Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of AmamiOshima T. flavoviridis venom led to isolation of a novel phospholipase A2 (PLA2). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA2, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD 50 for mice was 1.34 μg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the AmamiOshima and the Tokunoshima T. flavoviridis venomgland 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 II PLA2's based on the amino acid sequences revealed that neurotoxic PLA2's including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA2 groups such as PLA2 type, basic [Asp49]PLA2 type, and [Lys49]PLA2 type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA2's showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA2'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 PLA2'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.
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