Muscle phospholipid hydrolysis by Bothrops asper Asp49 and Lys49 phospholipase A2 myotoxins – distinct mechanisms of action

Abstract

Bothrops snakes are the major cause of ophidian envenomings in Latin America. Their venom contains myotoxins that cause prominent muscle damage, which may lead to permanent disability. These toxins include myotoxins Mt-I and Mt-II, which share the phospholipase A₂ (PLA₂) fold, but Mt-II lacks enzymatic activity because the essential active site Asp49 is replaced by Lys. Both myotoxins cause sarcolemma alterations, with Ca²⁺ entry and loss of ATP and K⁺ from muscle cells, but the molecular lesions at the basis of their cellular action are not known, particularly the role of phospholipid hydrolysis. Here we tested their PLA₂ activity in vivo, and evaluated the hypothesis that Ca²⁺-activated endogenous PLA₂s may be involved in the action of Mt-II. The time course of phospholipid hydrolysis by Mt-I and Mt-II in myotubes in culture and in tibialis anterior mouse muscles was determined. Mt-I rapidly hydrolyzed phosphatidylcholine and phosphatidylethanolamine but not phosphatidylserine, but no phospho-lipids were hydrolyzed in the presence of Mt-II. Whole Bothrops asper venom induced a higher extent of phospholipid hydrolysis than Mt-I alone. These results demonstrate in vivo PLA₂ activity of Mt-I for the first time, and indicate that it acts only on the external monolayer of the sarcolemma. They also exclude activation of endogenous PLA₂s in the action of Mt-II, implying that plasma membrane disruption by this toxin does not depend on phospholipid hydrolysis. Therefore, both Bothrops myotoxins induce Ca²⁺ entry and release of ATP and cause myonecrosis, but through different biochemical mechanisms.