Identification of linear B-cell epitopes on myotoxin II, a Lys49 phospholipase A₂ homologue from Bothrops asper snake venom.
Knowledge on toxin immunogenicity at the molecular level can provide valuable information for the improvement of antivenoms, as well as for understanding toxin structure-function relationships. The aims of this study are two-fold: first, to identify the linear B-cell epitopes of myotoxin II from Bothrops asper snake venom, a Lys49 phospholipase A₂ homologue; and second, to use antibodies specifically directed against an epitope having functional relevance in its toxicity, to probe the dimeric assembly mode of this protein in solution. Linear B-cell epitopes were identified using a library of overlapping synthetic peptides spanning its complete sequence. Epitopes recognized by a rabbit antiserum to purified myotoxin II, and by three batches of a polyvalent (Crotalidae) therapeutic antivenom (prepared in horses immunized with a mixture of B. asper, Crotalus simus, and Lachesis stenophrys venoms) were mapped using an enzyme-immunoassay based on the capture of biotinylated peptides by immobilized streptavidin. Some of the epitopes identified were shared between the two species, whereas others were unique. Differences in epitope recognition were observed not only between the two species, but also within the three batches of equine antivenom. Epitope V, located at the C-terminal region of this protein, is known to be relevant for toxicity and neutralization. Affinity-purified rabbit antibodies specific for this site were able to immunoprecipitate myotoxin II, suggesting that the two copies of epitope V are simultaneously available to antibody binding, which would be compatible with the mode of dimerization known as "conventional" dimer.
Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, SJ 11501, Costa Rica. bruno.lomonte@ucr.ac.cr
Bruno
B
Lomonte
D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood / Q000276:immunology; D000997:Antivenins / Q000235:genetics; D017837:Bothrops / Q000378:metabolism*; D003435:Crotalid Venoms / Q000201:enzymology*; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000235:genetics*; D054501:Group II Phospholipases A2 / Q000737:chemistry / Q000235:genetics*; D007106:Immune Sera / Q000276:immunology; D007124:Immunoenzyme Techniques; D008958:Models, Molecular; D011817:Rabbits; D030162:Reptilian Proteins / Q000737:chemistry / Q000235:genetics*; D013045:Species Specificity; D019809:Streptavidin
D057134:Antibodies, Neutralizing; D000997:Antivenins; D003435:Crotalid Venoms; D018985:Epitopes, B-Lymphocyte; D007106:Immune Sera; D030162:Reptilian Proteins; D019809:Streptavidin; D054501:Group II Phospholipases A2; C061056:myotoxin II, Bothrops asper