Activities of cobra venom cytotoxins toward heart and leukemic T-cells depend on localized amino acid differences

Several studies have suggested that along the concave surface of cobra venom cytotoxins, a hydrophobic region flanked by positively charged amino acid side-chains, as well as by tyrosine and/or serine/threonine, allows these toxins to depolarize muscle or cause cytolysis. Comparison of biological activities among structurally homologous toxins, however, has revealed significant functional diversity. The objective of the present study was to examine several toxins purified from different cobra venoms with regard to their ability to bind to and kill human T-lymphocytes and rat heart cell myoblasts. The activities observed were then correlated with differences in amino acid residues which occur in restricted regions of the toxins. The absence of an aromatic residue at position 11 (Loop 1) resulted in a lower cytolytic response at every concentration tested. A simple inversion of two residues in the amino acid sequence of toxin Loop 3 selectively impaired heart cell binding and cytolysis, but had no effect on T-cells. Loss of a positively charged residue in the tip of Loop 2 minimally affected binding but significantly reduced cytolysis. Replacement of valine at positions 27 and 32, along with the introduction of a negative charge at the tip of Loop 2, interfered with binding to either cell type and caused a reduction in cytolysis. The results of this study suggest that no one loop or region is solely responsible for the toxin's biological activity. However, because the binding and cytolytic sites within these toxins are distinct, it may become possible to develop toxin derivatives in which only selected activities are enhanced.