鲤胰蛋白酶原基因的电子克隆及生物信息学分析

In silico cloning and bioinformatics analysis of trypsinogen genes in common carp(Cyprinus carpio)

为深入研究胰蛋白酶在鱼类中的生理功能和作用机制,利用生物信息学的方法,成功获得了鲤3种胰凝乳蛋白酶原cDNA序列(ccTRP1、ccTRP2和ccTRP3)并对其进行了序列分析。结果表明,三者均含有一个长度为729bp的开放阅读框(open reading frame,ORF),编码由242个氨基酸组成的胰蛋白酶原,其中包括15个氨基酸组成的信号肽和5个氨基酸(LDDDK)组成的激活肽。总平均亲水性GRAVY(grand average of hydropathicity)分析表明,三者均是亲水性蛋白。氨基酸比对结果显示,三者具备胰蛋白酶原的保守结构特征,如含有催化三联体氨基酸(His-57、Asp-102和Ser-195),12个半胱氨酸,位于底物结合口袋底部Asp-189和口袋开口处的Gly-216、Gly-226等。同时,鲤3种胰蛋白酶之间具有90%以上的同源性。进化树结果显示,鲤3种胰蛋白酶均属于Group I(阴离子型胰蛋白酶),且三者的进化距离不一致。

Trypsin is one member of the serine protease family,which is synthesized as proenzyme trypsinogen by pancreas.After being secreted into the intestine,enterokinase removes the N-terminal activation peptide converting it into its active form,trypsin which can specifically cleave at the peptide bond on the carboxyl side of basic L-amino acids such as arginine or lysine residues.Meanwhile,trypsin is one of the most abundant proteases in the digestive system of teleost fish,and plays important roles in the protein absorption from the diet.For further studying the physiological funtions and mechanism of trypsinogen in common carp（Cyprinus carpio） ,one of the most economically important fish in China,we got three distinct trypsinogen cDNAs （ccTRP1,ccTRP2 and ccTRP3） successfully using in silico cloning technique,and then they were analysed by the method of bioinformatics.The results showed that three of them had an open reading frame of 729bp in length encoding 242 amino acids,the same sequence of a signal peptide consisted of 15 amino acids and an activation peptide of 5 amino acids,LDDDK.It also showed that they possessed the conserved domains of trypsin-like serine protease superfamily,and had the conversed structural characteristics,such as the catalytic triad（His-57,Asp-102,and Ser-195） ,12 cysteines forming 6 disulfide bonds,Asp-189 at the bottom of the substrate-binding pocket and Gly-216 and Gly-226 lining the sides of the binding pocket,the motif of Gly-AspSer-Gly-Gly-Pro in serine protease family,and so on.But,three of them also had some different characteristics with amino acid composition and phosphorylation sites.These results suggested that common carp trypsinogens we got in the present study may be functional,however,their funtions may be different to a certain degree.The analysis of GRAVY（grand average of hydropathicity） and the instability index indicated that they belonged to the hydrophilic and stable proteins.In the aspect of advanced structure,they mainly contained four secondary structural forms（alpha helix,extended strand,random coil and beta turn） by SOPMA software analysis,and the most abundant was random coil.The alignment and homology analysis based on the amino acid sequences revealed that it had the identity of beyond 90% among the three common carp trypsinogens,and ccTRP1 and ccTRP2 had the highest identity of 93% .Furhermore,compared to other species they had the highest identity with grass carp（Ctenopharyngodon idellus） .Although both common carp and topmouth culter（Culter alburnus） blonged to Cyprinidae,they only had the identity of 66% ,indicating that they may possess distinct property.The phylogenetic tree,based upon the alignment of amino acid sequences of trypsiongens,showed that three of them clustered with teleost anionic trypsinogen Group I,but the evolutional distances were not the same.These preliminary results from common carp trypsinogens by the method of bioinformatics can provide the foundations for further study of its expression profiles,the molecular characteristics and evolution of fish trypsinogen.