Analysis of a novel mode of action of the dipteran-specific insecticidal protein

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The course of processing of the 70-kDa protoxin of Cry11A was analysed. Cry11A is one of the dipteran-specific insecticidal protein produced by Bacillus thuringiensis. Upon tryptic digestion of the 70-kDa protoxin, a 36-kDa fragment and a 32-kDa fragment were produced. Based on the N-terminal amino acid sequences of the fragments, it was strongly suggested that the two fragments were produced by intramolecular cleavage of the 70-kDa protoxin. By engineering the cry11A gene, the genes encoding fusion proteins GST11A-36k and GST11A-32k were constructed, in which the GST tract was attached onto the N-termini of the 36-kDa and the 32-kDa fragments, respectively. Another fusion protein 32k-His was constructed, in which a histidine hexamer was attached onto the C-terminus of the 32-kDa fragment. Neither GST11A-36k nor GST11lA-32k was insecticidal toward the mosquito larvae. The two fragments proved to associate to form a heterodimer. Because the complex consisting of the 36-kDa and the 32-kDa fragments exhibited the significant insecticidal activity toward the mosquito larvae, it was suggested that the complex was one of the active forms of Cry11A. Moreover, we have found Cry11A-specific binding proteins in the brush border membrane vesicle from the mosquito larvae. It was also suggested that the Cry 11A-specific binding site on the target cell membrane is separate from the Cry4A-specific one. Thus, it was strongly suggested that the insecticidal mechanism of Cry11A is novel one different from that of Cry4A, a representative dipteran-specific insecticidal protein of B.thuringiensis.10.