牙鲆弹性蛋白酶cDNA全长和蛋白质结构分析

为研究牙鲆丝氨酸蛋白酶家族的功能和及其家族的分子进化规律，从本实验室已构建的牙鲆肝胰脏cDNA文库进行了部分测序，从而筛选出一个弹性蛋白酶新成员：弹性蛋白酶5。在此基础上，结合Genbank数据库中已经提交的胰凝乳蛋白酶和胰蛋白酶，对三者蛋白质进行了序列分析和三维结构的比较。牙鲆弹性蛋白酶cDNA包含一个完整的读码框（提交Genbank的登录号为EU873084）。其编码区平均GC含量为54％，推测编码的蛋白质包含296个氨基酸，分子量为29．04KD，等电点为6．14。蛋白序列比较表明它和牙鲆弹性蛋白酶3相似性最高。通过同源建模得到弹性蛋白酶5的三维结构和牛胰凝乳蛋白酶结构相似，包含了2个α螺旋、β个8折叠和13个转角结构。牙鲆弹性蛋白酶、胰凝乳蛋白酶和胰蛋白酶中底物结合区的3个关键氨基酸有明显的区别，这些氨基酸的变化改变了底物结合位点开口的大小，胰凝乳蛋白酶2的三个关键氨基酸和牛胰凝乳蛋白酶相同，该区域能接受结构较大的芳香族氨基酸；胰蛋白酶3能更好的结合阳性氨基酸Lys或Arg；而弹性蛋白酶开口很小，只能结合小的残基。上述结果证明了牙鲆丝氨酸蛋白酶家族中的弹性蛋白酶、胰凝乳蛋白酶和胰蛋白酶底物结合位点的结构差异决定了其对底物选择的特异性。

Full-length cDNA and Protein Structure Analysis of Elastase from Paralichthys Olivaceus

To investigate the function of serine protease family and its rule of molecular evolution, A part of the flounder hepatopancreas cDNA library established in our lab was sequenced. A new serine protease family member of elastase 5 was got from those experiments above. The sequences and 3D structure of chymotrypsinogen, trypsinogen and elastase 5 was compared though bioinformatics approaches. The full-length cDNA of elastase 5 contains the open reading frame (ORF) of P. olivaceus elastase gene (Genbank accession number: EU873084; nemed elastase 5). In the coding region, the average GC content is 54%. The deduced P. olivaceus elastase 5 polypeptide contains 296 amino acids (aa), with a predicted molecular size of 29.04 kD and pI at 6.14. This protein Multiple-alignment has shown that this peptide is highly identical to the corresponding homologous elastase 3 in P. olivaceus.. The modeled 3D structure of the cloned elastase 5 is similar to cow chymotrypsin. It contains 2 helices, 13 β strands and 13 turns. Three aa that are essential for serine protease substrate binding function were found to be different among elastase, chymotrypsinogen and trypsinogen of P. olivaceus. those key alterable aa changed the size of substrate binding opening,Three core aa(Ser, Gly and Gly are identical to cow CHYMOTRYPSIN) is conservative of chymotrypsinogen 2 in the substrate binding domain, which can accommodate a larger aromatic residues. trypsinogen 3 will be beneficial to combine with the positive charge lys and arg residues. elastase 5 has a shallow entrance of the pocket, so only small residues could enter into the pocket. the results above proved that nuances of substrate binding domain of elastase, chymotrypsinogen and trypsinogen decide the specificity of substrate.