CN1373808A - 新型钙蛋白酶及其应用 - Google Patents
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Abstract
本发明涉及新型钙蛋白酶及其应用。
Description
本发明涉及一种新型的哺乳动物钙蛋白酶CAPN11，其合成方法及其应用。
钙蛋白酶是相关蛋白质的超家族，据证实其中的一些起钙依赖的半胱氨酸蛋白酶的作用。在哺乳动物中鉴定出了八种不同的钙蛋白酶。
钙蛋白酶构成胞内钙依赖性半胱氨酸蛋白酶的一个家族。人们鉴定出的哺乳动物钙蛋白酶同系物的数量不断增加，而且各成员可根据物理结构和推测的性质分成四类。A类，“经典的钙蛋白酶”CAPN1，CAPN2，CAPN3(p-94)；CAPN8(nCL-2)和CAPN9(nCL-4)大约全部皆是具有蛋白酶活性和Ca2+依赖性的。它们由一大小可变的(80kDa)和一不变化的小亚基(30kDa)组成。B和D类的钙蛋白酶，CAPN5(6，15)和CAPN7(8)具有蛋白酶活性，但是很可能是Ca2+非依赖性的，C类的钙蛋白酶，CAPN6，可能不具有蛋白酶活性。钙蛋白酶可根据其表达模式分类，其中CAPN3，CAPN6，CAPN8和CAPN9具有一些组织特异性。钙蛋白酶的功能还不清楚，虽然它们与很多生理过程和病理状态有关(文献17中的综论)。为了弄清它们的功能和进化历程，必须鉴定钙蛋白酶家族成员的总谱。
本发明涉及新型的多肽CAPN11，在SEQ.ID.No.2中公开了其氨基酸序列。
该新的钙蛋白酶CAPN11具有典型的钙蛋白酶特性，包括潜在的蛋白酶和钙结合域。它具有较强局限性的组织分布并主要在睾丸中表达。籍助放射杂交作图(strahlungs-Hybrid-Kartierung)，已将该基因定位在染色体6上指定为p12的区域中。系统发生分析表明，哺乳动物中的CAPN11与CAPN1和CAPN2最密切相关。
然而，推测的CAPN11序列在可获得的钙蛋白酶序列中与鸡钙蛋白酶μ/m具有最大同源性。因此，CAPN11可能是μ/m钙蛋白酶的人体直向进化同源物(orthologon)。此新型钙蛋白酶的发现着重说明了钙蛋白酶家族的复杂性，其成员可根据蛋白酶活性，钙依赖性及组织表达加以区分。
CAPN11基因的cDNA核苷酸序列含有2338个核苷酸(SEQ.-ID.-No.1)。cDNA序列源于单一mRNA，借助侧翼引物由人体睾丸cDNA成功扩增整个推断编码区而获得的。对多个cDNA克隆进行了完全测序，以便排除任何PCR假象。
其中有一个大的开放阅读框，编码具有702个氨基酸的蛋白质(Mr80kDa)(图1)。氨基酸序列与钙蛋白酶家族成员的大亚基相似(图1)。该蛋白质可分成四个钙蛋白酶典型性的结构域。结构域II具有蛋白酶结构域的特性，其推测的氨基酸序列具有作为半胱氨酸蛋白酶活性位点部分的三个氨基酸基团(Cys102，His259和Asn283)(2)。对CAPN2报导的所有五个Ca2+结合序列(4，12)的氨基酸序列在一定程度上保守(图1)。因此，这种蛋白质可能具有蛋白酶特性和钙结合特性。推测的氨基酸序列与全部其它的钙蛋白酶相比表明，它与小鸡钙蛋白酶μ/m具有最大的序列同源性(57.5％)。在哺乳动物钙蛋白酶中，与人体CAPN1具有最大相似性(54.3％同源性)。具有最小相似性的人体钙蛋白酶是CAPN6，其同源性只有18.7％。对应于此cDNA的基因被人类基因命名委员会称为CAPN11。
对全部被鉴定的人体钙蛋白酶的完整氨基酸序列进行了系统发生分析。其结果可将人体钙蛋白酶分成四个主要进化组(图2)。第一组的代表为CAPN5，CAPN6，CAPN7和CAPN8，第二组的代表为CAPN1和CAPN2，第三组的代表为CAPN3和CAPN9，而第四组包括CAPN11。因此，系统发生分析暗示CAPN11可能代表另外的钙蛋白酶亚家族。
通过Northern和RNA斑点印迹分析对人体组织中CAPN11的表达进行了研究。在50种被研究的组织RNA中，CAPN11 mRNA在睾丸中表达最强(图3A)。这种信号的特异性已被Northern印迹分析证实，其对应于大约3kb大的mRNA(图3D)。在胸腺和乳腺中测出了弱得多的信号。但是这个发现的意义并未弄清，因为在通过Northern印迹分析对胸腺RNA进行的进一步研究中，虽然曝光时间较长仍未给出任何信号(图3D，数据未给出)。一种可能的解释是，这种弱信号归咎于与相关mRNA的交叉杂交。因此睾丸是CAPN11的主要表达位置，但我们不能排除该基因在其它尚未研究的组织中表达的可能性。
我们已经确定人体CAPN11基因定位在哪条染色体上。籍助PCR，用对人体CAPN11核苷酸序列特异的引物以及人/啮齿动物体细胞杂交作图板，我们已将该基因定位到染色体6上(Correll细胞文库)。籍助放射杂交作图，使用中等分辨的Stanford-G3板(Research Genefics Inc.)和Stanford Human GenomeCenter(Shgc-www.Stanford.edu)的数据库，已将该基因定位在该染色体上，距离标志物SHGC-32834有5centiray(LOD Score12.87)。这个标记物位于微卫星标记D6S1616(59.6cM)和D6S427(73.9cM)之间的间隔中(7)，该间隔中的一个标记物D6S269按细胞遗传定位在6p12上(5)。因此，CAPN11位于染色体6上p12附近。在此染色体上未定位任何其它的钙蛋白酶基因。
