摘要
为了研究合浦珠母贝(Pincatada fucata)的先天免疫调控机制,利用cDNA文库筛选和重测序技术克隆了1个合浦珠母贝组织蛋白酶L的全长cDNA序列(命名为poCL2)。poCL2cDNA全长1094bp,5′-非编码区(Untranslated region,UTR)长21bp,3′-UTR长80bp,开放阅读框(Open reading frame,ORF)为993bp,编码330个氨基酸组成的多肽链,分子量为37.1kD,理论等电点为6.9。poCL2蛋白由信号肽(Met1-Ala16)、前体域(Arg17-Asp113)和成熟域(Leu114-Val330)三部分组成,存在一个潜在的糖基化位点(Asn97),6个底物结合位点(Leu182,Met183,Ala249,Leu275,Gly278和Ser324),1个由半胱氨酸(Cys138)、组氨酸(His277)和天冬酰胺(Asn297)残基构成的催化位点和2个组织蛋白酶L签名序列(E42X3RX3WX2NX3IX3N61和G74X1NX1YX1D80)。同源性分析表明,poCL2氨基酸序列与其他物种高度保守,相似性在61.5%～73.9%之间,同源性在44.4%～59.8%之间。进化分析显示,poCL2与其他无脊椎动物的组织蛋白酶L聚为一支,和淡水螺(Radix peregra)亲缘关系最近。组织表达分析表明poCL2mRNA在消化腺、外套膜、性腺、闭壳肌、肠、血淋巴和鳃组织中均表达,外套膜表达量最高。应激实验表明,经溶藻弧菌(Vibrio alginolyticus)或脂多糖(Lipopolysaccharide,LPS)刺激后,poCL2mRNA在消化腺中的表达量显著上调,说明poCL2参与了合浦珠母贝的先天免疫应答反应,暗示其在合浦珠母贝先天免疫调控中发挥重要作用。
Pearl oyster Pinctada fucata is the most popular farming shellfish for seawater pearl production in Guangdong,Guangxi and Hainan province of China. In recent years,as other marine animals,higher frequencies of disease epidemics and the emergence of new diseases have been reported in artificial cultivation of Pearl oyster. Many researchers considered the reasons for high mortality were ocean pollution,disease outbreaks and stock degeneration. In order to control disease and enhance the yields and quality of seawater pearl,it is necessary to further research the innate immune mechanisms of pearl oyster. Cathepsin L is a member of cysteine protease family and involved in various biological functions,which is distributed widely in living organisms. In this study,we identified one cDNA encoding a cathepsin L cysteine proteases from EST library of pearl oyster Pinctada fucata（ designated as poCL2）. The poCL2 cDNA was 1 094 bp long and consisted of a 5′ -untranslated region（ UTR） of 21 bp,a 3′ -UTR of 80 bp with a polyadenylation signal（ AATAAA）,and an open reading frame（ ORF） of 993 bp encoding a polypeptide of 330 amino acids,which contained a typical signal peptide sequence（ Met1-Ala16）,a prodomain（ Arg17-Asp113）,and a mature domain（ Leu114-Val330）. The preprocathepsin contained a potential N-glycosylation site（ Asn97）,six substrate binding sites（ Leu182,Met183,Ala249,Leu275,Gly278,and Ser324）,three catalytic sites（ Cys113,His278,and Asn298）. The conserved E42X3RX3WX2NX3IX3N61 motif is discovered in the prodomain which may be play important function in the inhibition of proteolytic activity. The G74X1NX1YX1D80,which may be related to pH-dependent intramolecular processing,is also found in the prodomain of the poCL2. Position of prodomain cleavage site and conserved cysteine residues are based on the N-terminal sequence information from HumanCL1（ Homo sapiens,P07711）,RatCL1 （Rattus norvegicus,P07154）,BovineCS（ Bos taurus,P25326）,and ChickenCL（ Gallus gallus,P09648）,and the prodomain cleavage site was between Asp113 and Leu114.The number of N-glycosylation site is not conserved,there is one,two and three potential N-glycosylation sites in Ictalurus punctatus,Toxoplasma gondiis and Trypanosoma carassii cathepsin L,respectively,which revealed that the intracellular transport mechanism of cathepsin L proteases is different in living organisms. Homology analysis of poCL2 by MatGAT software revealed that the poCL2 shared 61.5-73.9% similarity and 44.4-59.8% identity to other known cathepsin L sequences. The phylogenetic tree showed that the poCL2 clustered with the invertebrate cathepsin L cysteine proteases and was closely related to Radix peregra cathepsin L,and a clear clade division was observed between vertebrates and invertebrates cathepsin L proteins. The previous study demonstrated that the expression pattern of cathepsin L was different in different species and exhibited specificity for some tissues in different species. The mRNA expression of the poCL2 in normal group could be detected in digestive gland,gonad,haemocytes,gills,mantle,adduct muscle and intestines,and with the higher level in mantle,the results indicated that the expression pattern of cathepsin L gene in various tissues was somewhat different,and had diverse functions. The digestive gland of mollusk was thought to be an important immune organ, which could secrete various enzymes to hydrolyze microorganisms and involve in digestive and defense functions. So we selected the digestive gland to research the temporal expression pattern of poCL2 after Vibrio alginolyticus and LPS challenge. The results showed that the expression level of the poCL2 was up-regulated in digestive gland not only in V. alginolyticus group but also in LPS group,and the highest expression was observed at 8 h after stimulation and was 1.73-fold,1.25-fold higher than the control group,respectively. These results suggested that the poCL2 was involved in the innate immune response of pearl oyster and might play an important function in immune regulation.
出处
《中国水产科学》
CAS
CSCD
北大核心
2010年第4期701-712,共12页
Journal of Fishery Sciences of China
基金
国家"863"计划项目(2009AA10Z106)
农业部公益性行业科研专项项目(200903028)
广东省科技兴海项目(A200701002)
中央级公益性科学院基础科研业务费专项资金项目(2009TS23
2010YD03)
