Detection and characterization of Cry1Ac binding molecules in brush border membrane vesicles isolated from the midguts of Manduca sexta larvae

Receptors for Bacillus thuringiensis Cry1A toxins are located on apical membranes in the midgut of susceptible lepidopteran larvae. The current model for Cry1 A mode of action is that toxins bind to glycoproteins present on the apical membrane allowing for toxin insertion and pore formation. The ion flux generated by toxin pore formation leads to a disruption of physiological functions, ultimately causing cell lysis and insect death. In vitro systems, using brush border membrane vesicles (BBMV) isolated from insect midguts, are used to study Cry1 A binding and identify potential receptors. For Manduca sexta, a susceptible insect model, the Cry1A binding molecules in BBMV are identified as a 120-kDa aminopeptidase N (APN) and a 210- kDa cadherin-like protein.; In this dissertation, the interactions of Cry1Ab and Cry1Ac toxins with M. sexta BBMV molecules were examined. On ligand blots, Cry1Ac bound to molecules of 120 and 2l0 kDa. Cry1Ab binding was also detected at 210-kDa however, this toxin also bound a 106-kDa molecule, different from the 120-kDa protein bound by Cry1Ac. Characterization of the 120-kDa APN in M. sexta BBMV revealed that it is attached to the membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Removal of the lipid moiety of APN's GPI-anchor converted the 120-kDa hydrophobic form of this protein to a 11 5-kDa hydrophilic molecule. On ligand blots of M. sexta BBMV treated with periodate, 125I-Cry1Ac did not bind to the 120- and 210-kDa molecules supporting the hypothesis that Cry1Ac binds to carbohydrate epitopes associated with these glycoproteins. 125I-Cry1Ac binding to the 120-kDa APN was not affected after chemical and enzymatic removal of the GPI-anchor, suggesting that the GPI-anchor is not the carbohydrate epitope for Cry1Ac.; The novel result from this dissertation is that in addition to glycoproteins, Cry1Ac toxin binds to glycolipids extracted from M. sexta BBMV. On thin-layer chromatography overlays, 125I-Cry1Ac bound up to 8 different glycolipids, depending on the solvent system used for separation, and this binding was reduced with addition of 1000-fold excess of unlabeled toxin. 125I-Soybean agglutinin bound to the same glycolipids as 125I-Cry1Ac, indicating the presence of N-acetylgalactosamine, a proposed component of the toxin binding epitope. Pore formation, a post-binding event, was detected in "protein-free" liposomes prepared from M. sexta lipids incubated with nanomolar amounts of Cry1Ac. The combined detection of Cry1Ac binding to glycolipids and pore formation in liposomes prepared from M. sexta midgut lipids support a functional role for these membrane molecules in Cry1Ac action.; The above results suggest refinement of current hypotheses on Bt toxin action to include membrane glycolipids as potential receptors on the apical membrane of midgut epithelial cells. on the apical membrane of midgut epithelial cells.