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

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 Cry1Ac 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 Cry1Ac-Sepharose affinity chromatography revealed that only APN-1 was able to bind Cry1Ac, The narrow specificity and kinetic binding characteristics of APN-1 for Cry1Ac were determined using a surface plasmon resonance-based optical biosensor, APN-1 from the gypsy moth possessed a single Cry1Ac toxin-binding site and did not interact with either Cry1Aa or Cry1Ab. The association and dissociation rate constants of Cry1Ac and APN-1 were determined to be 7.2 x 10(4)Ms(-1) and 2.3 x 10(-3)s(-1) respectively, with an apparent affinity constant of 3.2 x 10(-8)M, 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. Published by Elsevier Science Ltd.