| Upon digestion by gypsy moth (Lymantria dispar) midgut proteases, the first 49 amino acids at the N-terminus of Cry2Aa, a Bacillus thuringiensis δ-endotoxin, are removed from the 63-kDa protoxin. The toxin's potency was lost when further in vitro midgut protease processing continued due to the cleavage at the carboxyl end of Leu144. To prevent the production of the non-toxic fragment, L144D, L144A, L144G, L144H, and L144V mutant proteins were constructed. These mutant proteins were highly resistant to the midgut proteases. However, the mutant proteins were as toxic to the insects as the wild type protein, indicating that the cleaved fragment is associated with the rest of the entire protein molecule in the midgut environment.; The role of the 49-amino acid length of the N-terminal sequence of Cry2Aa was investigated. This short peptide sequence contains one extra α helix, called α0. Deletion of the cry2Aa gene at the α0 helix, and deletion of the entire N-terminus region led to production of the non-soluble inclusion bodies that were non-toxic to both L. dispar and Anopheles quadrimaculatus. However, expression cry2Aa gene that contains the α0 helix, but lacking the Val 3-Asn 17 region, gave the soluble, and toxic crystal inclusions. The melting temperature of the Cry2Aa protoxin and the mutant protein containing α0 were 7°C higher than the active Cry2Aa fragment. Gene deletion and site-directed mutagenesis results indicated that the presence of this α helix is necessary for the formation of biological active inclusion of Cry2Aa.; Cry2Aa is specific to both dipteran and lepidopteran insects. Results from intensive site-directed mutagenesis of amino acid residues on loop 1 and loop 2 of domain II and bioassays against A. quadrimaculatus and L. dispar revealed that specificity of Cry2Aa to dipteran insects was conferred by amino acid residues in loop 1, while the amino acid residues in loop 2 are responsible for Lepidoptera specificity. Results from dissociation experiments indicated that these mutant proteins were not involved in irreversible binding. The dramatic changes found in these mutant proteins were reversible binding. Therefore, initial binding of amino acids on loop 1 and loop 2 of Cry2Aa might be a factor that determines the toxin dual specificity. |
