Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N

binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to brush border me mbrane vesicle (BBMV) membranes and a purified putative receptor of the tar get insect Manduca sexta. Using hybrid proteins consisting of Cry1Ac and th e related Cry1C protein, it was shown that domain III of Cry1Ac is involved in specificity of binding as observed by all three techniques. In ligand b lotting experiments using SDS-PAGE- E-separated BBMV proteins as well as th e purified putative receptor aminopeptidase N (APN), the presence of domain III of Cry1Ac in a hybrid with Cry1C was necessary and sufficient for spec ific binding to APN. Using the surface plasmon resonance (SPR) technique wi th immobilized APN, it was shown that the presence of domain III of Cry1Ac in a hybrid is sufficient for binding to one of the two previously identifi ed Cry1Ac binding sites, whereas the second site requires the full Cry1Ac t oxin for binding. In addition, the role of domain III in the very specific inhibition of Cry1Ac binding by the amino sugar N-acetylgalactosamine (GalN Ac) was determined. Both in ligand blotting and in surface plasmon resonanc e experiments, as well as in binding assays using intact BBMVs, it was show n that the presence of domain III of Cry1Ac in a toxin molecule is sufficie nt for the inhibition of binding by GalNAc. These and other results strongl y suggest that domain III of delta-endotoxins play a role in insect specifi city through their involvement in specific binding to insect gut epithelial receptors.