DE19928021A1 - New nucleic acid encoding testis-specific calpain-11, useful for identifying specific inhibitors for treatment of fertility disorders - Google Patents

New nucleic acid encoding testis-specific calpain-11, useful for identifying specific inhibitors for treatment of fertility disorders

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DE19928021A1
DE19928021A1 DE19928021A DE19928021A DE19928021A1 DE 19928021 A1 DE19928021 A1 DE 19928021A1 DE 19928021 A DE19928021 A DE 19928021A DE 19928021 A DE19928021 A DE 19928021A DE 19928021 A1 DE19928021 A1 DE 19928021A1
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Abstract

Nucleic acid (1) encoding human calpain, designated CAPN11, having a 702 amino acid sequence (2). Independent claims are also included for: (A) CAPN11; (B) a nucleic acid comprising a 2338 base pair sequence described in the specification; and (C) identifying inhibitors (A) of CAPN11 comprising: (a) comparing the enzyme activity of CAPN11 in comparison to the activity of the enzyme activity in the absence of CAPN11; and (b) from this identifying the inhibitor.

Description

Die Erfindung betrifft ein neues Säugetier-Calpain CAPN11, seine Synthese sowie seine Verwendung.The invention relates to a new mammalian calpain CAPN11, his Synthesis as well as its use.

Calpaine sind eine Superfamilie verwandter Proteine, von denen einige nachgewiesenermaßen als calciumabhängige Cysteinproteasen wirken. In Säugetieren sind acht verschiedene Calpaine identifi­ ziert worden.Calpaine are a super family of related proteins, one of which some have been shown to be calcium dependent cysteine proteases Act. Eight different calpains are identified in mammals been decorated.

Calpaine bilden eine Familie intrazellulärer calciumabhängiger Cystein-Proteasen. Man hat eine steigende Zahl von Säugetier-Cal­ pain-Homologa identifiziert, und die einzelnen Mitglieder lassen sich auf der Basis der physikalischen Struktur und der vorherge­ sagten Eigenschaften in vier Klassen einteilen. Die Klasse A, die "klassischen Calpaine" CAPN1, CAPN2, CAPN3 (p94); CAPN8 (nCL-2) und CAPN9 (nCL-4) sind wahrscheinlich alle proteaseaktiv und Ca2+-abhängig. Sie bestehen aus einer variablen großen (80 kDa) und einer invarianten kleinen Untereinheit (30 kDa). Die Calpaine der Klasse B und D, CAPN5 (6, 15) und CAPN7 (8) sind proteaseak­ tiv, jedoch höchstwahrscheinlich Ca2+-unabhängig, das Calpain der Klasse C, CAPN6, besitzt wahrscheinlich keine Proteaseaktivität. Die Calpaine lassen sich auch auf der Basis ihrer Expressionsmu­ ster in Kategorien einteilen, wobei CAPN3, CAPN6, CAPN8 und CAPN9 etwas Gewebespezifität aufweisen. Die Funktion der Calpaine ist nicht bekannt, obwohl sie mit sehr vielen physiologischen Prozes­ sen und pathologischen Zuständen in Verbindung gebracht wurden (Überblick in Literaturstelle 17). Zur Aufklärung ihrer Funktion und Evolutionsgeschichte ist die Identifizierung des gesamten Spektrums der Calpain-Familienmitglieder notwendig.Calpains form a family of intracellular calcium-dependent cysteine proteases. An increasing number of mammalian cal pain homologs have been identified, and the individual members can be divided into four classes based on the physical structure and the predicted properties. Class A, the "classic Calpaine" CAPN1, CAPN2, CAPN3 (p94); CAPN8 (nCL-2) and CAPN9 (nCL-4) are probably all protease-active and Ca 2+ dependent. They consist of a variable large (80 kDa) and an invariant small subunit (30 kDa). The class B and D calpains, CAPN5 (6, 15) and CAPN7 (8) are protease-active, but most likely Ca 2+ -independent, the class C calpain, CAPN6, probably has no protease activity. The calpains can also be divided into categories based on their expression pattern, with CAPN3, CAPN6, CAPN8 and CAPN9 showing some tissue specificity. The function of calpains is not known, although they have been associated with a large number of physiological processes and pathological conditions (overview in reference 17). To clarify their function and evolutionary history, the identification of the entire spectrum of the Calpain family members is necessary.

Die Erfindung betrifft das neue Polypetid CAPN11 mit der in SEQ.- ID.-Nr. 2 offenbarten Aminosäuresequenz.The invention relates to the new polypeptide CAPN11 with the SEQ.- ID no. 2 disclosed amino acid sequence.

