| The goals of this research were to identify, purify and characterize B. thuringiensis Cry1Ac and Cry1Ca toxin-binding aminopeptidases from several lepidopteran insects including Manduca sexta, Heliothis virescens, and Plutella xylostella.; Receptors in the midgut brush border membrane catalyze binding and pore formation of B. thuringiensis Cry1 toxins, resulting in insect death. In the present study, I have identified aminopeptidase N (APN) as a major Cry1A and Cry1Ca toxin-binding protein in M. sexta, H. virescens, and P. xylostella. A 170 kDa APN purified from brush border membrane vesicles of H. virescens binds to Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1Ca and Cry1Ea toxins. When reconstituted into phospholipid vesicles containing {dollar}rmsp{lcub}86{rcub}Rbsp+,{dollar} the 170 kDa APN promoted toxin-induced {dollar}rmsp{lcub}86{rcub}Rbsp+{dollar} release from vesicles for three Cry1A toxins, but not Cry1Ca toxin. This correlation between toxin binding specificity and {dollar}rmsp{lcub}86{rcub}Rbsp+{dollar} release strongly suggests that the purified 170 kDa APN is a functional receptor in the brush border membrane of H. virescens. In M. sexta, I identified a 106 kDa form of APN as a Cry1Ca binding protein. Analyses of internal amino acid sequence and non-denaturing polyacrylamide gel electrophoresis revealed that the 106 kDa APN is distinct from a previously-described 115 kDa Cry1Ac-binding APN. These results show that Cry1Ca and Cry1Ac toxins recognize functionally related, but structurally distinct 106 kDa and 115 kDa isoforms of APN in the M. sexta midgut. These studies are evidence that APN functions as a Cry1 toxin receptor by mediating specific toxin binding and pore formation.; The following studies suggest that other membrane components, besides toxin-binding APN, modulate toxin action. For example, a 120 kDa aminopeptidase was detected in both susceptible and resistant P. xylostella. The presence of this Cry1Ac-binding aminopeptidase in the brush border membrane of P. xylostella is not sufficient to confer susceptibility to Cry1Ac. Similarly, the binding of B. thuringiensis Cry1 toxins to brush border membranes of Spodoptera exigua and Spodoptera frugiperda is necessary but not sufficient for toxin activity. Cry1Ac toxin, which is not toxic to S. exigua and S. frugiperda, bound to brush border membrane vesicles from both insects. However, pore formation assays revealed that Cry1Ac toxin did not alter the permeability of brush border membranes of S. exigua and S. frugiperda. In contrast, Cry 1Fa, which is highly toxic to S. exigua and S. frugiperda larvae, greatly increased the membrane permeability. These results demonstrate a positive correlation between Cry1 toxin-induced pore formation and toxicity in S. exigua and S. frugiperda. Furthermore, the data also suggest that some as-yet unidentified membrane components seem to be critical in determining Cry1Ac toxin-induced pore formation in the membrane. |
