Interisland mutation of a novel phospholipase A2 from Trimeresurus flavoviridis venom and evolution of crotalinae group II phospholipases A2

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A(2) (PLA(2)). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA(2), which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD50 for mice was 1.34 mug/g, which is comparable to that of trimucrotoxim. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group 11 PLA(2)'s based on the amino acid sequences revealed that neurotoxic PLA-(2)'s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA-(2), groups such as PLA(2) type, basic [Asp(49)]PLA(2) type, and [Lys(49)]PLA(2) type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA(2)'S showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA(2)'S, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA(2)'s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation.