Mi Kyong Lee

Brush border membrane aminopeptidase-n in the midgut of the gypsy moth serves as the receptor for the CryIA(c) δ-endotoxin of Bacillus thuringiensis

Aminopeptidase-N (AP-N) was purified from gypsy moth (Lymantria dispar, L.) brush border membrane vesicles (BBMV) proteins by mono-Q chromatography and Superdex-75 gel filtration in the presence of the zwitterionic detergent, CHAPS, using FPLC. The purified AP-N, identified by its enzymatic activity, had an apparent size of 100 kDa, and was identified as the unique Bacillus thuringiensis insecticidal toxin, CryIA(c), binding protein. AP-N clearly displayed strong binding to CryIA(c), exhibiting little or no binding to CryIA(a) or CryIA(b), and showing no binding for the coleopteran-specific toxin, CryIIIA. Protein blots of the BBMV proteins probed with biotin-labeled and 125I-labeled insecticidal proteins revealed that CryIAc binds only to 120 kDa protein which is a slightly larger size in comparison to purified AP-N. Antibodies raised against the gypsy moth AP-N demonstrated that the purified AP-N and the 120 kDa CryIA(c) binding protein of total BBMV proteins are antigenically identical.

Bacillus thuringiensis Insecticidal Proteins: Molecular Mode of Action

Growing interest in biorational pesticides has placed the Bacillus thuringiensis insecticidal crystal proteins at the forefront of pesticides for plant genetic engineering. The development of improvement pesticides, both in enhanced activity and broader host range, depends on an understanding of its mechanism of action. This review presents a complete overview of the bacterium and the group of insecticidal proteins known as Cry proteins or delta-endotoxins. The molecular mode of action is described in detail, including the mapping of receptor binding sites by site-directed mutagenesis, the known receptors, and the ion-channel activity of the toxins.

Binding of Bacillus thuringiensis Cry1Ac toxin to Manduca sexta aminopeptidase-N receptor is not directly related to toxicity

Bacillus thuringiensis Cry1Ac δ‐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.

Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensis delta-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.

Two 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.

Mutations at the arginine residues in α8 loop of Bacillus thuringiensis δ-endotoxin Cry1Ac affect toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N

The functional role of the α8 loop residues in domain II of Bacillus thuringiensis Cry1Ac toxin was examined. Alanine substitution mutations were introduced in the residues from 275 to 293. Among the mutant toxins, substitutions at R281 and R289 affected toxicity to Manduca sexta and Lymantria dispar. Loss of toxicity by these mutant toxins was well correlated with reductions in binding affinity for brush border membrane vesicles and the purified receptor, aminopeptidase N (APN), from both insects. These data suggest that the two arginine residues in the α8 loop region are important in toxicity and APN binding in L. dispar and M. sexta.