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 A₂ (PLA₂) isozymes were purified to homogeneity from P. elegans venom and sequenced. This led to a discovery of novel PLA₂ isozymes with Arg at position 49, that is, [Arg⁴⁹]PLA₂ 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 [Asp⁴⁹]PLA₂ named PePLA₂ was also obtained. In contrast to PLA₂ 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 PLA₂ 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 PmK49PLA₂, a [Lys⁴⁹]PLA₂, from P. mucrosquamatus (Taiwan) than BPI and BPII, both [Lys⁴⁹]PLA₂ forms, from P. flavoviridis (Amami-Oshima and Tokunoshima islands of Japan). Such evolutionary relationships are also seen in neutral [Asp⁴⁹]PLA₂ 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.