Das neue Calpain-Protein CAPN11 besitzt die für Calpaine typi­ schen Eigenschaften, einschließlich potentieller Protease- und Calciumbindungs-Domänen. Es weist eine stark eingeschränkte Gewe­ beverteilung auf und wird hauptsächlich im Hoden exprimiert. Mit Hilfe der Strahlungs-Hybrid-Kartierung wurde das Gen auf Chromo­ som 6 in einer Region lokalisiert, die p12 zugeordnet wird. Aus phylogenetischer Analyse geht hervor, daß CAPN11 in Säugetieren am engsteh mit CAPN1 und CAPN2 verwandt ist.The new calpain protein CAPN11 has the typi for calpaine properties, including potential protease and Calcium binding domains. It has a severely restricted tissue distribution and is mainly expressed in the testis. With Radiation hybrid mapping was used to map the gene on Chromo som 6 localized in a region assigned to p12. Out Phylogenetic analysis shows that CAPN11 in mammals is most closely related to CAPN1 and CAPN2.

Die vorhergesagte CAPN11-Sequenz weist jedoch von den verfügbaren Calpainsequenzen die größte Homologie zum Küken-Calpain µ/m auf. Somit kann CAPN11 das menschliche Orthologon des µ/m-Calpains sein. Die Entdeckung dieses neuen Calpains betont die Komplexität der Calpain-Familie, deren Mitglieder sich auf der Basis der Pro­ teaseaktivität, Calciumabhängigkeit und Gewebeexpression unter­ scheiden lassen.However, the predicted CAPN11 sequence points from the available ones Calpain sequences the greatest homology to chick calpain µ / m. CAPN11 can therefore be the human orthologon of µ / m calpain  his. The discovery of this new calpain emphasizes the complexity the Calpain family, whose members are based on the Pro tease activity, calcium dependence and tissue expression under divorce.

Die cDNA-Nukleotidsequenz des CAPN11-Gens enthält 2338 Nukleotide (SEQ.-ID.-Nr. 1). Die cDNA-Sequenz stammt von einer einzigen mRNA durch erfolgreiche Amplifizierung der gesamten mutmaßlichen co­ dierenden Region aus menschlicher Hoden-cDNA mittels flankieren­ der Primer. Mehrere cDNA-Klone wurden vollständig sequenziert, um jedwede PCR-Artefakte auszuschließen.The cDNA nucleotide sequence of the CAPN11 gene contains 2338 nucleotides (SEQ.ID.No. 1). The cDNA sequence comes from a single mRNA by successfully amplifying the entire putative co flanking region from human testicular cDNA the primer. Several cDNA clones were fully sequenced to to rule out any PCR artifacts.

Es gibt ein großes offenes Leseraster, das ein Protein mit 702 Aminosäuren codiert (Mr 80 kDa) (Fig. 1). Die Aminosäuresequenz ähnelt der großen Untereinheit von Mitgliedern der Calpain-Fami­ lie (Fig. 1). Das Protein läßt sich in die vier für Calpain typi­ schen Domänen unterteilen. Die Domäne II weist die Eigenschaften einer Protease-Domäne auf, und die vorhergesagte Aminosäure­ sequenz besitzt die drei Aminosäurereste (Cys102, His259 und Asn283), die Teil des aktiven Zentrums von Cysteinproteasen sind (2). Die Aminosäuresequenz sämtlicher fünf, für CAPN2 beschriebe­ nen Ca2+-bindenden Sequenzen (4, 12) sind in gewissem Ausmaß kon­ serviert (Fig. 1). Dieses Protein besitzt somit wahrscheinlich Protease- und Calciumbindungs-Eigenschaften. Ein Vergleich der vorhergesagten Aminosäuresequenz mit denen sämtlicher anderer Calpaine ergab die größte Sequenzhomologie (57,5%) zum Küken-Cal­ pain µ/m. Bei den Säugetier-Calpainen bestand die größte Ähnlich­ keit zum menschlichen CAPN1 (54,3% Homologie). Das am wenigsten ähnliche menschliche Calpain mit nur 18,7% Homologie war CAPN6. Das dieser cDNA entsprechende Gen wurde vom Human Gene Nomencla­ ture Committee als CAPN11 bezeichnet.There is a large open reading frame encoding a 702 amino acid protein (Mr 80 kDa) ( Fig. 1). The amino acid sequence is similar to the large subunit of members of the Calpain family ( Fig. 1). The protein can be divided into the four domains typical for calpain. Domain II has the properties of a protease domain and the predicted amino acid sequence has the three amino acid residues (Cys102, His259 and Asn283) which are part of the active center of cysteine proteases (2). The amino acid sequence of all five Ca2 + -binding sequences (4, 12) described for CAPN2 are served to a certain extent ( FIG. 1). This protein is likely to have protease and calcium binding properties. A comparison of the predicted amino acid sequence with that of all other calpains revealed the greatest sequence homology (57.5%) for the chick cal pain µ / m. In mammalian calpains, the greatest similarity to human CAPN1 was found (54.3% homology). The least similar human calpain with only 18.7% homology was CAPN6. The gene corresponding to this cDNA was designated CAPN11 by the Human Gene Nomenclature Committee.

