Spruce budworm elastase precipitates Bacillus thuringiensis delta-endotoxin by specifically recognizing the C-terminal region.
A gut juice protein from Choristoneura fumiferana (spruce budworm) larvae that precipitates certain delta-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)' 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 delta-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.
Milne
R
R
Canadian Forestry Service, Sault Ste. Marie, Ontario, Canada. rmilne@nrcan.gc.ca
Wright
T
T
Kaplan
H
H
Dean
D
D
D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001665:Binding Sites; D002250:Carbon Radioisotopes; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000378:metabolism*; D005782:Gels; D006460:Hemolysin Proteins; D005779:Immunodiffusion; D019476:Insect Proteins / Q000378:metabolism; D009036:Moths / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000378:metabolism*; D012685:Sepharose; D012967:Sodium Dodecyl Sulfate
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002250:Carbon Radioisotopes; D004731:Endotoxins; D005782:Gels; D006460:Hemolysin Proteins; D019476:Insect Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D012967:Sodium Dodecyl Sulfate; D012685:Sepharose; D010196:Pancreatic Elastase