小鸡μ/m钙蛋白酶是欲克隆的钙蛋白酶家族的第一个成员(16)。开始时它被称为m钙蛋白酶但在其它的鸡钙蛋白酶被鉴定出之后被重新分类，它们很可能是哺乳动物μ和m钙蛋白酶的直向进化同源物(18)。但是尚未测定到哺乳动物μ/m钙蛋白酶。由于CAPN11与小鸡μ/m钙蛋白酶的同源性比其它哺乳动物钙蛋白酶的大，它可能是其直向进化同源物。
迄今有五种钙蛋白酶具有一定程度的组织特异性-CAPN3(骨骼肌)，CAPN6(胎盘)，CAPN8(可能是平滑肌)，CAPN9(胃和小肠)和CAPN11(睾丸)。人们已经在睾丸中鉴定出多种蛋白酶，推测它们参与一些过程例如组织重组等(20)，精子发生调控(14)，精子渗入透明带(10)和受精(13)。但是这些活性中有许多与分泌蛋白酶有关，而APN11可能是胞内定位的。在睾丸中它可能参与其它组织中的钙蛋白酶参与的一些过程，例如生殖细胞凋亡(3)或睾丸特异性转录因子的调控。
本发明的另一主题涉及多肽CAPN11用于鉴定能抑制这种多肽酶活性的物质(即所谓的钙蛋白酶抑制剂)的用途，特别是对CAPN11具有选择性的这类钙蛋白酶抑制剂。选择性意味着，这类钙蛋白酶抑制剂抑制CAPN11的活性比抑制其它上面提及的钙蛋白酶更强，优选至少强10倍，更优选强25倍，或更多。CAPN11的酶活性是一种Ca依赖性的蛋白酶活性。
本发明的另一主题涉及一种鉴定可抑制权利要求1多肽的酶活性的化合物的方法，包括：
(a)将在该化合物存在时CAPN11的酶活性程度与无该化合物存在时CAPN11的酶活性程度进行比较，和
(b)选择与无该化合物存在时CAPN11的酶活性相比改变了CAPN11酶活性程度的化合物。
按上面提及的方法鉴定出的抑制性化合物适宜于治疗由于CAPN11活性非生理性提高所致或与此相关的疾病，例如男人的不育症。
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附图
图1，将CAPN11的推测氨基酸序列与其它人体钙蛋白酶进行分组比较。氨基酸序列的多次分组籍助CLUSTAL W(19)进行。CAPN11推测的起始甲硫氨酸(GGAatgG)相当于翻译起始位点的最小共有序列(RNNatgG，其中R为嘌呤，文献11)。在其它蛋白质中与CAPN11相同的氨基酸用阴影部分表示。虚线表示缺口，将之引入以便使比对最大化。箭头尖指三个保守的氨基酸，它们是钙蛋白酶活性位点的部分。CAPN8的潜在EF-Hand-钙结合结构域用下划线标出，并连续编号。给出了钙蛋白酶的任意结构域。分别只在大鼠和小鼠中鉴定出的CAPN4和CAPN7的序列未表示出来。已公布的CAPN6序列未包括在内，因为它只是部分序列。分组的钙蛋白酶的其它名称和编号数在图2的图例中给出。
图2，人体钙蛋白酶大亚基家族的无根系统发生树。分析用PAUP-程序进行，并使用海德堡德国癌症研究中心的HUSAR服务器中的CLUSTREE排列该树。水平线的长度与衍生的系统发生距离成比例；垂直线无任何意义。进行1000次引导指令(Bootstrapping)重复，其值在内部节点示出。CAPN7序列来源于小鼠，因为只有很少量的人体核苷酸和蛋白质序列可供使用。确实存在人体直向同源物(参见文献8)，因此用小鼠序列作这种比较是合理的。部分人体CAPN8序列是EST克隆AA026030的推测翻译(Hillier等人，1995，The Washu-Merck EST-Projekt，未发表的结果)。这一克隆的氨基酸翻译表现出与大鼠CAPN8序列有高的相似性。对这个序列未进行引导指令法，因为它比其它的短得多。因此，将引导指令值与全长序列比较的值进行比较无太大意义。本文将采用人类基因命名委员会规定的名称汇编。各种钙蛋白酶以前的名称为：CAPN1-m钙蛋白酶，CAPN2--m钙蛋白酶，CAPN3-p94，nCL-1，CAPN8，nCL-2，CAPN9，nCL-4，对于所用钙蛋白酶序列的EMBL编号为CAPN1(P17655)，CAPN2(P07384)，CAPN3(P20807)，CAPN5(Y10656)，CAPN6(Y12582)，CAPN7(AJ012475)和CAPN9(AF022799)。
图3是CAPN11的表达。将具有人体CAPN11cDNA编码序列的800碱基对区段的32P-标记DNA探针Master印迹(A)，带50种不同人体组织的RNA斑点印迹的尼龙膜或者Clontech-多组织Northern-印迹(D)上杂交。对膜进行高严谨度(6×SSC，65℃)的洗涤。不同的RNA在斑点印迹膜上的准确位置用图表示(C)。Northern印迹上的RNA在相应的泳道上方给出。斑点印迹和Northern印迹用人遍在蛋白(B)和β-肌动蛋白的DNA探针进行再杂交，以便测定所上样的poly(A+)RNA的量。对于Northern印迹给出了分子量标记物(以千碱基计)的位置。曝光时间为：A，72小时.；B，24小时.；D，48小时。PBL＝外周血白细胞。
序列表<110>BASF Aktiengesellschaft<120>Hoden-spezifisches Calpain<120>睾丸特异性钙蛋白酶<130>OZ0050/<140><141><160>2<170>PatentIn Ver. 2.0<210>1<211>2338<212>DNA<213>人(Homo sapiens)<220><221>5′UTR<222>(1)..(103)<220><221>CDS<222>(104)..(2212)<220><221>3′UTR<222>(2213)..(2338)<400>1ctccccaggg ccgagtcttc cggagtcagc agagagcctg gatggatcac aggaggataa 60gcctcggggc tcatgtgcgg agcccacttt tactgatacg gga atg gtg gct cac 115
