马氏珠母贝Pm-CTSC基因的克隆与表达分析
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摘要
组织蛋白酶C在无脊椎动物中具有重要的非特异性免疫防御功能。为了研究马氏珠母贝组织蛋白酶C(Pm-CTSC)的功能,本实验利用RACE技术克隆获得了Pm-CTSC,并对其结构和组织表达模式进行了分析。结果表明:Pm-CTSC基因cDNA序列全长为1 914 bp,其中开放式阅读框1 413 bp,5'UTR 25 bp,3'UTR 476 bp,共编码470个氨基酸,该蛋白理论分子量为53.41 k D,理论等电点为6.03。结构域分析发现Pm-CTSC具有组织蛋白酶C两个典型的结构域Cathepsin C exclusion和Peptidase_C1A_CathepsinC。多序列比对结果表明Pm-CTSC与太平洋牡蛎的同源性最高,为68.8%;系统进化分析发现,Pm-CTSC与无脊椎动物聚为一支。qRT-PCR表达分析发现,Pm-CTSC在肝胰腺中显著高表达(p<0.05),表明Pm-CTSC可能参与马氏珠母贝的免疫应答。本研究为进一步探讨组织蛋白酶C在马氏珠母贝天然免疫应答中的作用提供了基础资料。
Cathepsin C plays an important role in nonspecific immune defense function in invertebrates. To study the function of Cathepsin C in Pinctada fucata martensii(Pm-CTSC), the Pm-CTSC was cloned by rapid amplification of cDNA end(RACE) technology and the expression pattern of its structure and tissue was analyzed. The results showed that the full length of Pm-CTSC cDNA was 1 914 bp, of which the open reading frame(ORF) was 1 413 bp,5'UTR was 25 bp and 3'UTR was 476 bp, encoding 470 amino acids. The predicted molecular weight was 53.41 kD,and the isoelectric point was 6.03. The structural domain analysis proved that Pm-CTSC had two typical domains of Cathepsin C, including Cathepsin C exclusion and Peptidase_C1 A_Cathepsin C. The multiple alignment showed that Pm-CTSC had the highest homology with Pacific oyster(68.8%). Phylogenetic analysis found that Pm-CTSC was clustered with those in invertebrates. The qRT-PCR expression analysis showed that Pm-CTSC was highly expressed in the hepatopancreas(p<0.05), which suggested that Pm-CTSC might be involved in the immune response of the P. f.martensii. This study would provide basic information for further study on the role of Cathepsin C in the natural immune response in P. f. martensii.
Cathepsin C plays an important role in nonspecific immune defense function in invertebrates. To study the function of Cathepsin C in Pinctada fucata martensii(Pm-CTSC), the Pm-CTSC was cloned by rapid amplification of cDNA end(RACE) technology and the expression pattern of its structure and tissue was analyzed. The results showed that the full length of Pm-CTSC cDNA was 1 914 bp, of which the open reading frame(ORF) was 1 413 bp,5'UTR was 25 bp and 3'UTR was 476 bp, encoding 470 amino acids. The predicted molecular weight was 53.41 kD,and the isoelectric point was 6.03. The structural domain analysis proved that Pm-CTSC had two typical domains of Cathepsin C, including Cathepsin C exclusion and Peptidase_C1 A_Cathepsin C. The multiple alignment showed that Pm-CTSC had the highest homology with Pacific oyster(68.8%). Phylogenetic analysis found that Pm-CTSC was clustered with those in invertebrates. The qRT-PCR expression analysis showed that Pm-CTSC was highly expressed in the hepatopancreas(p<0.05), which suggested that Pm-CTSC might be involved in the immune response of the P. f.martensii. This study would provide basic information for further study on the role of Cathepsin C in the natural immune response in P. f. martensii.
