Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensis delta-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N

Two arginine residues (368-369) of Cry1Ab and Cry1Ac were mutated to alanin e, glutamic acid and lysine by site-directed mutagenesis. Insecticidal acti vities of the mutant toxins on Manduca sexta and Lymantria dispar larvae we re examined. Cry1Ac mutant toxins (c)RR-AA and (c)RR-EE and Cry1Ab mutant t oxins (b)RR-AA and (b)RR-EE showed great reductions in toxicity against bot h insects. In contrast, conservatively changed (c)RR-KK and (b)RR-KK mutant s did not alter toxicity to either insect. Binding assays with brush border membrane vesicles (BBMVs) prepared from L. dispar midguts demonstrated tha t (c)RR-AA, (c)RR-EE, (b)RR-AA and (b)RR-EE bound with lower affinities com pared with their respective wild-type toxins. To M. sexta BBMVs, (c)RR-AA a nd (c)RR-EE showed great reductions in BBMV binding. However, (b)RR-AA and (b)RR-EE did not alter BBMV competition patterns, despite their reduced tox icity. Further binding assays were performed with aminopeptidase N (APN) pu rified from L. dispar and M. sexta BBMVs using surface plasmon resonance (B IAcore). Direct correlation between toxicity and APN binding was observed f or the mutant toxins using this technique. The inconsistency between BBMV a nd APN binding data with Cry1Ab to M. sexta suggests the possibility of a d ifferent Cry1Ab toxin-binding mechanism or the importance of another recept or in M. sexta.