title: Activation and fragmentation of Bacillus thuringiensis delta-endotoxin by high concentrations of proteolytic enzymes.
abstract: Commercial enzymes and insect gut juice at various concentrations were used to digest Bacillus thuringiensis subsp. sotto Cry1Aa protoxin and examine the fragmentation pattern and effect on insecticidal activity. Trypsin at both high (5 mg/mL) and low (0.05 mg/mL) concentrations converted protoxin to toxin with no difference in insecticidal activity against Bombyx mori larvae. In both cases, the toxin protein had an apparent M(r) of 58.4 kDa (SDS-PAGE). Active toxin of identical M(r) was also produced with low concentrations of Pronase and subtilisin, but at high concentration, it was degraded into two protease-resistant fragments of apparent M(r) 31.8 and 29.6 kDa, and exhibited no insecticidal activity. Sequencing data established the primary cleavage site to be in domain II, the receptor-binding region of the toxin, in an exposed loop between two beta-sheet strands. Fragmentation was not observed, however, when the digests were analyzed by native protein techniques, but rather the toxin molecule appeared to be intact. The amount of activated toxin produced by Choristoneura fumiferana gut juice was markedly reduced when the gut-juice concentration was increased from 1 to 50% and correlated with a loss in insecticidal activity. However, no lower M(r) protease-resistant fragments were evident in the SDS-PAGE of these digests.
authors.affiliation: Canadian Forest Service, Great Lakes Forestry Centre, Sault Ste. Marie, ON, Canada. apang@nrcan.gc.ca
authors.forename: A S
authors.initials: AS
authors.lastname: Pang
authors.forename: J L
authors.initials: JL
authors.lastname: Gringorten
authors.forename: C
authors.initials: C
authors.lastname: Bai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001681:Biological Assay; D010450:Endopeptidases / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins; D009036:Moths / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011402:Pronase / Q000378:metabolism; D020860:Subtilisin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D010446:Peptide Fragments; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010450:Endopeptidases; D014357:Trypsin; D020860:Subtilisin; D011402:Pronase