引文
Dahl S.W.,Halkier T.,Lauritzen C.,Dolenc I.,Pedersen J.,Turk V.,and Turk B.,2001,Human recombinant pro-dipeptidyl peptidase I(cathepsin C)can be activated by cathepsins Land S but not by autocatalytic processing,Biochemistry,40(6):1671-1678
Dufour E.,1988,Sequence homologies,hydrophobic profiles and secondary structures of cathepsins B,H and L:comparison with papain and actinidin,Biochimie,70(10):1335-1342
Doughty M.J.,and Gruenstein E.I.,1987,Cell growth and substrate effects on characteristics of a lysosomal enzyme(cathepsin C)in Duchenne muscular dystrophy fibroblasts,Biochemistry and Cell Biology,65(7):617-625
Gai Y.,Qiu L.,Wang L.,Song L.,Mu C.,Zhao J.,Zhang Y.,and Li L.,2009,A clip domain serine protease(cSP)from the Chinese mitten cRNA Eriocheir sinensis:cDNA characterization and m RNA expression,Fish Shellfish Immunol,27(6):670-677
Gao K.,Deng X.Y.,and Guo X.J.,2010,Research progress on serine proteinase of immune system,Mianyixueza(Immunological Journal),(11):1003-1007(高坤,邓祥元,郭锡杰,2010,免疫系统中丝氨酸蛋白酶的研究进展,免疫学杂志,(11):1003-1007)
Gutmann H.R.,and Fruton J.S.,1948,On the proteolytic enzymes of animal tissuesⅧ:an intracellular enzyme related to chymotrypsin,Journal of Biological Chemistry,174(3):851
Hao R.J.,Zheng Z.,Wang Q.H.,Deng Y.W.,Jiao Y.,and Du X.D.,2015,Molecular characterization and expression analysis of PmCHST9 gene from sulfotransferase of Pinctada martensii,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(11):2387-2394(郝瑞娟,郑哲,王庆恒,邓岳文,焦钰,杜晓东,2015,马氏珠母贝磺基转移酶PmCHST9基因的分子特征与表达分析,基因组学与应用生物学,34(11):2387-2394)
Hudig D.,Allison N.J.,Pickett T.M.,Winkler U.,Kam C.M.,and Powers J.C.,1991,Th e function of lymphocyte proteases.Inhibition and restoration of granule-mediated lysis with isocoumarin serine protease inhibitors,Journal of Immunology,147(4):1360-1368
Jin K.,Niu D.H.,Wang L.,and Li J.L.,2013,Molecular characteristics and expression analysis of serine protease from Sinonovacula constricta,Shanghai Haiyangdaxue Xuebao(Journal of Shanghai Ocean University),22(4):481-487(金凯,牛东红,王劦,李家乐,2013,缢蛏丝氨酸蛋白酶基因的序列特征及其表达分析,上海海洋大学学报,22(4):481-487)
Kominami E.,Ishido K.,Muno D.,and Sato N.,1992,The primary structure and tissue distribution of cathepsin C,Biological Chemistry Hoppe-Seyler,373(2):367-374.
Li W.W.,Jin X.K.,He L.,Jiang H.,Xie Y.N.,and Wang Q.,2010,Molecular cloning,characterization and expression analysis of cathepsin C gene involved in the antibacterial response in Chinese mitten crab,Eriocheir sinensis,Developmental Comparative Immunology,34(11):1170-1174
Lynch G.W.,and Pfueller S.L.,1988,Thrombin-independent activation of platelet factor XⅡⅠby endogenous platelet acid protease,Thromb Haemost,59(3):372-377
Müller W.E.G.,Boreiko A.,Wang X.,Belikov S.I.,Wiens M.,Grebenjuk V.A.,Schlossmacher U.,and Schr觟dera H.C.,2007,Silicateins,the major biosilica forming enzymes present in demosponges:protein analysis and phylogenetic relationship,Gene,395(1-2):62-71
Niu D.,Xie S.,Bai Z.,Wang L.,Jin K.,and Li J.,2014,Identification,expression,and responses to bacterial challenge of the cathepsin C gene from the razor clam Sinonovacula constricta,Developmental&Comparative Immunology,46(2):241-245
Qiu L.,Jiang S.,Huang J.,Wang W.,Zhang D.,Wu Q.,and Yang K.,2008,Molecular cloning and m RNA expression of cathepsin C gene in black tiger shrimp(Penaeus monodon),Comparative Biochemistry Physiology Part A Molecular&Integrative Physiology,150(3):320-325
Rawlings N.D.,and Barrett A.J.,1994,Families of cysteine peptidases,Methods in Enzymology,244(1):461-486
Thiele D.L.,Mcguire M.J.,and Lipsky P.E.,1997,A selective inhibitor of dipeptidyl peptidase I impairs generation of CD8+T cell cytotoxic effector func tion,Journal of Immunology,158(11):5200-5210