Met Val Ala His
1ata aac aac agc cgg ctc aag gcc aag ggc gtg ggc cag cac gac aac 163Ile Asn Asn Ser Arg Leu Lys Ala Lys Gly Val Gly Gln His Asp Asn5 10 15 20gcc cag aac ttt ggt aac cag agc ttt gag gag ctg cga gca gcc tgt 211Ala Gln Asn Phe Gly Asn Gln Ser Phe Glu Glu Leu Arg Ala Ala Cys
25 30 35cta aga aag ggg gag ctc ttc gag gac ccc tta ttc cct gct gaa ccc 259Leu Arg Lys Gly Glu Leu Phe Glu Asp Pro Leu Phe Pro Ala Glu Pro
40 45 50agc tca ctg ggc ttc aag gac ctg ggc ccc aac tcc aaa aat gtg cag 307Ser Ser Leu Gly Phe Lys Asp Leu Gly Pro Asn Ser Lys Asn Val Gln
55 60 65aac atc tcc tgg cag cgg ccc aag gat atc ata aac aac cct cta ttc 355Asn Ile Ser Trp Gln Arg Pro Lys Asp Ile Ile Asn Asn Pro Leu Phe
70 75 80atc atg gat ggg att tct cca aca gac atc tgc cag ggg atc ctc ggg 403Ile Met Asp Gly Ile Ser Pro Thr Asp Ile Cys Gln Gly Ile Leu Gly85 90 95 100gac tgc tgg ctg ctg gct gcc atc ggc tcc ctt acc acc tgc ccc aaa 451Asp Cys Trp Leu Leu Ala Ala Ile Gly Ser Leu Thr Thr Cys Pro Lys
105 110 115ctg cta tac cgc gtg gtg ccc aga gga cag agc ttc aag aaa aac tat 499Leu Leu Tyr Arg Val Val Pro Arg Gly Gln Ser Phe Lys Lys Asn Tyr
120 125 130gct ggc atc ttc cat ttt cag att tgg cag ttt gga cag tgg gtg aac 547Ala Gly Ile Phe His Phe Gln Ile Trp Gln Phe Gly Gln Trp Val Asn
135 140 145gtg gtg gta gat gac cgg ctg ccc aca aag aat gac aag ctg gtg ttt 595Val Val Val Asp Asp Arg Leu Pro Thr Lys Asn Asp Lys Leu Val Phe
150 155 160gtg cac tca acc gaa cgc agt gag ttc tgg agt gcc ctg ctg gag aag 643Val His Ser Thr Glu Arg Ser Glu Phe Trp Ser Ala Leu Leu Glu Lys165 170 175 180gcg tat gcc aag ctg agt ggg tcc tat gaa gca ttg tca ggg ggc agt 691Ala Tyr Ala Lys Leu Ser Gly Ser Tyr Glu Ala Leu Ser Gly Gly Ser
185 190 195acc atg gag ggc ctt gag gac ttc aca gga ggc gtg gcc cag agc ttc 739Thr Met Glu Gly Leu Glu Asp Phe Thr Gly Gly Val Ala Gln Ser Phe
200 205 210caa ctc cag agg ccc cct cag aac ctg ctc agg ctc ctt agg aag gcc 787Gln Leu Gln Arg Pro Pro Gln Asn Leu Leu Arg Leu Leu Arg Lys Ala
215 220 225gtg gag cga tcc tcc ctc atg ggt tgc tcc att gaa gtc acc agt gat 835Val Glu Arg Ser Ser Leu Met Gly Cys Ser Ile Glu Val Thr Ser Asp
