Structural and Functional Characterization of γ-Type Phospholipase A2 Inhibitor from Bothrops jararacussu Snake Plasma

Phospholipases A(2) (PLA(2)s) from snake venoms comprise a group of 14-18 kDa proteins, responsible for several toxic effects induced by the whole venom. Considering this, studies aiming at the search for natural inhibitors of these proteins are very important. The present work had as objectives the isolation and functional/structural characterization of a gamma-type phospholipase A(2) inhibitor (PLI) from Bothrops jararacussu snake plasma, named gamma BjussuMIP. This acidic glycoprotein was isolated in a high purity level through affinity chromatography on CNBr-Sepharose 4B coupled with BthTX-II, showing a pI similar to 5.5 and molecular weight of 23,500 for the monomer (determined by SDS-PAGE), and 160,000 for the oligomer (determined by molecular exclusion chromatography on Sephacryl S-200). The interaction between gamma BjussuMIP (MIP) and Phospholipase A(2) (PLA(2)) was confirmed using circular dichroism (CD) and emission fluorescence techniques. The helical content of the 1: 1 molar mixture was higher than that calculated for the addition of the spectra of the unbound proteins indicating binding. The emission fluorescence experiments pointed that Trp residues in PLA(2) participate in proteins interaction as blue shift of 4 nm was observed. The gamma BjussuMIP cDNA, obtained by PCR of the liver of B. jararacussu snake, revealed 543 bp codifying for a mature protein of 181 amino acid residues. Alignment of its amino acid sequence with those of other snake gamma PLIs showed 89-94% of similarity. gamma BjussuMIP mainly inhibited the pharmacological properties of Asp49 PLA(2)s, such as phospholipase, anticoagulant, myotoxic, edema inducing, cytotoxic, bactericidal and lethal activities. In addition, it showed to be able to supplement Bothrops antivenom, potentiating its antimyotoxic effect. The aspects broached in this work will be able to provide complementary information on possible mechanisms of action, relating structure and function, which could result in a better understanding of the inhibitory effects induced by gamma BjussuMIP.