Cytotoxic and inflammatory potential of a phospholipase A2 from Bothrops jararaca snake venom

Rafhaella C A Cedro,Norival A Santos-Filho,Karina F Zoccal,Suely V Sampaio,Tássia R Costa,Danilo L Menaldo,Marco A Sartim,Lúcia H Faccioli

doi:10.1186/s40409-018-0170-y

Abstract

BackgroundSnake venom phospholipases A2 (PLA2s) have been reported to induce myotoxic, neurotoxic, hemolytic, edematogenic, cytotoxic and proinflammatory effects. This work aimed at the isolation and functional characterization of a PLA2 isolated from Bothrops jararaca venom, named BJ-PLA2-I.Methods and ResultsFor its purification, three consecutive chromatographic steps were used (Sephacryl S-200, Source 15Q and Mono Q 5/50 GL). BJ-PLA2-I showed acidic characteristics, with pI~ 4.4 and molecular mass of 14.2 kDa. Sequencing resulted in 60 amino acid residues that showed high similarity to other Bothrops PLA2s, including 100% identity with BJ-PLA2, an Asp49 PLA2 previously isolated from B. jararaca venom. Being an Asp49 PLA2, BJ-PLA2-I showed high catalytic activity, and also inhibitory effects on the ADP-induced platelet aggregation. Its inflammatory characterization showed that BJ-PLA2-I was able to promote leukocyte migration in mice at different concentrations (5, 10 and 20 μg/mL) and also at different response periods (2, 4 and 24 h), mainly by stimulating neutrophil infiltration. Furthermore, increased levels of total proteins, IL-6, IL-1β and PGE2 were observed in the inflammatory exudate induced by BJ-PLA2-I, while nitric oxide, TNF-α, IL-10 and LTB4 levels were not significantly altered. This toxin was also evaluated for its cytotoxic potential on normal (PBMC) and tumor cell lines (HL-60 and HepG2). Overall, BJ-PLA2-I (2.5–160 μg/mL) promoted low cytotoxicity, with cell viabilities mostly varying between 70 and 80% and significant values obtained for HL-60 and PBMC only at the highest concentrations of the toxin evaluated.ConclusionsBJ-PLA2-I was characterized as an acidic Asp49 PLA2 that induces acute local inflammation and low cytotoxicity. These results should contribute to elucidate the action mechanisms of snake venom PLA2s.

Highlights

Snake venom phospholipases A2 (PLA2s) have been reported to induce myotoxic, neurotoxic, hemolytic, edematogenic, cytotoxic and proinflammatory effects
BJ-PLA2-I was characterized as an acidic Asp49 PLA2 that induces acute local inflammation and low cytotoxicity
Isolation of BJ-PLA2-I B. jararaca venom fractionation was initiated with a molecular exclusion chromatography on Sephacryl S-200, which resulted in several protein fractions that were named A to G1 (Fig. 1a)

Summary

Introduction

Snake venom phospholipases A2 (PLA2s) have been reported to induce myotoxic, neurotoxic, hemolytic, edematogenic, cytotoxic and proinflammatory effects. Phospholipases A2 (PLA2s) catalyze the hydrolysis of fatty acids at the sn-2 position of the phospholipid membranes, and release lysophospholipids and free fatty acids, especially polyunsaturated ones, such as arachidonic acid Based on their structure, In general, snake venom PLA2s are acidic or basic enzymes with molecular masses ranging from 13 to 15 kDa, and structure consisting of about 120 amino acid residues stabilized by 7 disulfide bonds, making them very stable molecules. Commonly reported in the literature is the existence of PLA2 homologues with a mutation at position 49 that exchanges the aspartic acid residue for a lysine These toxins are called Lys PLA2-like molecules, and this amino acid substitution leads to partial or total loss of their catalytic activity [4, 5]. Asp PLA2s present high catalytic activity, while Lys PLA2-like molecules do not, but can still induce several biological effects, such as myonecrosis, inflammation and cytotoxicity [4, 6,7,8]

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