Characterization of a phospholipase A2 inhibitor from the Crotalus durissus terrificus venom gland: a possible adjuvant in the antivenom therapy

Abstract

Snake envenoming is a public health problem in many countries. In Brazil, more than 170 thousand cases have been recorded, being the majority due to snakebites from Bothrops genus. However, the Crotalus genus is the most lethal one. Snakes from this genus are popularly known as rattlesnakes, and there is only one species (Crotalus durissus) in the Brazilian territory. The C. durissus is subdivided into 6 subspecies and C. d. terrificus is one of the most prevalent. Crotalic venom presents mainly neurotoxic and myotoxic actions and activity on blood coagulation. The major component of this venom is crotoxin, which is a complex comprising a phospholipase A2 (PLA2) catalytically inactive (acid subunit) that acts as chaperone for a PLA2 catalytically active (basic subunit). The acid subunit directs the acid one to specific sites that cause specially neurotoxic actions. Crotoxin is the main responsible for deaths in crotalic envenoming due to the blockage of neuromuscular transmission consequently leading the victim to a respiratory failure. The actual therapy for snake envenoming is the antivenom produced from the purification of horse immunoglobulins previously immunized with snake venom(s). Despite its efficacy, the antivenom can cause many adverse reactions (early or late) in the victim, which includes anaphylactic shock and the development of the "serum sickness". PLA2 inhibitors (PLIs), that are present in snake blood as a part of an innate defense mechanism against the self-venom and/or venoms from other snakes, have already demonstrated anti-myotoxic and anti-neurotoxic activities. These inhibitors are classified into 3 groups (alpha, beta or gamma) according to their structural domains. This study aims the characterization of a beta PLI, named CdtPLI2, recently detected in the C. d. terrificus venom gland, focusing on an adjuvant use in the antivenom therapy. For this purpose, a synthetic gene will be designed with the CdtPLI2 cDNA sequence and further expressed in Pichia pastoris. The recombinant inhibitor will be isolated from the culture supernatant using protein liquid chromatography. The recombinant CdtPLI2 will be extensively structurally characterized. This characterization will be focused on molecular mass, post-translational modifications and tridimensional structure. In vitro studies (phospholipase activity inhibition and kinetics of enzyme inhibition) will be performed to characterize the inhibition of crotoxin and PLA2 from C. d. terrificus venom and inhibition of phospholipase activity from different medically important Brazilian snake venoms. On the other hand, in vivo assays will evaluate the inhibition of sistemic effects and edema induction caused by crotoxin. Other in vivo assays (alteration of biochemical parameters and cytokines releasing) will evaluate possible toxic activity caused by the recombinant CdtPLI2 administration. This study aims the use of the recombinant CdtPLI2 as a complement in the snake antivenom therapy due to the inhibition of systemic effects caused by crotoxin. This study will be the first detailed study about a ² PLI. Furthermore, this work paves the way to possible applications of phospholipase inhibitors in diseases related to an increase in the endogenous phospholipase A2 activity in the organism. (AU)