230 235 240agt gaa ctg gaa tcc atg act gac aag atg ctg gtg aga ggg cac gct 883Ser Glu Leu Glu Ser Met Thr Asp Lys Met Leu Val Arg Gly His Ala245 250 255 260tac tct gtg act ggc ctt cag gat gtc cac tac aga ggc aaa atg gaa 931Tyr Ser Val Thr Gly Leu Gln Asp Val His Tyr Arg Gly Lys Met Glu
265 270 275aca ctg att cgg gtc cgg aat ccc tgg ggc cgg att gag tgg aat gga 979Thr Leu Ile Arg Val Arg Asn Pro Trp Gly Arg Ile Glu Trp Asn Gly
280 285 290gct tgg agt gac agt gcc agg gag tgg gaa gag gtg gcc tca gac atc 1027Ala Trp Ser Asg Ser Ala Arg Glu Trp Glu Glu Val Ala Ser Asp Ile
295 300 305cag atg cag ctg ctg cac aag acg gag gac ggg gag ttc tgg atg tcc 1075Gln Met Gln Leu Leu His Lys Thr Glu Asp Gly Glu Phe Trp Met Ser
310 315 320tac caa gat ttc ctg aac aac ttc acg ctc ctg gag atc tgc aac ctc 1123Tyr Gln Asp Phe Leu Asn Asn Phe Thr Leu Leu Glu Ile Cys Asn Leu325 330 335 340acg cct gat aca ctc tct ggg gac tac aag agc tac tgg cac acc acc 1171Thr Pro Asp Thr Leu Ser Gly Asp Tyr Lys Ser Tyr Trp His Thr Thr
345 350 355ttc tac gag ggc agc tgg cgc aga ggc agc tcc gca ggg ggc tgc agg 1219Phe Tyr Glu Gly Ser Trp Arg Arg Gly Ser Ser Ala Gly Gly Cys Arg
360 365 370aac cac cct ggc acg ttc tgg acc aac ccc cag ttt aag atc tct ctt 1267Asn His Pro Gly Thr Phe Trp Thr Asn Pro Gln Phe Lys Ile Ser Leu
375 380 385cct gag ggg gat gac cca gag gat gac gca gag ggc aat gtt gtg gtc 1315Pro Glu Gly Asp Asp Pro Glu Asp Asp Ala Glu Gly Asn Val Val Val
390 395 400tgc acc tgc ctg gtg gcc cta atg cag aag aac tgg cgg cat gca cgg 1363Cys Thr Cys Leu Val Ala Leu Met Gln Lys Asn Trp Arg His Ala Arg405 410 415 420cag cag gga gcc cag ctg cag acc att ggc ttt gtc ctc tac gcg gtc 1411Gln Gln Gly Ala Gln Leu Gln Thr Ile Gly Phe Val Leu Tyr Ala Val
425 430 435cca aaa gag ttt cag aac att cag gat gtc cac ttg aag aag gaa ttc 1459Pro Lys Glu Phe Gln Asn Ile Gln Asp Val His Leu Lys Lys Glu Phe
440 445 450ttc acg aag tat cag gac cac ggc ttc tca gag atc ttc acc aac tca 1507Phe Thr Lys Tyr Gln Asp His Gly Phe Ser Glu Ile Phe Thr Asn Ser
455 460 465cgg gag gtg agc agc caa ctc cgg ctg cct ccg ggg gaa tat atc att 1555Arg Glu Val Ser Ser Gln Leu Arg Leu Pro Pro Gly Glu Tyr Ile Ile
470 475 480att ccc tcc acc ttt gag cca cac aga gat gct gac ttc ctg ctt cgg 1603Ile Pro Ser Thr Phe Glu Pro His Arg Asp Ala Asp Phe Leu Leu Arg485 490 495 500gtc ttc acc gag aag cac agc gag tca tgg gaa ttg gat gaa gtc aac 1651Val Phe Thr Glu Lys His Ser Glu Ser Trp Glu Leu Asp Glu Val Asn
