Poster 126 - Structural determinants of PLA2s-Lys49 biological activities: A site directed mutagenesis

Poster 126. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.485.

Structural Determinants of PLA2s-Lys49 Biological Activities: A Site Directed Mutagenesis Study.

1 Depto. de Bioquímica e Imunologia, FMRP-USP, Ribeirão Preto-SP; 2 Depto de Química, FFCLRP-USP, Ribeirão Preto-SP.

Phospholipases A2 (EC 3.1.1.4; PLA2) are involved in a wide variety of physiological process, including phospholipid digestion, membrane turnover, and host defense. PLA2 mediated biosynthesis of proinflammatory lipid mediators such as prostaglandins and leukotrienes also play a role in pathophysiologicals processes. The venoms of Agkistrodon, Bothrops e Trimeresurus snake species contain Lys49-PLA2 homologues that lack catalytic activity, yet nevertheless display a wide range of biological effects, including myotoxic, edema-inducing, citotoxic, and bactericidal activities. The absence of a clear correlation between catalysis and pharmacological activity together with the diversity of biological effects make Lys49-PLA2s an interesting target to study the structural bases of these diverse activities. We have investigated the structural determinants of biological activities by site directed mutagenesis of bothropstoxin-I (BthTX-I), a Lys49-PLA2 isolated from Bothropsjararacussu venom. Thirty six recombinant proteins mutated at the homodimer interfacial binding site or the C-terminal extension were produced, expressed in E. coli BL21(DE3){pLysS} as inclusion bodies, refolded and purified by cationic exchange chromatography. Biological activity assays showed that the majority of residues that influence the myotoxic effect are located in the C terminal region of the protein. The physical-chemical properties of the amino acids at the interfacial binding site and C-terminal extension also influence the membrane-damaging activity. Although superposed, the protein motifs responsible for the myotoxic and membrane–damaging activities are different. Furthermore, the hydrophobicity conferred by the Y117, Y119, F125 residues of the C-terminal region is critical to the bactericidal effect presented against Gram-negative organisms, however other positions and/or regions also participate in the bactericidal mechanism. This study demonstrates the potential of site directed mutagenesis as a tool to further the understanding of the structural determinants of biological activities in venom PLA2s.