Turk D.,JanjiéV.,tern I.,Podobnik M.,Lamba D.,Dahl S.W.,Lauritzen C.,Pedersen J.,Turk V.,and Turk B.,2001,Structure of human dipeptidyl peptidaseⅠ(cathepsin C):exclusion domain added to an endopeptidase framework creates the machine for activation of granular serine proteases,Embo Journal,20(23):6570-6582
Wang S.,Shi L.J.,Liu N.,Chen A.J.,Zhao X.F.,and Wang J.X.,2012,Involvement o f Fenn eropenaeus chinensis Cathepsin C in antiviral immunity,Fi sh&Shellfish Immunology,33(4):821-828
Zhang G.,Fang X.,Guo X.,Li L.,Luo R.,Xu F.,Yang P.,Zhang L.,Wang X.,Qi H.,Xiong Z.,Que H.,Xie Y.,Holland P.W.,Paps J.,Zhu Y.,Wu F.,Chen Y.,Wang J.,Peng C.,Meng J.,Yang L.,Liu J.,Wen B.,Zhang N.,Huang Z.,Zhu Q.,Feng Y.,Mount A.,Hedgecock D.,Xu Z.,Liu Y.,Domazet-Lo觢o T.,Du Y.,Sun X.,Zhang S.,Liu B.,Cheng P.,Jiang X.,Li J.,Fan D.,Wang W.,Fu W.,Wang T.,Wang B.,Zhang J.,Peng Z.,Li Y.,Li N.,Wang J.,Chen M.,He Y.,Tan F.,Song X.,Zheng Q.,Huang R.,Yang H.,Du X.,Chen L.,Yang M.,Gaffney P.M.,Wang S.,Luo L.,She Z.,Ming Y.,Huang W.,Zhang S.,Huang B.,Zhang Y.,Qu T.,Ni P.,Miao G.,Wang J.,Wang Q.,Steinberg C.E.,Wang H.,Li N.,Qian L.,Zhang G.,Li Y.,Yang H.,Liu X.,Wang J.,Yin Y.,and Wang J.,2012,The oyster genome reveals stress adaptation and complexity of shell formation,Nature,490(7418):49-54
Zhang M.M.,Ning J.J.,Xu S.L.,Wang C.L.,and Wang D.L.,2015,Effect of Vibrio alginolyticus infection on immune function of Charybdis Japonica hepatopancreas,Shichan Xuebao(Journal of Fisheries of China),39(12):1863-1871(张萌萌,宁俊佳,徐善良,王春琳,王丹丽,2015,感染溶藻弧菌对日本蟳肝胰腺免疫功能的影响,水产学报,39(12):1863-1871)
Zhang Y.X.,Zou Y.H.,Manchu R.G.,and Zhou Y.C.,2010,An-timicrobial activity in gills of shellfish Pinctada martensii,Shuichan Kexue(Fisheries Science),29(8):465-468(张英霞,邹瑗徽,满初日嘎,周永灿,2010,马氏珠母贝鳃组织抗菌活性的研究,水产科学,29(8):465-468)
Dufour E.,1988,Sequence homologies,hydrophobic profiles and secondary structures of cathepsins B,H and L:comparison with papain and actinidin,Biochimie,70(10):1335-1342
Doughty M.J.,and Gruenstein E.I.,1987,Cell growth and substrate effects on characteristics of a lysosomal enzyme(cathepsin C)in Duchenne muscular dystrophy fibroblasts,Biochemistry and Cell Biology,65(7):617-625
Gai Y.,Qiu L.,Wang L.,Song L.,Mu C.,Zhao J.,Zhang Y.,and Li L.,2009,A clip domain serine protease(cSP)from the Chinese mitten cRNA Eriocheir sinensis:cDNA characterization and m RNA expression,Fish Shellfish Immunol,27(6):670-677
Gao K.,Deng X.Y.,and Guo X.J.,2010,Research progress on serine proteinase of immune system,Mianyixueza(Immunological Journal),(11):1003-1007(高坤,邓祥元,郭锡杰,2010,免疫系统中丝氨酸蛋白酶的研究进展,免疫学杂志,(11):1003-1007)
Gutmann H.R.,and Fruton J.S.,1948,On the proteolytic enzymes of animal tissuesⅧ:an intracellular enzyme related to chymotrypsin,Journal of Biological Chemistry,174(3):851
Hao R.J.,Zheng Z.,Wang Q.H.,Deng Y.W.,Jiao Y.,and Du X.D.,2015,Molecular characterization and expression analysis of PmCHST9 gene from sulfotransferase of Pinctada martensii,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(11):2387-2394(郝瑞娟,郑哲,王庆恒,邓岳文,焦钰,杜晓东,2015,马氏珠母贝磺基转移酶PmCHST9基因的分子特征与表达分析,基因组学与应用生物学,34(11):2387-2394)
Hudig D.,Allison N.J.,Pickett T.M.,Winkler U.,Kam C.M.,and Powers J.C.,1991,Th e function of lymphocyte proteases.Inhibition and restoration of granule-mediated lysis with isocoumarin serine protease inhibitors,Journal of Immunology,147(4):1360-1368
Jin K.,Niu D.H.,Wang L.,and Li J.L.,2013,Molecular characteristics and expression analysis of serine protease from Sinonovacula constricta,Shanghai Haiyangdaxue Xuebao(Journal of Shanghai Ocean University),22(4):481-487(金凯,牛东红,王劦,李家乐,2013,缢蛏丝氨酸蛋白酶基因的序列特征及其表达分析,上海海洋大学学报,22(4):481-487)
Kominami E.,Ishido K.,Muno D.,and Sato N.,1992,The primary structure and tissue distribution of cathepsin C,Biological Chemistry Hoppe-Seyler,373(2):367-374.
