Construção e caracterização de uma molécula recombinante híbrida (disintegrina/fosfolipase D) e avaliação de sua atividade biológica

Abstract

The phospholipases D (PLDs) of the venom of the spider Loxosceles gaucho are the main toxins responsible for the observed effects in the poisoning. These toxins can exert a high hydrolytic activity on sphingomyelins, releasing ceramide 1-phosphate (C1P) and cyclic phosphates (cPA). cPA affects numerous cellular functions, including inhibition of tumor cell invasion and metastasis. This feature could be interesting for biotechnological use, however, PLDs promote strong platelet aggregation. On the other hand, the disintegrins present in Viperidae venom venoms can inhibit platelet aggregation and can bind to tumor cells because of their ability to interact with the highly expressed integrins (αvβ3) in these cells. Thus, a fusion of a snake disintegrin with a spider PLD may be an interesting option to eliminate unwanted platelet aggregation caused by PLD, and further act as a toxin carrier for tumor cells. Thus, in order to exploit the antitumor potential of a L. gaucho PLD called LgRec1 and the particularity of an E. carinatus snake disintegrin called Echistatin to bind to tumor cells, we designed a hybrid toxin composed by the fusion of these two molecules which was called Rechistatin. This sequence was cloned into the pAE vector and transformed into BL21 (DE3) bacterium for protein expression, which was then purified by IMAC. Mass spectrometry and circular dichroism analyzed the molecular mass and secondary structure of the hybrid molecule, respectively. Platelet aggregation was measured by aggregometry assay. The cytotoxicity of Rechistatin was analyzed in tumor cells. The results showed that the hybrid molecule was cloned and successfully expressed in the soluble form. Rechistatin showed an expected band size of 38 kDa as confirmed by mass spectrometry. Circular dichroism revealed an expected pattern of conformation. Fusion of Echistatin to LgRec1 was effective to abolish the aggregation properties of LgRec1 PLD and ELISA tests on cells showed that Echistatin was efficient in directing LgRec1 to tumor cells U87-MG and RD. Additionally, the results showed that Rechistatin exerts cytotoxicity on U87-MG and RD cells. Taken together, these results indicate that the fusion of Echistatin with LgRec1 did not impair the biological activity of the chimeric toxin and was effective in abolishing the strong platelet aggregation of LgRec1. In addition, Echistatin was efficient in delivery LgRec1 to tumor cells, promoting cytotoxic activity.