海参i型溶菌酶基因及其编码产物的结构特点

Characterization and Structure Analysis of a Gene Encoding i-Type Lysozyme from Sea Cucumber Stichopus japonicus

通过RT-PCR和RACEPCR技术,从海参(Stichopus japonicus)体壁中克隆得到一种溶菌酶基因(GenBank:EF036468).生物信息软件分析表明,其中全长cDNA为713bp,5′非编码区(UTR)246bp,3′UTR29bp,开放阅读框438bp,编码145个氨基酸,包括溶菌酶成熟肽124个氨基酸和信号肽21个氨基酸.对海参溶菌酶与多种无脊椎动物的c、g和i型溶菌酶进行分析比较,发现它与i型溶菌酶有较高的同源性,并具有i型溶菌酶高度保守的2个活性位点,即Glu34和Ser50.活性位点附近具有i型溶菌酶的一段特有的氨基酸保守序列MDVGSLSCG(P/Y)(Y/F)QIK,所以推断克隆的海参溶菌酶为i型.另外,通过搜索蛋白保守结构域数据库,发现海参溶菌酶与医用水蛭失稳酶相似性最高,并且这2个酶的三级结构模型也极其相似.因此推测,海参i型溶菌酶具有双功能特性,既能作用于细菌细胞壁的糖苷键使细胞裂解,又具有失稳酶的一些生化功能,能够水解纤维蛋白,这些特点在海参自溶过程中发挥重要的作用.

The lysozyme cDNA from the body wall of the sea cucumber Stichopus japonicus was cloned by RT- PCR and RACE PCR methods. Bioinformatic software analysis showed that the full length cDNA was 713 bp with 246 bp 5＇ UTR, 29 bp 3＇ UTR and 438 bp ORF （GenBank：EF036468）. The ORF encoded 145 amino acids （aa） including a signal peptide of 21 aa at the N-terminus and a mature peptide of 124 aa. Based on the sequence analysis, the lysozyme gene from Stichopus japonicus showed significant homology with the i-type lysozyme genes in invertebrate. Two catalytic residues （Glu34 and Ser50） conserved in i-type lysozyme and a highly conserved sequence region near the active sites, MDVGSLSCG（P/Y）（Y/F）QIK, were detected in the amino acid sequence from Stichopus japonicus. These results indicated that the cDNA sequence cloned from Stichopus japonicus was a member of i-type lysozyme family. In addition, the analysis of the lysozyme sequence from Stichopus japonicus in the Conserved Domain Database showed that it was highly similar to the medicinal leech destabilase. Furthermore, the Stichopus japonicus lysozyme displayed an extreme similarity in 3D MODEL with destabilase of the medicinal leech. So it can be speculated that the Stichopus japonicus lysozyme has a dual biological function： hydrolysing β-1,4-glycosidic bonds of bacterial cell walls to cause bacterial lysis, as well as hydrolysing fibrin which acts as destabilase.