Interaction analyses of Bacillus thuringiensis Cry1A toxins with two aminopeptidases from gypsy moth midgut brush border membranes

Abstract

A 100 kDa aminopeptidase N isolated from Lymantria dispar (gypsy moth) larval midgut brush border membrane vesicles (BBMVs) has previously been reported to function as a surface binding protein for the entomocidal protein toxin CrylAc from Bacillus thuringiensis (Valaitis et al., 1995; Lee et al., 1996). Fractionation of detergent-solubilized, phosphatidylinositol-specific phospholipase C-digested BBMV membrane proteins by ion-exchange chromatography revealed two distinct peaks of aminopeptidase activity from which two proteins, APN-1 and APN-2, were purified. Western blot immunoanalysis revealed that the previously reported 100 kDa APN (APN-1 in this study) was antigenically distinct from the newly identified 105 kDa APN-2. Both ligand blots and CrylAc-Sepharose affinity chromatography revealed that only APN-1 was able to bind CrylAc. The narrow specificity and kinetic binding characteristics of APN-1 for CrylAc were determined using a surface plasmon resonance-based optical biosensor. APN-1 from the gypsy moth possessed a single CrylAc toxin-binding site and did not interact with either CrylAa or CrylAb. The association and dissociation rate constants of CrylAc and APN-1 were determined to be 7.2 × 10 4 Ms −1 and 2.3 × 10 −3s −1, respectively, with an apparent affinity constant of 3.2 × 10 −8M. Toxin binding to APN-1 was directly inhibited with N-acetylgalactosamine, suggesting that this aminosugar forms an integral part of the binding site. The absence of recognition of all Cry toxins by APN-2 suggests that either APN-2 recognizes an untested subclass of Cry toxins, or alternatively, not all APN molecules in larval midguts serve to function as toxin-binding proteins.