Mapping the epitope in cadherin-like receptors involved in Bacillus thuringiensis Cry1A toxin interaction using phage display

In susceptible lepidopteran insects, aminopeptidase N and cadherin-like pro teins are the putative receptors for Bacillus thuringiensis (Bt) toxins. Us ing phage display, we identified a key epitope that is involved in toxin-re ceptor interaction. Three different scFv molecules that bind Cry1Ab toxin w ere obtained, and these scFv proteins have different amino acid sequences i n the complementary determinant region 3 (CDR3). Binding analysis of these scFv molecules to different members of the Cry1A toxin family and to Escher ichia coli clones expressing different Cry1A toxin domains showed that the three selected scFv molecules recognized only domain 11. Heterologous bindi ng competition of Cry1Ab toxin to midgut membrane vesicles from susceptible Manduca sexta larvae using the selected scFv molecules showed that scFv73 competed with Cry1Ab binding to the receptor. The calculated binding affini ties (Kd) of scFv73 to Cry1Aa, Cry1Ab, and Cry1Ac toxins are in the range o f 20-51 nm. Sequence analysis showed this scFv73 molecule has a CDR3 signif icantly homologous to a region present in the cadherin-like protein from M. sexta (Bt-R-1), Bombyx mori (Bt-R-175), and Lymantria dispar. We demonstra ted that peptides of 8 amino acids corresponding to the CDR3 from scFv73 or to the corresponding regions of Bt-R-1 or Bt-R-175 are also able to compet e with the binding of Cry1Ab and Cry1Aa toxins to the Bt-R-1 or Bt-R-175 re ceptors. Finally, we showed that synthetic peptides homologous to Bt-R-1 an d scFv73 CDR3 and the scFv73 antibody decreased the in vivo toxicity of Cry 1Ab to M. sexta larvae. These results show that we have identified the amin o acid region of Bt-Ri and Bt-R-175 involved in Cry1A toxin interaction.