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 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.