Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1Atoxins with high affinity

BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule tha t binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was pu rified by preparative gel electrophoresis. Rabbit antibodies specific for t he Bt toxin-binding molecule were raised. Attempts to label BTR-270 by prot ein-directed techniques were futile, but it was degraded by proteases with broad specificity indicating the presence of a peptide. Carbohydrate was de tected by labeling with digoxigenin hydrazide following periodate oxidation . Mild alkaline hydrolysis destroyed toxin and antibody binding, suggesting O-linked glycans are involved in the activity. GC/MS composition analysis showed that the predominant sugars were galactose, glucose, and N-acetyl ga lactosamine with lesser amounts of N-acetyl glucosamine, glucuronic acid, x ylose, and fucose. The carbohydrate moiety accounted for 73% of its total m ass. Amino acid analysis showed a high content of aspartic/asparagine, thre onine, and serine residues in the protein moiety. The purified glycoconjuga te was not visualized using Coomassie or silver staining procedures, but st ained "blue" using the cationic dye Stains-all. BTR-270 was labeled with bi otin and used as a diagnostic probe for screening and identifying toxins th at bind to the receptor. Toxin-binding kinetics obtained using a biosensor demonstrated that the receptor binds Cry1Aa and Cry1Ab toxins with high aff inity, and displays a weaker affinity for Cry1Ac, in correlation with the t oxicity of these toxins towards gypsy moth. (C) 2001 Wiley-Liss, Inc.