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Biochemical and functional studies of ColTx-I, a new myotoxic phospholipase A2 isolated from Crotalus oreganus lutosus (Great Basin rattlesnake) snake venom

Abstract:

Commonly, phospholipases A<inf>2</inf> (PLA<inf>2</inf>s) play key roles in the pathogenesis of the local tissue damage characteristic of crotaline and viperine snake envenomations. Crotalus oreganus lutosus snake venom has not been extensively studied; therefore, the characterization of its components represents a valuable biotechnological tool for studying pathophysiological processes of envenoming and for gaining a deeper understanding of its biological effects. In this study, for the first time, a basic PLA<inf>2</inf> myotoxin, ColTx-I, was purified from C. o. lutosus through two chromatographic steps. ColTx-I is monomeric with calculated molecular mass weight (Mw) of 14,145 Da and a primary structure closely related to basic PLA<inf>2</inf>s from viperid venoms. The pure enzyme has a specific activity of 15.87 ± 0.65 nmol/min/mg at optimal conditions (pH 8.0 and 37°C). ColTx-I activity was found to be dependent on Ca<sup>2+</sup>, as its substitution by other ionic species as well as the addition of chelating agents significantly reduced its phospholipase activity. In vivo, ColTx-I triggered dose-dependent inflammatory responses, measured using the paw edema model, with an increase in IL-6 levels, systemic and local myotoxicity, characterized by elevated plasma creatine kinase activity. ColTx-I induced a complex series of degenerative events associated with edema, inflammatory infiltrate and skeletal muscle necrosis. These biochemical and functional results suggest that ColTx-I, a myotoxic and inflammatory mediator, plays a relevant role in C. o. lutosus envenomation. Thus, detailed studies on its mechanism of action, such as evaluating the synergism between ColTx-I and other venom components may reveal targets for the development of more specific and effective therapies.