Specificity for target insects of Bacillus thuringiensisinsecticidal Cry toxins is largely determined by toxin affinity for insect midgut receptors. The mode of binding for one such toxin-receptor complex was investigated by extensive toxin mutagenesis, followed by realtime receptor binding analysis using an optical biosensor (BIAcore). Wild-type Cry1Ac, a three-domain, lepidopteran-specific toxin, bound purified gypsy moth (Lymantria dispar) aminopeptidase N (APN) biphasically. Site 1 displayed fast association and dissociation kinetics, while site 2 possessed slower kinetics, yet tighter affinity. We empirically determined that two

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The insecticidal activity of toxins CryIAa, CryIAb, and CryIAc against Lymantria dispar (gypsy moth) and Bombyx mori (silkworm) was examined by force-feeding bioassays. Toxin CryIAa exhibited higher toxicity than toxins CryIAb and CryIAc for L. dispar and B. mori. To evaluate possible synergism among these toxins, bioassays were performed with mixtures of CryIAa and CryIAb, CryIAb and CryIAc, and CryIAa and CryIAc. Expected toxicity was calculated from the activity of each individual toxin and its proportion in the mixture by using the equation described by Tabashnik (B. E. Tabashnik, Appl. Environ. Microbiol. 58:3343-3346, 1992). Observed 50% growth-inhibitory doses were calculated from mixing experiments by probit analysis. In L. dispar bioassays, synergism was observed with a mixture of CryIAa and CryIAc while a mixture of CryIAa and CryIAb exhibited an antagonistic effect. No synergistic effect on B. mori was observed with any toxin combination. Voltage clamping assays of isolated L. dispar midguts also demonstrated that the mixture of CryIAa and CryIAc induced a greater slope of inhibition of short circuit current than did other toxin combinations.

Bacillus thuringiensis Cry1Ac N N-endotoxin specifically binds a 115-kDa aminopeptidase-N purified from Manduca sexta midgut. Cry1Ac domain III mutations were constructed around a putative sugar-binding pocket and binding to purified aminopeptidase-N and brush border membrane vesicles (BBMV) was compared to toxicity. Q509A, R511A, Y513A, and 509^511 (QNR-AAA) eliminated aminopeptidase-N binding and reduced binding to BBMV. However, toxicity decreased no more than two-fold, indicating activity is not directly correlated with aminopeptidase-N binding. Analysis of toxin binding to aminopeptidase-N in M. sexta is therefore insufficient for predicting toxicity. Mutants retained binding, however, to another BBMV site, suggesting alternative receptors may compensate in vivo.z 1999 Federation of European Biochemical Societies.

BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were futile, but it was degraded by proteases with broad specificity indicating the presence of a peptide. Carbohydrate was detected 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 galactosamine with lesser amounts of N-acetyl glucosamine, glucuronic acid, xylose, and fucose. The carbohydrate moiety accounted for 73% of its total mass. Amino acid analysis showed a high content of aspartic/asparagine, threonine, and serine residues in the protein moiety. The purified glycoconjugate was not visualized using Coomassie or silver staining procedures, but stained "blue" using the cationic dye Stains-all. BTR-270 was labeled with biotin and used as a diagnostic probe for screening and identifying toxins that bind to the receptor. Toxin-binding kinetics obtained using a biosensor demonstrated that the receptor binds Cry1Aa and Cry1Ab toxins with high affinity, and displays a weaker affinity for Cry1Ac, in correlation with the toxicity of these toxins towards gypsy moth. Arch.

SummaryTwo arginine residues (368±369) of Cry1Ab and Cry1Ac were mutated to alanine, glutamic acid and lysine by site-directed mutagenesis. Insecticidal activities of the mutant toxins on Manduca sexta and Lymantria dispar larvae were examined. Cry1Ac mutant toxins (c)RR-AA and (c)RR-EE and Cry1Ab mutant toxins (b)RR-AA and (b)RR-EE showed great reductions in toxicity against both insects. In contrast, conservatively changed (c)RR-KK and (b)RR-KK mutants did not alter toxicity to either insect. Binding assays with brush border membrane vesicles (BBMVs) prepared from L. dispar midguts demonstrated that (c)RR-AA, (c)RR-EE, (b)RR-AA and (b)RR-EE bound with lower affinities compared with their respective wild-type toxins. To M. sexta BBMVs, (c)RR-AA and (c)RR-EE showed great reductions in BBMV binding. However, (b)RR-AA and (b)RR-EE did not alter BBMV competition patterns, despite their reduced toxicity. Further binding assays were performed with aminopeptidase N (APN) purified from L. dispar and M. sexta BBMVs using surface plasmon resonance (BIAcore). Direct correlation between toxicity and APN binding was observed for the mutant toxins using this technique. The inconsistency between BBMV and APN binding data with Cry1Ab to M. sexta suggests the possibility of a different Cry1Ab toxin-binding mechanism or the importance of another receptor in M. sexta.