Protein Engineering and Gene Analysis of Trimeresurus flavoviridis Phospholipase A_2 Isozymes

点击翻译按钮获取中文摘要

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.