Denaturation of Either Manduca sexta Aminopeptidase N or Bacillus thuringiensis Cry1A Toxins Exposes Binding Epitopes Hidden under Nondenaturing Conditions

Abstract

ABSTRACT The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with 125 I-labeled Cry1Ac, Cry1Ac mutant 509 QNR-AAA 511 (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N -acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both 125 I-Cry1Ac and 125 I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots 125 I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, 125 I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. 125 I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured 125 I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) 125 I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.