Immunochemical characterization and role in toxic activities of region 115-129 of myotoxin II, a Lys49 phospholipase A2 from Bothrops asper snake venom

Abstract

The region 115–129 of myotoxin II, a catalytically inactive Lys49 phospholipase A2, was previously shown to constitute a heparin-binding site and to be involved in its cytolytic action in vitro. An immunochemical approach was utilized to further explore the role of this region in the toxic activities of myotoxin II. By using a carrier-linked 13-mer synthetic peptide as immunogen, rabbit polyclonal antibodies against region 115– 129 were obtained. These antibodies were able to bind to the native protein and to inhibit its myotoxic and cytolytic effects in preincubation-type neutralization experiments. Antibodies to peptide 115–129 formed precipitating macromolecular complexes in gel immunodiffusion, demonstrating the oligomeric state of myotoxin II not only in its crystalline structure (dimeric), but also in solution. Analyses of the antibody response to carrier-linked peptide 115–129 and native myotoxin II suggest that region 115–129, although potentially immunogenic, is not an immunodominant B-cell epitope of this protein, failing to elicit significant antibody responses in animals immunized with the native toxin. Antibodies to peptide 115–129 cross-reacted with 15 purified class II myotoxic phospholipases A2 found in snake venoms of the genera Bothrops, Agkistrodon, Trimeresurus, and Vipera, but not with the recombinant human class II phospholipase A2, for which no toxic actions have been described. Myotoxic phospholipases of the class I (notexin) and class III (bee venom) groups were not recognized by antibodies to p115–129. These results demonstrate that the overall antigenic structure of region 115–129 is conserved among class II myotoxic phospholipases A2, despite differences in their corresponding amino acid sequences. Based on the accumulated experimental evidence, a model of the myotoxic region of myotoxin II, and possibly of related class II Lys49 phospholipase A2 myotoxins, is proposed.