Die vollständige Aminosäuresequenz sämtlicher identifizierter menschlicher Calpaine wurde phylogenetisch analysiert. Die Ergeb­ nisse ermöglichen die Klassifizierung der menschlichen Calpaine in vier Haupt-Evolutionsgruppen (Fig. 2). Die erste Gruppe wird durch CAPN5, CAPN6, CAPN7 und CAPN8 vertreten, die zweite durch CAPN1 und CAPN2, die dritte Gruppe durch CAPN3 und CAPN9, und die vierte umfaßt CAPN11. Die phylogenetische Analyse legt somit nahe, daß CAPN11 eine eigene Calpain-Subfamilie darstellt.The complete amino acid sequence of all identified human calpains was analyzed phylogenetically. The results allow the human calpains to be classified into four main evolution groups ( Fig. 2). The first group is represented by CAPN5, CAPN6, CAPN7 and CAPN8, the second by CAPN1 and CAPN2, the third group by CAPN3 and CAPN9, and the fourth group comprises CAPN11. The phylogenetic analysis thus suggests that CAPN11 is a separate calpain subfamily.

Die Expression von CAPN11 in menschlichen Geweben wurde durch Northern- und RNA-DOt-Blot-Analyse untersucht. Von den 50 unter­ suchten Gewebe-RNAs wurde die CAPN11-mRNA am stärksten im Hoden exprimiert (Fig. 3A). Die Spezifität dieses Signals wurde durch Northern-Blot-Analyse bestätigt und entsprach einer etwa 3 kb großen mRNA (Fig. 3D). Im Thymus und in der Brustdrüse wurden viel schwächere Signale nachgewiesen. Die Signifikanz dieses Be­ fundes ist jedoch unklar, da eine weitere Untersuchung von Thy­ mus-RNA mit Northern-Blot-Analyse trotz langer Expositionszeiten kein Signal ergab (Fig. 3D und Daten nicht gezeigt). Eine mögli­ che Erklärung wäre, daß dieses schwache Signal auf eine Kreuzhy­ bridisierung mit verwandten mRNAs zurückzuführen ist. Somit ist der Hoden die Haupt-Expressionsstelle von CAPN11, obwohl wir die Möglichkeit nicht ausschließen können, daß das Gen in anderen, nicht untersuchten Geweben exprimiert wird.The expression of CAPN11 in human tissues was examined by Northern and RNA DOt blot analysis. Of the 50 tissue RNAs examined, the CAPN11 mRNA was most strongly expressed in the testis ( FIG. 3A). The specificity of this signal was confirmed by Northern blot analysis and corresponded to an approximately 3 kb mRNA ( FIG. 3D). Much weaker signals were detected in the thymus and in the mammary gland. However, the significance of this finding is unclear, since a further examination of Thymus RNA with Northern blot analysis did not produce any signal despite long exposure times ( FIG. 3D and data not shown). A possible explanation would be that this weak signal is due to cross hybridization with related mRNAs. Thus, the testis is the major expression site of CAPN11, although we cannot rule out the possibility that the gene may be expressed in other, unexamined tissues.

Wir haben bestimmt, auf welchem Chromosom das menschliche CAPN11-Gen lokalisiert ist. Mittels PCR mit Primern, die spezi­ fisch für die menschliche CAPN11-Nukleotidsequenz sind, haben wir mit einem somatischen Mensch/Nagetier-zellhybrid-Kärtierungspanel das Gen dem Chromosom 6 zugeordnet (Correll Cell Repositories). Mit Hilfe der Strahlungs-Hybridkartierung mit dem Stanford-G3-Pa­ nel mittlerer Auflösung (Research Genetics Inc.) und der Daten­ bank am Stanford HumanGenöme Center (shgc-www.stanford.edu) wurde das Gen 5 Centiray vom Marker SHGC-32834 entfernt auf die­ sem Chromosom lokalisiert (LOD-Score 12, 87). Dieser Marker befin­ det sich im Zwischenraum zwischen den Mikrosatelliten-Markern D6S1616 (59,6 cM) und D65427 (73,9 cM) (7), und ein Marker in diesem Zwischenraum, D65269, ist cytogenetisch 6p12 zugeordnet worden (5). Somit befindet sich CAPN11 auf Chromosom 6 in der Nähe von p12. Auf diesem Chromosom ist kein anderes Calpain-Gen lokalisiert worden.We have determined on which chromosome the human CAPN11 gene is localized. Using PCR with primers that are speci are fish for the human CAPN11 nucleotide sequence, we have with a somatic human / rodent cell hybrid curing panel the gene is assigned to chromosome 6 (Correll Cell Repositories). With the help of radiation hybrid mapping with the Stanford G3-Pa nel medium resolution (Research Genetics Inc.) and data bank at Stanford HumanGenöme Center (shgc-www.stanford.edu) the 5 Centiray gene was removed from the SHGC-32834 marker on the This chromosome localized (LOD score 12, 87). This marker is is in the space between the microsatellite markers D6S1616 (59.6 cM) and D65427 (73.9 cM) (7), and a marker in this space, D65269, is cytogenetically assigned to 6p12 been (5). CAPN11 is thus located on chromosome 6 in the Near p12. There is no other calpain gene on this chromosome been localized.

