Protein Engineering and Gene Analysis of Trimeresurus flavoviridis Phospholipase A_2 Isozymes

从竹叶青蛇（Trimeresurus flavoviridis）蛇毒中分离到6个PLA_2同工酶基因，并进行了序列测定。比较这些cDNA的核苷酸序列发现，5'和3'非翻译区的同源性远高于蛋白质编码区的同源性，并且蛋白质编码区中密码子的三个位置的碱基替换似乎以相似的速率发生。从分子进化的角度来看，这些新发现具有极大的意义。为了进一步了解这一进化事件，我们分离并测序了6个T。黄病毒PLA_2同工酶基因。每个基因由4个外显子和3个内含子组成，编码138个氨基酸残基的蛋白质，其中包括16个氨基酸残基的信号序列。核苷酸序列比较表明，除第1外显子的信号肽编码区外，内含子的同源性远高于外显子的蛋白质编码区。内含子的每个位点的核苷酸替换数（K_n）约为蛋白质编码区每个同义位点的核苷酸替换数（K_s）的四分之一，表明内含子是异常保守的。在内含子中没有明显的功能作用表明，蛋白质编码区，除了信号肽编码结构域，已经在更大的取代率比内含子的演变。每个非同义位点的核苷酸替换数（K_a）接近或大于相关基因对的K_s值，表明外显子的蛋白质编码区发生了达尔文型加速替换。这与蛇毒中存在具有不同生理活性的PLA_2种类是一致的。融合蛋白的溶解和连续切割成成熟蛋白是目前尚待解决的主要难题。

Six cDNAs encoding Trimeresurus flavoviridis venom phospholipase A_2(PLA_2)isozymes were isolated and sequenced. Comparison of the nucleotide sequences of these cDNAs revealed that the homologies of the 5' and 3' untranslated regions were much higher than that of the protein-coding regions and that base substitutions appeared to have occurred at similar rates for the three positions of codons in the protein-coding regions. Such novel findings are of great interest from the viewpoint of molecular evolution. To gain a further insight into this evolutional event, we have isolated and sequenced six T. flavoviridis PLA_2 isozyme genes. Each gene consisted of four exons and three introns and encoded protein of 138 amino acid residues including the signal sequence of 16 amino acid residues. Comparison of the nucleotide sequences showed that the introns are much more homologous than the protein-coding regions of exons except for the signal peptide-coding region of the 1st exon. The number of nucleotide substitutions per site (K_n) for introns were approximately one-fourth of the numbers of nucleotide substitutions per synonymous site (K_s) for the protein-coding regions, indicating that the introns are unusually conserved. The absence of apparent functional role in the introns suggested that protein-coding regions, except for the signal peptide-coding domains, have evolved at greater substitution rates than introns. The fact that the numbers of nucleotide substitutions per nonsynonymous site (K_a) are close to or larger than K_s values for relevant pairs of genes revealed that Darwinian-type accelerated substitutions have occurred in the protein-coding regions of exons. This is compatible with the presence of PLA_2 species with diverse physiological activities in the venom.Expressions of PLA_2s using cDNAs are under way. Solubilization of fusion-proteins and successive cleavage to mature proteins are the major difficulties to be solved.