Li W.W.,Jin X.K.,He L.,Jiang H.,Xie Y.N.,and Wang Q.,2010,Molecular cloning,characterization and expression analysis of cathepsin C gene involved in the antibacterial response in Chinese mitten crab,Eriocheir sinensis,Developmental Comparative Immunology,34(11):1170-1174
Lynch G.W.,and Pfueller S.L.,1988,Thrombin-independent activation of platelet factor XⅡⅠby endogenous platelet acid protease,Thromb Haemost,59(3):372-377
Müller W.E.G.,Boreiko A.,Wang X.,Belikov S.I.,Wiens M.,Grebenjuk V.A.,Schlossmacher U.,and Schr觟dera H.C.,2007,Silicateins,the major biosilica forming enzymes present in demosponges:protein analysis and phylogenetic relationship,Gene,395(1-2):62-71
Niu D.,Xie S.,Bai Z.,Wang L.,Jin K.,and Li J.,2014,Identification,expression,and responses to bacterial challenge of the cathepsin C gene from the razor clam Sinonovacula constricta,Developmental&Comparative Immunology,46(2):241-245
Qiu L.,Jiang S.,Huang J.,Wang W.,Zhang D.,Wu Q.,and Yang K.,2008,Molecular cloning and m RNA expression of cathepsin C gene in black tiger shrimp(Penaeus monodon),Comparative Biochemistry Physiology Part A Molecular&Integrative Physiology,150(3):320-325
Rawlings N.D.,and Barrett A.J.,1994,Families of cysteine peptidases,Methods in Enzymology,244(1):461-486
Thiele D.L.,Mcguire M.J.,and Lipsky P.E.,1997,A selective inhibitor of dipeptidyl peptidase I impairs generation of CD8+T cell cytotoxic effector func tion,Journal of Immunology,158(11):5200-5210
Turk D.,JanjiéV.,tern I.,Podobnik M.,Lamba D.,Dahl S.W.,Lauritzen C.,Pedersen J.,Turk V.,and Turk B.,2001,Structure of human dipeptidyl peptidaseⅠ(cathepsin C):exclusion domain added to an endopeptidase framework creates the machine for activation of granular serine proteases,Embo Journal,20(23):6570-6582
Wang S.,Shi L.J.,Liu N.,Chen A.J.,Zhao X.F.,and Wang J.X.,2012,Involvement o f Fenn eropenaeus chinensis Cathepsin C in antiviral immunity,Fi sh&Shellfish Immunology,33(4):821-828
Zhang G.,Fang X.,Guo X.,Li L.,Luo R.,Xu F.,Yang P.,Zhang L.,Wang X.,Qi H.,Xiong Z.,Que H.,Xie Y.,Holland P.W.,Paps J.,Zhu Y.,Wu F.,Chen Y.,Wang J.,Peng C.,Meng J.,Yang L.,Liu J.,Wen B.,Zhang N.,Huang Z.,Zhu Q.,Feng Y.,Mount A.,Hedgecock D.,Xu Z.,Liu Y.,Domazet-Lo觢o T.,Du Y.,Sun X.,Zhang S.,Liu B.,Cheng P.,Jiang X.,Li J.,Fan D.,Wang W.,Fu W.,Wang T.,Wang B.,Zhang J.,Peng Z.,Li Y.,Li N.,Wang J.,Chen M.,He Y.,Tan F.,Song X.,Zheng Q.,Huang R.,Yang H.,Du X.,Chen L.,Yang M.,Gaffney P.M.,Wang S.,Luo L.,She Z.,Ming Y.,Huang W.,Zhang S.,Huang B.,Zhang Y.,Qu T.,Ni P.,Miao G.,Wang J.,Wang Q.,Steinberg C.E.,Wang H.,Li N.,Qian L.,Zhang G.,Li Y.,Yang H.,Liu X.,Wang J.,Yin Y.,and Wang J.,2012,The oyster genome reveals stress adaptation and complexity of shell formation,Nature,490(7418):49-54
Zhang M.M.,Ning J.J.,Xu S.L.,Wang C.L.,and Wang D.L.,2015,Effect of Vibrio alginolyticus infection on immune function of Charybdis Japonica hepatopancreas,Shichan Xuebao(Journal of Fisheries of China),39(12):1863-1871(张萌萌,宁俊佳,徐善良,王春琳,王丹丽,2015,感染溶藻弧菌对日本蟳肝胰腺免疫功能的影响,水产学报,39(12):1863-1871)
Zhang Y.X.,Zou Y.H.,Manchu R.G.,and Zhou Y.C.,2010,An-timicrobial activity in gills of shellfish Pinctada martensii,Shuichan Kexue(Fisheries Science),29(8):465-468(张英霞,邹瑗徽,满初日嘎,周永灿,2010,马氏珠母贝鳃组织抗菌活性的研究,水产科学,29(8):465-468)