Das Küken-µ/m-Calpain war das erste Mitglied der zu klonierenden Calpain-Familie (16). Es wurde ursprünglich als m-Calpain be­ zeichnet, jedoch umklassifiziert, nachdem andere Küken-Calpaine identifiziert wurden, die sehr wahrscheinlich zu den µ- und m- Calpainen aus Säugetier ortholog sind (18). Ein Säugetier-µ/m- Calpain muß jedoch noch bestimmt werden. Da aber CAPN11 eine grö­ ßere Homologie zum Küken-µ/m-Calpain aufweist als zu anderen Säu­ getier-Calpainen, kann es sich dabei um dessen Orthologon han­ deln.The chick µ / m calpain was the first member of the clone to be cloned Calpain family (16). It was originally called m-Calpain records but reclassified after other chick-calpaine have been identified that are very likely to belong to the µ- and m- Calpaines from mammals are orthologous (18). A mammal µ / m Calpain has yet to be determined. But since CAPN11 is a big shows greater homology to the chick µ / m calpain than to other sau animal calpaines, it may be its ortholog deln.

Es gibt bisher 5 Calpaine, die einen gewissen Grad von Gewebespe­ zifität aufweisen - CAPN3 (Skelettmuskel), CAPN6 (Plazenta), CAPN8 (möglicherweise glatte Muskeln), CAPN9 (Magen und Dünndarm) und CAPN11 (Hoden). Man hat zahlreiche Proteasen im Hoden identi­ fiziert, und es wird angenommen, daß sie an Prozessen beteiligt sind, wie Gewebereorganisation (20), Regulation der Spermatoge­ nese (14), Durchdringung der Zona pellucida durch Sperma (10) und Fruchtbarkeit (13). Viele dieser Aktivitäten sind jedoch von se­ zernierten Proteasen abhängig, und CAPN11 ist wahrscheinlich in­ trazellulär lokalisiert. Im Hoden könnte es an Prozessen betei­ ligt sein, an denen Calpaine in anderen Geweben beteiligt sind, wie Keimzellen-Apoptose (3) oder Regulation von hodenspezifischen Transkriptionsfaktoren.So far there are 5 calpains that have a certain degree of tissue spike have specificity - CAPN3 (skeletal muscle), CAPN6 (placenta), CAPN8 (possibly smooth muscles), CAPN9 (stomach and small intestine) and CAPN11 (testicles). Numerous proteases have been identified in the testis infected, and is believed to be involved in processes are, like tissue reorganization (20), regulation of the spermatogen nese (14), penetration of the zona pellucida by sperm (10) and Fertility (13). However, many of these activities are from se harvested proteases, and CAPN11 is likely in Tracellularly localized. There could be processes in the testicles  in which calpains are involved in other tissues, such as germ cell apoptosis (3) or regulation of testicular-specific Transcription factors.

Ein weiterer Aspekt dieser Erfindung betrifft die Verwendung des Polypeptides CAPN11 zur Identifizierung von Substanzen, die die enzymatische Aktivität dieses Polypeptides hemmen können, soge­ nannte Calpain-Inhibitoren, insbesondere solche Calpain-Inhibito­ ren, die für CAPN11 selektiv sind. Selektivität bedeutet, daß solche Calpain-Inhibitoren die Aktivität von CAPN11 stärker hem­ men als die Aktivität der anderen vorstehend genannten Calpaine, und zwar vorzugsweise mindestens 10mal, stärker bevorzugt 25mal stärker. Die Enzymaktivität von CAPN11 ist eine Ca-abhängige Pro­ teaseaktivität.Another aspect of this invention relates to the use of the Polypeptides CAPN11 for the identification of substances that the can inhibit enzymatic activity of this polypeptide, so-called called calpain inhibitors, especially those calpain inhibito that are selective for CAPN11. Selectivity means that such calpain inhibitors inhibit CAPN11 activity more men as the activity of the other calpains mentioned above, preferably at least 10 times, more preferably 25 times stronger. The enzyme activity of CAPN11 is a Ca-dependent pro tease activity.

Ein weiterer Aspekt der Erfindung betrifft ein Verfahren zur Identifizierung von Verbindungen, die die Enzymaktivität eines Polypeptides nach Anspruch 1 hemmen, umfassend:
Another aspect of the invention relates to a method for identifying compounds which inhibit the enzyme activity of a polypeptide according to claim 1, comprising:

  • a) das Vergleichen des Ausmaßes der enzymatischen Aktivität von CAPN11 in Gegenwart der Verbindung mit dem Ausmaß der enzyma­ tischen Aktivität von CAPN11 in Abwesenheit der Verbindung unda) comparing the level of enzymatic activity of CAPN11 in the presence of the compound with the extent of the enzyma activity of CAPN11 in the absence of connection and
  • b) das Auswählen von Verbindungen, die das Ausmaß der enzymati­ schen Aktivität von CAPN11 gegenüber der enzymatischen Aktivität von CAPN11 in Abwesenheit der Verbindung ändern.b) selecting compounds that reflect the extent of the enzymati activity of CAPN11 against the enzymatic Change CAPN11 activity in the absence of the connection.

