Abstract
Two subtypes of phospholipases A2 (PLA2s) with the ability to induce myonecrosis, ‘Asp49’ and ‘Lys49’ myotoxins, often coexist in viperid snake venoms. Since the latter lack catalytic activity, two different mechanisms are involved in their myotoxicity. A synergism between Asp49 and Lys49 myotoxins from Bothrops asper was previously observed in vitro, enhancing Ca2+ entry and cell death when acting together upon C2C12 myotubes. These observations are extended for the first time in vivo, by demonstrating a clear enhancement of myonecrosis by the combined action of these two toxins in mice. In addition, novel aspects of their synergism were revealed using myotubes. Proportions of Asp49 myotoxin as low as 0.1% of the Lys49 myotoxin are sufficient to enhance cytotoxicity of the latter, but not the opposite. Sublytic amounts of Asp49 myotoxin also enhanced cytotoxicity of a synthetic peptide encompassing the toxic region of Lys49 myotoxin. Asp49 myotoxin rendered myotubes more susceptible to osmotic lysis, whereas Lys49 myotoxin did not. In contrast to myotoxic Asp49 PLA2, an acidic non-toxic PLA2 from the same venom did not markedly synergize with Lys49 myotoxin, revealing a functional difference between basic and acidic PLA2 enzymes. It is suggested that Asp49 myotoxins synergize with Lys49 myotoxins by virtue of their PLA2 activity. In addition to the membrane-destabilizing effect of this activity, Asp49 myotoxins may generate anionic patches of hydrolytic reaction products, facilitating electrostatic interactions with Lys49 myotoxins. These data provide new evidence for the evolutionary adaptive value of the two subtypes of PLA2 myotoxins acting synergistically in viperid venoms.
Highlights
Phospholipases A2 (PLA2s) are widespread enzymes in snake venoms, where they play major toxic roles in the immobilization and/or killing of prey [1,2]
A higher effect of these toxins was observed in myotubes than in myoblasts, and this difference was more conspicuous in the case of the Asp49 myotoxin, which was extremely weak against myoblasts (Fig. 1A)
A comprehensive examination of the bioactivities displayed by myotoxic Asp49 and Lys49 variants does not provide evident clues on the possible evolutionary advantages conferred by the emergence of the latter, since both types of myotoxins share similar toxicological profiles and often coexist in viperid venoms [41]
Summary
Phospholipases A2 (PLA2s) are widespread enzymes in snake venoms, where they play major toxic roles in the immobilization and/or killing of prey [1,2]. Among their diverse activities, myotoxicity is a clinically relevant effect which may lead to severe tissue damage and associated sequelae in envenomings [3,4,5]. Through a process of accelerated evolution [7], these genes accumulated mutations that converted their corresponding non-toxic proteins into potent toxins, most notably displaying neurotoxicity and/or myotoxicity. A growing body of knowledge has been gathered on the characterization of PLA2 toxins, but the structural bases for their toxicity and precise modes of action remain only partially understood, leaving opened a number of challenging questions [10]
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