THE INTERACTION OF SNAKE VENOM LYS49-PHOSPHOLIPASES A2 WITH MEMBRANES STUDIED BY PROTEIN ENGINEERING

Ward, R. J.(1)

(1)Departamento de Química, FFCLRP-USP

Bothropstoxin-I (BtxTx-I) is a myotoxic, homodimeric PLA₂ homologue from B. jararacussu venom which shows a Asp49Lys substitution. Although lacking detectable hydrolytic activity, BtxTx-I causes rapid Ca²⁺-independent release of liposome entrapped markers. Flexibility at the dimer interface results in "open" and "closed" dimer conformations, and a transition between these two forms suggests a mechanism for the Ca²⁺-independent membrane damaging activity. Site-directed BthTx-I mutants expressed as inclusion bodies in E. coli, refolded and purified by reverse phase chromatography, were used to evaluate the Ca²⁺-independent membrane damaging and myotoxic activities. Phospholipid hydrolysis, myotoxic activity and entrapped fluorescent marker release from liposomes were measured for several active site mutants. No catalytic activity was detected with the native, wild type recombinant and His48Gln proteins, yet myotoxic and membrane damaging activities were unaffected. These results demonstrate that neither the myotoxic nor the Ca²⁺-independent membrane damage by BtxTx-I involves phospholipid hydrolysis. In contrast, substitution of  positively charged amino acids between positions 115-122 abolished or significantly reduced membrane damaging activity, although no effect was observed by substitution of the lysines between positions 123-133. This suggests a purely physico-chemical explanation for the membrane damaging activity which involves alternative functions for the distal and proximal regions of the C-terminal loop. The myotoxicity of these mutants were also altered, although in a different pattern, suggesting overlapping yet distinct structural determinants in the C-terminus. Finally, the model predicts that the stability of the homodimer is important in the Ca²⁺-independent membrane damaging mechanism. Therefore, the monomer/dimer equilibrium constant of the native BthTx-I was compared to dimer interface mutants, and which revealed a direct correlation between dimer stability and membrane damaging activity.

Supported by FAPESP and CNPq.

CORRESPONDENCE TO:

Ward, R. J. Departamento de Química, FFCLRP-USP, Av. Bandeirantes 3900, Ribeirão Preto, 14040-903, SP, Brazil. Email: rjward@fmrp.usp.br