EFFECTS ON TOXICITY OF ELIMINATING A CLEAVAGE SITE IN A PREDICTED INTERHELICAL LOOP IN BACILLUS-THURINGIENSIS CRYIVB DELTA-ENDOTOXIN

When activated by treatment with mosquito (Aedes aegypti) gut extract, the Bacillus thuringiensis CryIVB delta-endotoxin lysed A. aegypti cells in vitro. SDS-PAGE and N-terminal sequence determination showed that in addition to removal of the C-terminal half of the molecule, the activated toxin had undergone proteolytic cleavage at two internal regions producing 47-48-kDa and 16-18-kDa polypeptides. Aligning the CryIVB protein sequence with the crystallographic structure of the CryIIIA toxin suggested that one set of cleavages occurred in a region before the start of the N-terminal helical bundle and the second cleavage site occurred in a predicted loop between helices 5 and 6 in the bundle at arginine-203. To investigate the suggestion by Li et al. [8] that interhelical proteolysis is important in the cytolytic mechanism of these toxins, arginine-203 was substituted by alanine. The mutated toxin now resisted proteolysis at this position and showed a marked decrease in cytolysis in vitro but an increase in larvicidal activity.