Die durch das vorstehend erwähnte Verfahren identifizierten hem­ menden Verbindungen eignen sich zur Behandlung von Erkrankungen, die mit einer unphysiologisch erhöhten CAPN11-Aktivität einherge­ hen oder damit verknüpft sind, wie Unfruchtbarkeit bei Männern.The hem identified by the above-mentioned method compounds are suitable for the treatment of diseases, which is associated with an unphysiologically increased CAPN11 activity hen or related to it, like male infertility.

Die Dosierung und das Behandlungsschema dieser Inhibitoren müssen durch Routineverfahren bestimmt werden, die von anderen Protease­ inhibitoren bekannt sind.The dosage and treatment regimen of these inhibitors must be be determined by routine procedures used by other protease inhibitors are known.

LITERATURLITERATURE

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6. Dear, N., Mätena, K., Vingron, M. und Boehm, T. (1997). A new subfamily of vertebrate calpains lacking a calmodulin-like domain: Implications for calpain regulation and evolution. Genomics 45: 175-184.
7. Deloukas, P. et al. (1998). A physical map of 30,000 human genes. Science 282: 744-746.
8. Franz, T., Vingron, N., Boehm, T. und Dear, T. N. (1999). Capn7: A highly divergent vertebrate calpain with a novel C-terminal domain. Mamm. Genome, im Druck.
9. Frohman, M. A., Dush, M. K. und Martin, G. R. (1988). Rapid production of full-length cDNAs from rare transcripts: ampli­ fication using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85: 8998-9002.
10. Kohno, N., Yamagata, K., Yamada, S., Kasiwabara, S., Sakal, Y. und Baba, T. (1998). Two novel testicular serine protea­ ses, TESP1 and TESP2, are present in the mouse sperm. acro­ some. Biochem. Biophys. Res. Commun. 245: 658-665.
11. Kozak, M. (1996). Interpreting cDNA sequences: some insights from studies on translation. Mamm. Genome 7: 563-574.
12. Lin, G.-D., Chattopadhyay, D., Maki, M., Wang, K. K. W., Carson, M., Jin, L., Yuen, P.-W., Takano, E., Hatanaka, M., DeLucas, L. J. und Narayana, S. V. L. (1997). Crystal structure of calcium bound domain VI of calpain at 1.9 Ai.resolution and its role in enzyme assembly, regulation, and inhibitor bind­ ing. Nature Structural Biology 4: 539-547.
13. Mbikay, M., Tadros, H., Ishida, N., Lerner, C. P., De Lami­ rande, E., Chen, A., E1-Alfy, M., Clermont, Y., Seidah, N. G., Chretien, N., Gagnon, C. und Simpson, E. M. (1997). Impaired fertility in mice deficient for the testicular germ-cell protease PC4. Proc. Natl. Acad. Sci. USA 94: 6842-6846.
14. Monsees, T. K., Gornig, N., Schill, W. B. und Miska, W. (1998). Possible involvement of proteases in the regulation of sper­ matogenesis. Andrologia 30: 185-191.
15. Mugita, N., Kimura, Y., Ogawa, M., Saya, H. und Nakao, M. (1997), Identification of a novel, tissue-specific calpain htra-3; a human homologue of the Caenorhabditis elegans sex determination gene. Biochern. Biophys. Res. Comm. 239: 845-850.
16. Ohno, 5., Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M. und Suzuki, K. (1984). Evolutionary origin of a calcium-de­ pendent protease by fusion of genes for a thiol protease and a calcium-binding protein? Nature 312: 566-570.
17. Ono, Y., Sorimachi, H. und Suzuki, K. (1998). Structure and physiology of calpain, an enigmatic protease. Biochem. Biophys. Res. Commun. 245: 289-294.
18. Sorimachi, H., Tsukahara, T., Okada-Ban, M., Sugita, H., Ishiura, S. und Suzuki, K. (1995). Identification of a third ubiquitous calpain species - chicken muscle expresses four distinct calpains. Biochim. Biophys. Acta 1261: 381-393.
19. Thompson J. D., Higgins D. G. und Gibson T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22: 4673-4680.
20. Tohonen, V., Osterlund, C. und Nordqvist, K. (1998). Testa­ tin: A cystatin-related gene expressed during early testis development. Proc Natl. Acad. Sci. USA 95: 14208-14213.-induced conformational changes. Nature structural Biology 4: 532-538.
5. Bray-Ward, P., Menninger, J., Lieman, T., Desai, T., Mokady, N., Banks, A. and Ward, DC (1996). Integration of the cyto genetic, genetic and physical maps of the human genome by FISH mapping of CEPH YAC clones. Genomics 32: 1-14.
6. Dear, N., Matena, K., Vingron, M. and Boehm, T. (1997). A new subfamily of vertebrate calpains lacking a calmodulin-like domain: Implications for calpain regulation and evolution. Genomics 45: 175-184.
7. Deloukas, P. et al. (1998). A physical map of 30,000 human genes. Science 282: 744-746.
8. Franz, T., Vingron, N., Boehm, T. and Dear, TN (1999). Capn7: A highly divergent vertebrate calpain with a novel C-terminal domain. Mom. Genomes, in press.
9. Frohman, MA, Dush, MK and Martin, GR (1988). Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85: 8998-9002.
10. Kohno, N., Yamagata, K., Yamada, S., Kasiwabara, S., Sakal, Y. and Baba, T. (1998). Two novel testicular serine protea ses, TESP1 and TESP2, are present in the mouse sperm. acro some. Biochem. Biophys. Res. Commun. 245: 658-665.
11. Kozak, M. (1996). Interpreting cDNA sequences: some insights from studies on translation. Mom. Genome 7: 563-574.
12. Lin, G.-D., Chattopadhyay, D., Maki, M., Wang, KKW, Carson, M., Jin, L., Yuen, P.-W., Takano, E., Hatanaka, M ., DeLucas, LJ and Narayana, SVL (1997). Crystal structure of calcium bound domain VI of calpain at 1.9 Ai.resolution and its role in enzyme assembly, regulation, and inhibitor bind ing. Nature Structural Biology 4: 539-547.
13. Mbikay, M., Tadros, H., Ishida, N., Lerner, CP, De Lami rande, E., Chen, A., E1-Alfy, M., Clermont, Y., Seidah, NG, Chretien , N., Gagnon, C. and Simpson, EM (1997). Impaired fertility in mice deficient for the testicular germ-cell protease PC4. Proc. Natl. Acad. Sci. USA 94: 6842-6846.
14. Monsees, TK, Gornig, N., Schill, WB and Miska, W. (1998). Possible involvement of proteases in the regulation of sper matogenesis. Andrologia 30: 185-191.
15. Mugita, N., Kimura, Y., Ogawa, M., Saya, H. and Nakao, M. (1997), Identification of a novel, tissue-specific calpain htra-3; a human homologue of the Caenorhabditis elegans sex determination gene. Biochern. Biophys. Res. Comm. 239: 845-850.
16. Ohno, 5th, Emori, Y., Imajoh, S., Kawasaki, H., Kisaragi, M. and Suzuki, K. (1984). Evolutionary origin of a calcium-de pendent protease by fusion of genes for a thiol protease and a calcium-binding protein? Nature 312: 566-570.
17. Ono, Y., Sorimachi, H. and Suzuki, K. (1998). Structure and physiology of calpain, an enigmatic protease. Biochem. Biophys. Res. Commun. 245: 289-294.
18. Sorimachi, H., Tsukahara, T., Okada-Ban, M., Sugita, H., Ishiura, S. and Suzuki, K. (1995). Identification of a third ubiquitous calpain species - chicken muscle expresses four distinct calpains. Biochim. Biophys. Acta 1261: 381-393.
19. Thompson JD, Higgins DG and Gibson TJ (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22: 4673-4680.
20. Tohonen, V., Osterlund, C. and Nordqvist, K. (1998). Testa tin: A cystatin-related gene expressed during early testis development. Proc Natl. Acad. Sci. USA 95: 14208-14213.

