Structures and functions of crotoxin-like heterodimers and acidic phospholipases A2 from Gloydius intermedius venom: Insights into the origin of neurotoxic-type rattlesnakes☆

Abstract The cDNAs encoding four major phospholipases A 2 (PLA 2 s) were sequenced while the expressed sequence tags of Gloydius intermedius venom glands were constructed. These PLA 2 s were designated as Gintexin-A precursor, Gintexin-B, Gin-E6a and Gin-E6b, respectively. The deduced amino acid sequences of the former two PLA 2 s are 80% and 90% identical to those of crotoxin-A-precursor and crotoxin-B1, respectively. We also purified Gintexin-A, Gintexin-B, Gin-E6a and Gin-E6b like PLA 2 from the venom. The latter three PLA 2 s are enzymatically active but not strongly anticoagulant for human plasma. Gin-E6a and E6b-like PLA 2 s induced mouse platelet aggregation but inhibited rabbit platelet aggregation. The isolated Gintexin, a 1:1 complex of Gintexin-A and Gintexin-B, blocked the twitch of chick biventer cervicis tissue presynaptically. Results of N -terminal sequencing and peptide mass fingerprinting reveal that Gintexin-A undergoes proteolytic processing similar to crotoxin-A. This is the first time heterodimeric β-neurotoxins are found in Asian pitviper venom, and incompatible neurotoxic- and hemorrhagic-type venoms are found to evolve in parallel within the genus Gloydius , like in Crotalus . Thus, G. intermedius probably is the ancestor of rattlesnakes with type-II venom, and characterization of its venomics helps us to understand the evolution of heterodimeric neurotoxic PLA 2 s and the paedomorphic trend observed in Neotropical rattlesnake venoms. Biological significance For the first time, a heterodimeric neurotoxic PLA 2 (designated as Gintexin) has been isolated from the venom of an Asian pitviper, which shows a characteristic venom gland transcriptome similar to those of the neurotoxic type rattlesnakes. The fact that the venom of G. intermedius is less hemorrhagic than those of other Gloydius species, reveals that incompatible neurotoxic- and hemorrhagic-type venoms have evolved in parallel within the genus Gloydius , like the genus Crotalus . Our findings suggest that G. intermedius is the most probable ancestor of some Neotropical rattlesnakes. The results may revolutionize the theory regarding the origin of type-II rattlesnakes and assist with the diagnosis and clinical management of G. intermedius bites. Furthermore, the possibility of using the currently available antivenoms of Neotropical rattlesnakes to treat G. intermedius bites seems feasible.