Banca de DEFESA: ANDERSON MACIEL DE LIMA

Antiparasitic activity of acid proteins isolated from Bothrops jararacussu snake venom: Phospholipase A2 (BthA-I-PLA2) and Type C Lectin (BjcuL) with antimalarial potential

Snake venoms. Phospholipases A2. Lectin type-c. Antiplasmodial activity.

Malaria is an infection caused by protozoa of the genus Plasmodium, and stands out for its high number of cases in tropical regions. In addition, it is characterized by having a therapy that in some localities have strains that have developed resistance to the drugs used. New strategies have been adopted to identify molecules that are effective for treating this disease. The present work aimed to evaluate the in vitro antiplasmodial activity of BjcuL (lectin type C) and BthA-I-PLA2 (phospholipase A2), both isolated from Bothrops jararacussu snake venom. The purification of lectin was performed using as the first chromatographic step an affinity chromatography on agarose-lactose resin, followed by reverse phase C18 column. Electrophoretic and mass spectrometric analysis showed that lectin has a dimeric structure, bind by disulfide bonds, with molecular weight of 16,117 Da for each monomer, constituting a dimeric protein with 32,642 Da. Lectin as well as its inhibition was verified by lactose, galactosamine and EDTA, indirectly demonstrating its affinity for carbohydrate glucose and the dependence by Ca2 + ions with the use of EDTA chelator that inhibited the maintenance of this biological activity. PLA2 purification was performed in 2 chromatographic steps, the first one being ion exchange in CM-Sepharose resin, followed by reverse phase chromatography in C-18 resin, extracting an acid fraction of interest and rechromatographed under the same conditions of the latter one. SDS-PAGE showed the presence of a 15 kDa apparent molecular weight protein compatible with snake phospholipases A2. Enzyme activity was evaluated using the chromogenic substrate 4N3OBA and demonstrated that BthA-I-PLA2 is an enzymatically active phospholipase A2. Next, the antiparasitic potential of venom and isolated proteins were evaluated in vitro against intraerythrocytic forms of Plasmodium falciparum. The results of antiparasitic inhibition against P. falciparum, venom, lectin and PLA2 presented IC50 of 0.31, 0.11 and 0.028 μg/mL, respectively. The evaluation of cytotoxic activity against the HepG2 cell line presented by Lectin and PLA2 showed that the molecules were selective for the parasite and not for the cells, denoting CC50 ≥ 10 with a selectivity index (IS) of 90,9 for Lectin and 357,14 for PLA2. The results underscore the relevance of the study of snake venom as a promising source of active molecules against tropical parasitic diseases that affect mankind and in particular demonstrate the inhibitory potential of P. falciparum parasite growth by the acid proteins isolated from the B. jararacussu snake venom.