505 510 515tat gct gag caa ctc caa gag gaa aag gtc tct gag gat gac atg gac 1699Tyr Ala Glu Gln Leu Gln Glu Glu Lys Val Ser Glu Asp Asp Met Asp
520 525 530cag gac ttc cta cat ttg ttt aag ata gtg gca gga gag ggc aag gag 1747Gln Asp Phe Leu His Leu Phe Lys Ile Val Ala Gly Glu Gly Lys Glu
535 540 545ata ggg gtg tat gag ctc cag agg ctg ctc aac agg atg gcc atc aaa 1795Ile Gly Val Tyr Glu Leu Gln Arg Leu Leu Asn Arg Met Ala Ile Lys
550 555 560ttc aaa agc ttc aag acc aag ggc ttt ggc ctg gat gct tgc cgc tgc 1843Phe Lys Ser Phe Lys Thr Lys Gly Phe Gly Leu Asp Ala Cys Arg Cys565 570 575 580atg atc aac ctc atg gat aaa gat ggc tct ggc aag ctg ggg ctt cta 1891Met Ile Asn Leu Met Asp Lys Asp Gly Ser Gly Lys Leu Gly Leu Leu
585 590 595gag ttc aag atc ctg tgg aaa aaa ctc aag aaa tgg atg gac atc ttc 1939Glu Phe Lys Ile Leu Trp Lys Lys Leu Lys Lys Trp Met Asp Ile Phe
600 605 610aga gag tgt gac cag gac cat tca ggc acc ttg aac tcc tat gag atg 1987Arg Glu Cys Asp Gln Asp His Ser Gly Thr Leu Asn Ser Tyr Glu Met
615 620 625cgc ctg gtt att gag aaa gca ggc atc aag ctg aac aac aag gta atg 2035Arg Leu Val Ile Glu Lys Ala Gly Ile Lys Leu Asn Asn Lys Val Met
630 635 640cag gtc ctg gtg gcc agg tat gca gat gat gac ctg atc ata gac ttt 2083Gln Val Leu Val Ala Arg Tyr Ala Asp Asp Asp Leu Ile Ile Asp Phe645 650 655 660gac agc ttc atc agc tgt ttc ctg agg cta aag acc atg ttc aca ttc 2131Asp Ser Phe Ile Ser Cys Phe Leu Arg Leu Lys Thr Met Phe Thr Phe
665 670 675ttt cta acc atg gac ccc aag aat act ggc cat att tgc ttg agc ctg 2179Phe Leu Thr Met Asp Pro Lys Asn Thr Gly His Ile Cys Leu Ser Leu
680 685 690gaa cag tgg ctg cag atg acc atg tgg gga tag aggcgctgta ggagcctggt 2232Glu Gln Trp Leu Gln Met Thr Met Trp Gly
695 700catctctacc agcagcagca gcagcgaggt tctagcccag gagggtgggg tgcttcttgt 2292agccctcagc tctccagtct ctgctgatga aatgggatcc aggtgg 2338<210>2<211>702<212>PRT<213>人(Homo sapiens)<400>2Met Val Ala His Ile Asn Asn Ser Arg Leu Lys Ala Lys Gly Val Gly1 5 10 15Gln His Asp Asn Ala Gln Asn Phe Gly Asn Gln Ser Phe Glu Glu Leu
20 25 30Arg Ala Ala Cys Leu Arg Lys Gly Glu Leu Phe Glu Asp Pro Leu Phe
35 40 45Pro Ala Glu Pro Ser Ser Leu Gly Phe Lys Asp Leu Gly Pro Asn Ser
