1 (en) * 2019-10-11 2022-11-17 The Board Of Trustees Of The Leland Stanford Junior University Compositions and methods for measuring and inhibiting calpain-5 activity

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