Spruce budworm elastase precipitates Bacillus thuringiensis δ-endotoxin by specifically recognizing the C-terminal region

Abstract

A gut juice protein from Choristoneura fumiferana (spruce budworm) larvae that precipitates certain δ-endotoxins shows a unique specificity for the C-terminal amino acid sequence. Using homolog scanning mutants, we have identified a contiguous region of the Cry1Aa toxin which interacts with the 75-kDa toxin precipitating protein (TPP-75) 1 Abbreviations used: BAPNA, Nα-benzoyl-DL-arginine p-nitroanilide; BBMV, brush border membrane vesicles; CAPS, 3-(cyclohexylamino)-propanesulfonic acid; CBB, Coomassie brilliant blue R250; Cf, Choristoneura fumiferana; DFP, diisopropyl flurophosphate; DIP, diisopropyl phosphate; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; FITC, fluorescein isothiocyanate; HPLC, high pressure liquid chromatography; OSU, Ohio State University; SDS PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TOAD, toxin overlay agarose diffusion; TPP-75, 75-kDa toxin precipitating protein. 1 resulting in precipitation. The contiguous region from Cry1Aa can be transferred to Cry1Ac and results in an identical precipitation reaction. The precipitation reaction occurs rapidly and is unique in that the ratio of precipitating protein to toxin is low (estimated at 0.01), unlike antibody–antigen reactions which exhibit mole ratios close to 1. TPP-75 has been characterized as an elastase-like serine protease. We have taken advantage of this serine protease character and incorporated a radiolabel using an irreversible inhibitor. The radiolabel has allowed us to show the coincidence of the catalytically-inhibited TPP-75 with the toxin in a blotting assay and to follow the degradation of TPP-75 during storage. TPP-75 represents the first evidence that gut juice proteins may selectively attenuate the activity of δ-endotoxins, prior to binding to putative receptors on susceptible cells. TPP-75 should be evaluated as a possible resistance mechanism for those larvae that do not exhibit a receptor-based resistance.