Discovery of novel [Arg 49]phospholipase A 2 isozymes from Protobothrops elegans venom and regional evolution of Crotalinae snake venom phospholipase A 2 isozymes in the southwestern islands of Japan and Taiwan

Abstract

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.