50 55 60Lys Asn Val Gln Asn Ile Ser Trp Gln Arg Pro Lys Asp Ile Ile Asn65 70 75 80Asn Pro Leu Phe Ile Met Asp Gly Ile Ser Pro Thr Asp Ile Cys Gln
85 90 95Gly Ile Leu Gly Asp Cys Trp Leu Leu Ala Ala Ile Gly Ser Leu Thr
100 105 110Thr Cys Pro Lys Leu Leu Tyr Arg Val Val Pro Arg Gly Gln Ser Phe
115 120 125Lys Lys Asn Tyr Ala Gly Ile Phe His Phe Gln Ile Trp Gln Phe Gly
130 135 140Gln Trp Val Asn Val Val Val Asp Asp Arg Leu Pro Thr Lys Asn Asp145 150 155 160Lys Leu Val Phe Val His Ser Thr Glu Arg Ser Glu Phe Trp Ser Ala
165 170 175Leu Leu Glu Lys Ala Tyr Ala Lys Leu Ser Gly Ser Tyr Glu Ala Leu
180 185 190Ser Gly Gly Ser Thr Met Glu Gly Leu Glu Asp Phe Thr Gly Gly Val
195 200 205Ala Gln Ser Phe Gln Leu Gln Arg Pro Pro Gln Asn Leu Leu Arg Leu
210 215 220Leu Arg Lys Ala Val Glu Arg Ser Ser Leu Met Gly Cys Ser Ile Glu225 230 235 240Val Thr Ser Asp Ser Glu Leu Glu Ser Met Thr Asp Lys Met Leu Val
245 250 255Arg Gly His Ala Tyr Ser Val Thr Gly Leu Gln Asp Val His Tyr Arg
260 265 270Gly Lys Met Glu Thr Leu Ile Arg Val Arg Asn Pro Trp Gly Arg Ile
275 280 285Glu Trp Asn Gly Ala Trp Ser Asp Ser Ala Arg Glu Trp Glu Glu Val
290 295 300Ala Ser Asp Ile Gln Met Gln Leu Leu His Lys Thr Glu Asp Gly Glu305 310 315 320Phe Trp Met Ser Tyr Gln Asp Phe Leu Asn Asn Phe Thr Leu Leu Glu
325 330 335Ile Cys Asn Leu Thr Pro Asp Thr Leu Ser Gly Asp Tyr Lys Ser Tyr
340 345 350Trp His Thr Thr Phe Tyr Glu Gly Ser Trp Arg Arg Gly Ser Ser Ala
355 360 365Gly Gly Cys Arg Asn His Pro Gly Thr Phe Trp Thr Asn Pro Gln Phe
370 375 380Lys Ile Ser Leu Pro Glu Gly Asp Asp Pro Glu Asp Asp Ala Glu Gly385 390 395 400Asn Val Val Val Cys Thr Cys Leu Val Ala Leu Met Gln Lys Asn Trp
405 410 415Arg His Ala Arg Gln Gln Gly Ala Gln Leu Gln Thr Ile Gly Phe Val
420 425 430Leu Tyr Ala Val Pro Lys Glu Phe Gln Asn Ile Gln Asp Val His Leu
435 440 445Lys Lys Glu Phe Phe Thr Lys Tyr Gln Asp His Gly Phe Ser Glu Ile
450 455 460Phe Thr Asn Ser Arg Glu Val Ser Ser Gln Leu Arg Leu Pro Pro Gly465 470 475 480Glu Tyr Ile Ile Ile Pro Ser Thr Phe Glu Pro His Arg Asp Ala Asp
485 490 495Phe Leu Leu Arg Val Phe Thr Glu Lys His Ser Glu Ser Trp Glu Leu
500 505 510Asp Glu Val Asn Tyr Ala Glu Gln Leu Gln Glu Glu Lys Val Ser Glu
515 520 525Asp Asp Met Asp Gln Asp Phe Leu His Leu Phe Lys Ile Val Ala Gly
530 535 540Glu Gly Lys Glu Ile Gly Val Tyr Glu Leu Gln Arg Leu Leu Asn Arg545 550 555 560Met Ala Ile Lys Phe Lys Ser Phe Lys Thr Lys Gly Phe Gly Leu Asp
