title: Bacillus thuringiensis Cry1A toxin-binding glycoconjugates present on the brush border membrane and in the peritrophic membrane of the Douglas-fir tussock moth are peritrophins.
abstract: Bacillus thuringiensis (Bt) Cry1A toxin-binding sites in the Douglas fir tussock moth (DFTM) larval gut were localized using immunofluorescence microscopy. Cry1Aa, Cry1Ab and Cry1Ac all bound strongly to the DFTM peritrophic membrane (PM); weaker binding of the Cry1A toxins was observed along the apical brush border of the midgut epithelium. Comparative analysis of the Cry1A toxin-binding molecules in the PM and brush border membrane vesicles (BBMVs) showed that a similar toxin-binding complex was present in both. The Cry1A toxin-binding substance, a broad band with an apparent size of 180kDa, consisted of a closely spaced doublet. The doublet was present in peritrophins, proteins tightly bound to the PM. Lectin binding studies of the PM and BBMV toxin-binding components revealed that they are glyconjugates with terminal α-GalNAc residues comprised exclusively of O-linked oligosaccharides in their glycan structures. Mild periodate oxidation, release of O-linked glycans by β-elimination, and enzymatic removal of terminal α-linked GalNAc residues with N-acetyl-α-D-galactosaminidase digestion abolished Cry1A toxin-binding to the PM and BBMV components. These data provide strong evidence that O-linked glycans are the target structures on the toxin-binding glycoconjugates for the Cry1A class of insecticidal proteins in DFTM larvae.
authors.affiliation: USDA Forest Service, Northern Research Station, 359 Main Road, Delaware, OH 43015, USA. avalaitis@fs.fed.us
authors.forename: Algimantas P
authors.initials: AP
authors.lastname: Valaitis
authors.forename: John D
authors.initials: JD
authors.lastname: Podgwaite
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D004064:Digestive System / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006001:Glycoconjugates / Q000378:metabolism; D006460:Hemolysin Proteins / Q000378:metabolism*; D007814:Larva; D008871:Microvilli / Q000378:metabolism; D009036:Moths / Q000469:parasitology*; D010572:Pest Control, Biological / Q000379:methods; D010935:Plant Diseases / Q000469:parasitology / Q000517:prevention & control; D011134:Polysaccharides / Q000378:metabolism*; D028224:Pseudotsuga
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006001:Glycoconjugates; D006460:Hemolysin Proteins; D011134:Polysaccharides; C030956:insecticidal crystal protein, Bacillus Thuringiensis