FIGURENCHARACTERS

Fig. 1. Gruppierung der vorhergesagten Aminosäuresequenz von CAPN11 mit anderen menschlichen Calpainen. Die Mehrfach-Gruppie­ rung der Aminosäuresequenzen wurde mittels CLUSTAL W (19) durch­ geführt. Das mutmaßliche Start-Methionin für CAPN11 (GGAatgG) entspricht der minimalen Konsensussequenz für die Translations­ startstelle (RNNatgG, wobei R ein Purin ist, Lit. 11). Aminosäu­ ren, die in den anderen Proteinen mit denen von CAPN11 identisch sind, sind schattiert. Striche bedeuten Lücken, die zur Maximie­ rung des Alignments eingefügt worden sind. Pfeilspitzen deuten auf die drei konservierten Aminosäuren hin, die Teil des aktiven Zentrums der Calpaine sind. Die potentiellen EF-Hand-Calcium-Bin­ dungsdomänen von CAPN8 sind unterstrichen und nacheinander durch­ nummeriert. Es sind die willkürlichen Domänen von Calpain angege­ ben. Die Sequenzen für CAPN4 und CAPN7, die nur in Ratte bzw. Maus identifiziert worden sind, sind nicht gezeigt. Die ver­ öffentlichte CAPN6-Sequenz wurde nicht mit aufgenommen, da sie nur eine Teilsequenz ist. Die Alternativnamen und Zugangsnummern für die gruppierten Calpaine sind in der Legende von Fig. 2 an­ gegeben. Figure 1. Grouping of the predicted amino acid sequence of CAPN11 with other human calpains. The multiple grouping of the amino acid sequences was carried out using CLUSTAL W (19). The putative start methionine for CAPN11 (GGAatgG) corresponds to the minimal consensus sequence for the translation start site (RNNatgG, where R is a purine, ref. 11). Amino acids that are identical to those of CAPN11 in the other proteins are shaded. Dashes indicate gaps that have been added to maximize alignment. Arrowheads indicate the three conserved amino acids that are part of the active center of the Calpaine. The potential EF hand calcium binding domains of CAPN8 are underlined and numbered consecutively. The arbitrary domains of Calpain are given. The sequences for CAPN4 and CAPN7, which were only identified in rats and mice, are not shown. The published CAPN6 sequence was not included because it is only a partial sequence. The alternative names and accession numbers for the grouped calpains are given in the legend of FIG. 2.

