Discovery of novel [Arg49]phospholipase A2 isozymes from Protobothrops elegans venom and regional evolution of Crotalinae snake venom phospholipase A2 isozymes in the southwestern islands of Japan and Taiwan.

Protobothrops (formerly Trimeresurus) elegans, a Crotalinae snake, inhabits Ishigaki and Iriomote islands of the Sakishima Islands of Japan which are located between Okinawa island of Japan and Taiwan. Two phospholipase A(2) (PLA(2)) isozymes were purified to homogeneity from P. elegans venom and sequenced. This led to a discovery of novel PLA(2) isozymes with Arg at position 49, that is, [Arg(49)]PLA(2) forms, named PeBP(R)-I and PeBP(R)-II. They are polymorphic at position 3, Val for PeBP(R)-I and Ile for PeBP(R)-II. The cDNAs encoding PeBP(R)-I and PeBP(R)-II were cloned. The cDNA encoding an [Asp(49)]PLA(2) named PePLA(2) was also obtained. In contrast to PLA(2) isozymes from Protobothrops genus with 122 amino acid residues, PeBP(R)-I and PeBP(R)-II are composed of 121 amino acid residues due to lack of Pro at position 90. They exhibited necrotic and edema-inducing activities but no hemorrhagic activity was detected. A phylogenetic tree constructed for venom PLA(2) isozymes of Protobothrops genus and of related genera in the southwestern islands of Japan and Taiwan revealed that PeBP(R)-I and PeBP(R)-II of P. elegans are evolutionarily much closer to PmK49PLA(2), a [Lys(49)]PLA(2), from P. mucrosquamatus (Taiwan) than BPI and BPII, both [Lys(49)]PLA(2) forms, from P. flavoviridis (Amami-Oshima and Tokunoshima islands of Japan). Such evolutionary relationships are also seen in neutral [Asp(49)]PLA(2) isozymes from the three Protobothrops species. Thus, P. elegans is the species much closer to P. mucrosquamatus than P. flavoviridis. Their evolutionary distances seem to be well related to geological history of the islands where they have lived. In addition, it was clearly noted that Ovophis okinavensis (Amami-Oshima), which had formerly belonged to the Trimeresurus genus, and Trimeresurus stejnegeri (Taiwan) are the species fairly distant from Protobothrops genus.