Cloning and characterization of Trimeresurus gracilis venom phospholipases A(2): comparison with Ovophis okinavensis venom and the systematic implications

Abstract

This study focuses on the structural and functional characterizations of novel venom phospholipases A(2) (PLA(2)s) from Trimeresurus gracilis, an endemic Taiwanese pitviper. The PLA(2) cDNAs were cloned from venom glands and sequenced. The majority of the clones encoded a Glu6-containing PLA(2) (designated as Tgc-E6) whose deduced amino acid sequence resembled those of other Crotalinae acidic PLA(2)s. Tgc-E6 was also purified and constituted about 6% (w/w) of the total venom proteins. For human platelet rich plasma, Tgc-E6 inhibited the ADP- and collagen-induced aggregation with an IC(50) of 272 nM and 518 nM, respectively. Like Ovophis okinavensis venom, T. gracilis venom did not contain any Lys49-PLA(2)s, although a cDNA encoding Lys49-PLA(2) has been cloned from each of the species. Their predicted protein sequences are 94% identical, and their pI values 8.3 are lower than those of other Lys49-PLA(2)s, mainly due to the acidic substitutions within positions 78-111, which are apparently more similar to those in Tgc-E6 than to those in other Lys49-PLA(2)s. This unique structural feature of the venom PLA(2)s thus render evidence for close phylogenetic relationship between both species. The structural variations in the venom acidic PLA(2)s of the two species possibly have resulted from adaptation to different prey ecology.