Fig. 2. Wurzelloser phylogenetischer Stammbaum der Familie der großen Untereinheit menschlicher Calpaine. Die Analyse wurde mit dem PAUP-Programm durchgeführt, und der Stammbaum mit CLUSTREE vom HUSAR-Server des Deutschen Krebsforschungszentrums, Heidel­ berg (www.dkfz-heidelberg.de) angeordnet. Die Längen der horizon­ talen Linien sind proportional zu den abgeleiteten phylogeneti­ schen Entfernungen; die vertikalen Linien haben keine Bedeutung. Es wurden 1000 Bootstrapping-Wiederholungen durchgeführt, und die Werte sind an den inneren Knoten gezeigt. Die CAPN7-Sequenz stammt von der Maus, da nur wenig menschliche Nukleotid- und Pro­ teinsequenz verfügbar ist. Allerdings existiert das menschliche Orthologon (siehe Lit. 8), so daß die Verwendung der Maussequenz für diesen Vergleich gerechtfertigt ist. Die partielle menschli­ che CAPN8-Sequenz ist die vorhergesagte Translation des EST-Klo­ nes AA026030 (Hillier et al., 1995, The WashU-Merck EST-Projekt, unveröffentlichte Ergebnisse). Eine Aminosäuretranslation dieses Klons zeigt hohe Ähnlichkeit zur Ratten-CAPN8-Sequenz. Mit dieser Sequenz wurde kein Bootstrapping durchgeführt, da sie viel kürzer als die anderen ist. Somit kann der Bootstrapping-Wert nicht sinnvoll mit den Werten aus Vergleichen von Vollängen-Sequenzen verglichen werden. Es wird die vom Human Gene Nomenclature Committee spezifizierte Nomenklatur verwendet. Vorherige Namen für die verschiedenen Calpaine sind: CAPN1 - m-Calpain, CAPN2 - m-Calpain, CAPN3 - p94, nCl-1, CAPN8, nCL-2, CAPN9, nCL-4. Die EMBL-Zugangsnummern für die verwendeten Calpain-Sequenzen sind:
CAPN1 (P17655), CAPN2 (P07384), CAPN3 (P20807), CAPN5 (Y10656), CAPN6 (Y12582), CAPN7 (AJ012475) und CAPN9 (AF022799). Fig. 2. Rootless phylogenetic family tree of the family of the large subunit of human calpaine. The analysis was carried out with the PAUP program and the family tree with CLUSTREE from the HUSAR server of the German Cancer Research Center, Heidelberg (www.dkfz-heidelberg.de) arranged. The lengths of the horizontal lines are proportional to the derived phylogenetic distances; the vertical lines have no meaning. 1000 bootstrapping retries were performed and the values are shown on the inner nodes. The CAPN7 sequence comes from the mouse, since only a little human nucleotide and protein sequence is available. However, the human orthologue exists (see ref. 8), so the use of the mouse sequence for this comparison is justified. The partial human CAPN8 sequence is the predicted translation of the EST clone AA026030 (Hillier et al., 1995, The WashU-Merck EST project, unpublished results). An amino acid translation of this clone shows high similarity to the rat CAPN8 sequence. No bootstrapping was done with this sequence because it is much shorter than the others. The bootstrapping value can therefore not be meaningfully compared with the values from comparisons of full-length sequences. The nomenclature specified by the Human Gene Nomenclature Committee is used. Previous names for the different calpains are: CAPN1 - m-Calpain, CAPN2 - m-Calpain, CAPN3 - p94, nCl-1, CAPN8, nCL-2, CAPN9, nCL-4. The EMBL accession numbers for the calpain sequences used are:
CAPN1 (P17655), CAPN2 (P07384), CAPN3 (P20807), CAPN5 (Y10656), CAPN6 (Y12582), CAPN7 (AJ012475) and CAPN9 (AF022799).

