Role of proteolysis in determining potency of Bacillus thuringiensis Cry1Ac delta-endotoxin

Bacillus thuringiensis protein delta -endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin, Here, we investigated the role of proteolytic processing in determining the potency of the B, thu ringiensis Cry1Ac delta -endotoxin towards Pieris brassicae (family: Pierid ae) and Mamestra brassicae (family: Noctuidae), In bioassays, Cry1Ac was ov er 2,000 times more active against P, brassicae than against M, brassicae l arvae. Using gut juice purified from both insects, we processed Cry1Ac to s oluble forms that had the same N terminus and the same apparent molecular w eight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With pro teases from P, brassicae, the Cry1Ac-susceptible insect, Cry1Ac was process ed to an insoluble product with a molecular mass of similar to 56 kDa, wher eas proteases from M, brassicae, the non-susceptible insect, generated prod ucts with molecular masses of similar to 58, similar to 40, and similar to 20 M)a, N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but M, brassicae proteases also cle aved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gin or Ser, the resulting m utant toxins resisted degradation by M, brassicae proteases, However, this mutation had little effect on toxicity to M, brassicae, Differential proces sing of membrane-bound Cry1Ac was also observed in qualitative binding expe riments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.