title: Effects on toxicity of eliminating a cleavage site in a predicted interhelical loop in Bacillus thuringiensis CryIVB delta-endotoxin.
abstract: When activated by treatment with mosquito (Aedes aegypti) gut extract, the Bacillus thuringiensis CryIVB delta-endotoxin lysed A. aegypti cells in vitro. SDS-PAGE and N-terminal sequence determination showed that in addition to removal of the C-terminal half of the molecule, the activated toxin had undergone proteolytic cleavage at two internal regions producing 47-48-kDa and 16-18-kDa polypeptides. Aligning the CryIVB protein sequence with the crystallographic structure of the CryIIIA toxin suggested that one set of cleavages occurred in a region before the start of the N-terminal helical bundle and the second cleavage site occurred in a predicted loop between helices 5 and 6 in the bundle at arginine-203. To investigate the suggestion by Li et al. that interhelical proteolysis is important in the cytolytic mechanism of these toxins, arginine-203 was substituted by alanine. The mutated toxin now resisted proteolysis at this position and showed a marked decrease in cytolysis in vitro but an increase in larvicidal activity.
authors.affiliation: Department of Biochemistry, University of Cambridge, UK.
authors.forename: C
authors.initials: C
authors.lastname: Angsuthanasombat
authors.forename: N
authors.initials: N
authors.lastname: Crickmore
authors.forename: D J
authors.initials: DJ
authors.lastname: Ellar
mesh_terms: D000330:Aedes; D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000633:toxicity*; D001427:Bacterial Toxins; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004731:Endotoxins / Q000235:genetics* / Q000633:toxicity*; D006460:Hemolysin Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010572:Pest Control, Biological; D017434:Protein Structure, Tertiary
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis