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 A 2 (PLA 2 ) fold, but Mt-II lacks enzymatic activity because the essential active site Asp49 is replaced by Lys. Both myotoxins cause sarcolemma alterations, with Ca 2+ 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 PLA 2 activity in vivo, and evaluated the hypothesis that Ca 2+ -activated endogenous PLA 2 s 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 phospholipids 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 PLA 2 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 PLA 2 s 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 Ca 2+ entry and release of ATP and cause myonecrosis, but through different biochemical mechanisms.

Role of enzymatic activity in muscle damage and cytotoxicity induced byBothrops asperAsp49 phospholipase A2myotoxins: are there additional effector mechanisms involved?

“…These observations have led to the suggestion that viperid Asp49 PLA 2 myotoxins might possess mechanisms additional to their catalytic activity, to exert toxicity (Díaz et al, 1991; Bultrón, Thelestam & Gutiérrez, 1993; Díaz-Oreiro & Gutiérrez, 1997; Andrião-Escarso et al, 2000; Soares et al, 2001a; Soares et al, 2001b). The present results strengthen the concept that the effector mechanism of toxicity of viperid Asp49 PLA 2 myotoxins, i.e., the capacity to disrupt the integrity of plasma membrane, essentially relies on their ability to hydrolyze phospholipids on the membrane of target cells (Fernández et al, 2013). On the other hand, p -BPB, besides blocking the catalytic center of Asp49 PLA 2 s, may induce subtle conformational changes at distant sites.…”

“…Altogether, current evidence on the mode of action of the two divergent subtypes of group II PLA 2 myotoxins of viperids supports the notion that the basic Asp49 enzymes acquired this toxic activity by changes that directed their catalytic action towards the membrane phospholipids of muscle, whereas the Lys49 proteins, which lost catalytic activity along their evolutionary history due to critical substitutions (Petan, Križaj & Pungerčar, 2007), acquired changes that enabled them to directly alter membrane permeability via their specialized C-terminal region (Lomonte & Gutiérrez, 2011; Fernández et al, 2013). …”

“…A number of studies have demonstrated that Lys49 PLA 2 homologues induce myonecrosis by a catalytic-independent mechanism, involving amino acids at their C-terminal region which affect the integrity of the sarcolemma (Lomonte et al, 1994; Núñez, Angulo & Lomonte, 2001; Chioato et al, 2002; Chioato & Ward, 2003; Lomonte, Angulo & Calderón, 2003; dos Santos et al, 2009; Lomonte & Rangel, 2012; Fernandes et al, 2013; Fernández et al, 2013). On the other hand, Asp49 PLA 2 s have been shown to depend on their catalytic activity to disrupt the integrity of this membrane (Lomonte & Gutiérrez, 2011).…”

Rastreamento em células in vitro de uma fosfolipase A2 da peçonha de Bothrops alternatus por meio de pontos quânticos de tamanhos mágicos

“…O mecanismo de indução de danos celulares pelas svPLA 2 Asp-49 e svPLA 2 Lys-49 ocorre de forma diferente. A hidrólise de fosfolipídeos de membrana ocasionada pela atuação das svPLA 2 Asp-49 geram alterações irreversíveis às células, sendo que essa ação enzimática libera derivados lisofosfolipídicos, agentes que causam a desestruturação da membrana plasmática, aumentando a permeabilidade da mesma a íons (MORA-OBANDO et al, 2014;FERNÁNDEZ et al, 2013;HARRIS et al, 2013). Esse evento altera de Figura 4.…”

“…As svPLA 2 Lys-49 independem de ação enzimática para causarem danos a nível celular. Essas proteínas atuam por meio de seus resíduos de aminoácidos catiônicos e hidrofóbicos/aromáticos presentes na posição C-terminal (FERNÁNDEZ et al, 2013;LOMONTE & GUTIÉRREZ, 2011), além de serem capazes de adentrar as células e interagir com componentes intracelulares, seguido de alterações no funcionamento celular (MASSIMINO et al, 2018).…”