565 570 575Ala Cys Arg Cys Met Ile Asn Leu Met Asp Lys Asp Gly Ser Gly Lys
580 585 590Leu Gly Leu Leu Glu Phe Lys Ile Leu Trp Lys Lys Leu Lys Lys Trp
595 600 605Met Asp Ile Phe Arg Glu Cys Asp Gln Asp His Ser Gly Thr Leu Asn
610 615 620Ser Tyr Glu Met Arg Leu Val Ile Glu Lys Ala Gly Ile Lys Leu Asn625 630 635 640Asn Lys Val Met Gln Val Leu Val Ala Arg Tyr Ala Asp Asp Asp Leu
645 650 655Ile Ile Asp Phe Asp Ser Phe Ile Ser Cys Phe Leu Arg Leu Lys Thr
660 665 670Met Phe Thr Phe Phe Leu Thr Met Asp Pro Lys Asn Thr Gly His Ile
675 680 685Cys Leu Ser Leu Glu Gln Trp Leu Gln Met Thr Met Trp Gly
690 695 700
Claims (6)
1.具有SEQ.ID.No.2.氨基酸序列的多肽。
2.编码权利要求1的多肽的多核苷酸序列。
3.具有SEQ.ID.No.1序列的多核苷酸序列。
4.权利要求1的多肽用于鉴定能抑制这类多肽酶活性的物质的应用。
5.可抑制权利要求1多肽的酶活性的化合物的鉴定方法，包括：
(a)将在该化合物存在时CAPN11的酶活性程度与在无该化合物存在时CAPN11的酶活性程度比较，和
(b)选择与无该化合物存在时CAPN11的酶活性相比改变了CAPN11酶活性程度的化合物。
6.通过权利要求5的方法鉴定出的化合物对治疗男人生育紊乱的应用。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19928021A DE19928021A1 (de) | 1999-06-18 | 1999-06-18 | Neue Calpaine und deren Verwendung |
| DE19928021.5 | 1999-06-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1373808A true CN1373808A (zh) | 2002-10-09 |
Family
ID=7911777
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN00808932A Pending CN1373808A (zh) | 1999-06-18 | 2000-06-07 | 新型钙蛋白酶及其应用 |
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| Country | Link |
|---|---|
| EP (1) | EP1185673A2 (zh) |
| JP (1) | JP2003503023A (zh) |
| KR (1) | KR20020011140A (zh) |
| CN (1) | CN1373808A (zh) |
| AR (1) | AR024573A1 (zh) |
| AU (1) | AU5970600A (zh) |
| BG (1) | BG106186A (zh) |
| BR (1) | BR0011721A (zh) |
| CA (1) | CA2375477A1 (zh) |
| DE (1) | DE19928021A1 (zh) |
| HU (1) | HUP0201800A2 (zh) |
| IL (1) | IL146940A0 (zh) |
| MX (1) | MXPA01012937A (zh) |
| NO (1) | NO20015922L (zh) |
| PL (1) | PL353004A1 (zh) |
| SK (1) | SK18262001A3 (zh) |
| TR (1) | TR200103662T2 (zh) |
| WO (1) | WO2000078933A2 (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6433153B1 (en) | 1999-09-02 | 2002-08-13 | Lexicon Genetics Incorporated | Human calcium dependent proteases and polynucleotides encoding the same |