Fig. 3. Expression von CAPN11. Eine 32P-markierte DNA-Sonde mit einem 800-Basenpaar-Segment der codierenden Sequenz der menschli­ chen CAPN11-cDNA wurde an einen Master-Blot (A), einen Nylonfil­ ter mit Dot-Blots von RNAs von 50 verschiedenen menschlichen Ge­ weben oder einen Clontech-Mehrgewebe-Northern-Blot (D) hybridi­ siert. Die Filter wurden hochstringent (6x SSC, 65°C) gewaschen. Die genaue Stelle der verschiedenen RNAS auf dem Dot-B1ot-Filter ist schematisch gezeigt (C). Die ENAs auf dem Northern-Blot sind über den entsprechenden Spuren angegeben. Dot-Blot- und Northern- Blot wurden mit DNA-Sonden für menschliches Ubiquitin (B) und b-Aktin rehybridisiert, um die Mengen der aufgetragenen Poly(A+)-RNA zu bestimmen. Für den Northern-Blot sind die Stellen der Größenmarker (in Kilobasen) angegeben. Die Expositionszeiten waren: A, 72 Std.; B, 24 Std.; D, 48 Std. PBL = Periphere Blut- Leukocyten. Figure 3. Expression of CAPN11. A 32P-labeled DNA probe with an 800 base pair segment of the coding sequence of the human CAPN11 cDNA was attached to a master blot (A), a nylon filter with dot blots of RNAs from 50 different human tissues or a Clontech multi-tissue Northern blot (D) hybridized. The filters were washed with high stringency (6x SSC, 65 ° C). The exact location of the different RNAS on the Dot B1ot filter is shown schematically (C). The ENAs on the Northern blot are indicated above the corresponding lanes. Dot blot and Northern blot were re-hybridized with human ubiquitin (B) and b-actin DNA probes to determine the amounts of poly (A +) RNA applied. The positions of the size markers (in kilobases) are given for the Northern blot. The exposure times were: A, 72 hours; B, 24 hours; D, 48 hrs. PBL = peripheral blood leukocytes.

SEQUENZPROTOKOLL SEQUENCE LOG

Claims (6)

1. Polypeptid mit der Aminosäuresequenz SEQ.-ID.-Nr. 2.1. Polypeptide with the amino acid sequence SEQ.ID.No. 2nd 2. Polynukleotidsequenz, die ein Polypeptid nach Anspruch 1 codiert.2. A polynucleotide sequence comprising a polypeptide according to claim 1 coded. 3. Polynukleotidsequenz mit der Sequenz SEQ.-ID.-Nr. 1.3. Polynucleotide sequence with the sequence SEQ.ID.No. 1. 4. Verwendung eines Polypeptides nach Anspruch 1 zur Identifi­ zierung von Substanzen, die die enzymatische Aktivität dieses Polypeptides hemmen können.4. Use of a polypeptide according to claim 1 for identification ornamentation of substances that have the enzymatic activity of this Can inhibit polypeptides. 5. Verfahren zur Identifizierung von Verbindungen, die die enzy­ matische Aktivität eines Polypeptides nach Anspruch 1 hemmen, umfassend:
  • a) das Vergleichen des Ausmaßes der enzymatischen Aktivität von CAPN11 in Gegenwart der Verbindung mit dem Ausmaß der enzymatischen Aktivität von CAPN11 in Abwesenheit der Verbindung und
  • b) das Auswählen von Verbindungen, die das Ausmaß der enzy­ matischen Aktivität von CAPN11 gegenüber der enzymati­ schen Aktivität von CAPN11 in Abwesenheit der Verbindung ändern.
5. A method of identifying compounds that inhibit the enzymatic activity of a polypeptide according to claim 1, comprising:
  • a) comparing the level of enzymatic activity of CAPN11 in the presence of the compound with the level of enzymatic activity of CAPN11 in the absence of the compound and
  • b) selecting compounds that change the level of the enzymatic activity of CAPN11 over the enzymatic activity of CAPN11 in the absence of the compound.
6. Verwendung von Verbindungen, die durch das Verfahren nach Anspruch 5 identifiziert worden sind, zur Behandlung von Fruchtbarkeitsstörungen bei Männern.6. Use of compounds by following the procedure Claim 5 have been identified for the treatment of Fertility disorders in men.
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IL14694000A IL146940A0 (en) 1999-06-18 2000-06-07 Novel calpains and their use
AU59706/00A AU5970600A (en) 1999-06-18 2000-06-07 Novel calpains and their use
BR0011721-8A BR0011721A (en) 1999-06-18 2000-06-07 Polypeptide, polynucleotide sequence, use of a polypeptide, process to identify compounds that inhibit the enzymatic activity of a polypeptide, and use of compounds
EP00945716A EP1185673A2 (en) 1999-06-18 2000-06-07 Novel calpains and their use
CA002375477A CA2375477A1 (en) 1999-06-18 2000-06-07 Novel calpains and their use
TR2001/03662T TR200103662T2 (en) 1999-06-18 2000-06-07 New calpains and their use
PCT/EP2000/005261 WO2000078933A2 (en) 1999-06-18 2000-06-07 Novel calpains and their use
CN00808932A CN1373808A (en) 1999-06-18 2000-06-07 Calpains and their use
PL00353004A PL353004A1 (en) 1999-06-18 2000-06-07 Novel calpains and their use
KR1020017016244A KR20020011140A (en) 1999-06-18 2000-06-07 Novel Calpains and Their Use
JP2001505676A JP2003503023A (en) 1999-06-18 2000-06-07 New calpain and its use
HU0201800A HUP0201800A2 (en) 1999-06-18 2000-06-07 Novel calpains and their use
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