Expression and characterization of a sphingomyelinase from Loxosceles gaucho spider venom and its peptides generated by the action of MMPs

Abstract

Loxosceles venom is composed of a mixture of toxins capable of causing an intense inflammatory reaction, dermonecrotic lesions and systemic manifestations such as platelet aggregation, hemolytic anemia and acute renal failure. Regarding the chemical composition, studies have shown that Loxosceles venom is rich in hialurinidases, peptidases, collagenases, alkaline phosphates, 5'-ribonucleotidase, fosfohidrolases, metalloproteinases and phospholipases D among others. The phospholipase D (FLDs) also called dermonecrotic toxins are the most studied because it has been shown to be responsible for the main effects observed in loxoscelism. However, since the total amount of poison is small, it becomes difficult to obtain the necessary amount of these toxins for biological studies and production of antiserum. Therefore, the strategy of cloning and expression of these toxins in recombinant form has been used. Despite the importance of poisoning by L. gaucho, to date no FLDS of this spider was cloned and expressed in order to obtain more quantity to perform functional and comparative studies. Therefore in this project, we aim to express an FLD of L. gaucho in bacterial system and evaluate its biological activity in vivo and in vitro. Moreover, since preliminary results indicated a possible processing by metalloproteinases FLDS, we intend to isolate and characterize these peptides and generate antibodies against them to evaluate its neutralizing action against the total venom. Thus, the studies proposed here will be important for understanding the mechanisms of action of the FLDS to contribute to the understanding of the processes involved in the poisoning caused by the spiders of the genus Loxosceles. (AU)