| GB2505572B (en) | 2010-04-30 | 2014-08-06 | Abbott Cardiovascular Systems | Catheter system providing step reduction for postconditioning |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19737105A1 (de) * | 1997-08-26 | 1999-03-04 | Basf Ag | Neue gewebsspezifische Calpaine, ihre Herstellung und Verwendung |
| AU3774500A (en) * | 1999-03-31 | 2000-10-16 | Curagen Corporation | Nucleic acids including open reading frames encoding polypeptides; "orfx" |
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1999
- 1999-06-18 DE DE19928021A patent/DE19928021A1/de not_active Withdrawn
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2000
- 2000-06-07 AU AU59706/00A patent/AU5970600A/en not_active Abandoned
- 2000-06-07 WO PCT/EP2000/005261 patent/WO2000078933A2/de not_active Application Discontinuation
- 2000-06-07 SK SK1826-2001A patent/SK18262001A3/sk unknown
- 2000-06-07 JP JP2001505676A patent/JP2003503023A/ja active Pending
- 2000-06-07 MX MXPA01012937A patent/MXPA01012937A/es unknown
- 2000-06-07 BR BR0011721-8A patent/BR0011721A/pt not_active IP Right Cessation
- 2000-06-07 EP EP00945716A patent/EP1185673A2/de not_active Withdrawn
- 2000-06-07 HU HU0201800A patent/HUP0201800A2/hu unknown
- 2000-06-07 CN CN00808932A patent/CN1373808A/zh active Pending
- 2000-06-07 CA CA002375477A patent/CA2375477A1/en not_active Abandoned
- 2000-06-07 PL PL00353004A patent/PL353004A1/xx unknown
- 2000-06-07 TR TR2001/03662T patent/TR200103662T2/xx unknown
- 2000-06-07 KR KR1020017016244A patent/KR20020011140A/ko not_active Application Discontinuation
- 2000-06-07 IL IL14694000A patent/IL146940A0/xx unknown
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Also Published As
| Publication number | Publication date |
|---|---|
| PL353004A1 (en) | 2003-09-22 |
| JP2003503023A (ja) | 2003-01-28 |
| BG106186A (bg) | 2002-12-29 |
| WO2000078933A2 (de) | 2000-12-28 |
| KR20020011140A (ko) | 2002-02-07 |
| HUP0201800A2 (en) | 2002-09-28 |
| AU5970600A (en) | 2001-01-09 |
| MXPA01012937A (es) | 2002-07-30 |
| EP1185673A2 (de) | 2002-03-13 |
| NO20015922L (no) | 2002-02-12 |
| TR200103662T2 (tr) | 2002-05-21 |
| SK18262001A3 (sk) | 2002-09-10 |
| NO20015922D0 (no) | 2001-12-04 |
| BR0011721A (pt) | 2002-03-05 |
| DE19928021A1 (de) | 2000-12-21 |
| IL146940A0 (en) | 2002-08-14 |
| AR024573A1 (es) | 2002-10-16 |
| WO2000078933A3 (de) | 2001-07-26 |
| CA2375477A1 (en) | 2000-12-28 |
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