Intramolecular cleavage at the loop between α3-helix and α4-helix is critical for cytotoxic activity of Cry8Da

Abstract: Cry8Da from Bacillus thuringiensis galleriae SDS-502 has the toxicity against both larvae and adult P. japonica. Cry8Da is processed into three fragments (64 kDa, 54 kDa and 8 kDa) by gut juice of P. japonica. Fragments of 54 kDa and 8 kDa are derived from the cleavage of 64 kDa fragment at the loop between α3-helix and α4-helix in Domain I. Binding assays showed that the 54 kDa fragment bound to both larvae and adult P. japonica brush-border membrane vesicles while the 64 kDa and 8 kDa fragments did not. We constructed a protease-resistant mutant, 8Da-R163A, in which R163 on the loop was changed to A163. To directly investigate whether intramolecular cleavage is critical for insecticidal activity of Cry8Da, we performed cytotoxic assays against midgut epithelial cells (MECs) prepared from adult P. japonica using purified uncleaved (64 kDa) and intramolecular cleaved (mixture of 54 kDa and 8 kDa) Cry8Da toxin. Cytotoxic assay showed MECs were destroyed by only intramolecular cleaved Cry8Da toxin. Intramolecular cleaved Cry8Da toxin also formed oligomeric structure after incubation with MGCs. These results strongly support our idea that the cleavage at the loop between α3-helix and α4-helix is critical for toxicity of Cry8Da.