Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity.
BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were futile, but it was degraded by proteases with broad specificity indicating the presence of a peptide. Carbohydrate was detected by labeling with digoxigenin hydrazide following periodate oxidation. Mild alkaline hydrolysis destroyed toxin and antibody binding, suggesting O-linked glycans are involved in the activity. GC/MS composition analysis showed that the predominant sugars were galactose, glucose, and N-acetyl galactosamine with lesser amounts of N-acetyl glucosamine, glucuronic acid, xylose, and fucose. The carbohydrate moiety accounted for 73% of its total mass. Amino acid analysis showed a high content of aspartic/asparagine, threonine, and serine residues in the protein moiety. The purified glycoconjugate was not visualized using Coomassie or silver staining procedures, but stained "blue" using the cationic dye Stains-all. BTR-270 was labeled with biotin and used as a diagnostic probe for screening and identifying toxins that bind to the receptor. Toxin-binding kinetics obtained using a biosensor demonstrated that the receptor binds Cry1Aa and Cry1Ab toxins with high affinity, and displays a weaker affinity for Cry1Ac, in correlation with the toxicity of these toxins towards gypsy moth. Arch.
Valaitis
A P
AP
USDA Forest Service, Delaware, Ohio, USA. avalaitis@fs.fed.us
Jenkins
J L
JL
Lee
M K
MK
Dean
D H
DH
Garner
K J
KJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001681:Biological Assay; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity / Q000379:methods; D010450:Endopeptidases / Q000378:metabolism; D004731:Endotoxins / Q000378:metabolism*; D006001:Glycoconjugates / Q000302:isolation & purification* / Q000378:metabolism; D006460:Hemolysin Proteins; D006868:Hydrolysis; D019476:Insect Proteins / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism; D009036:Moths; D010636:Phenols; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
D000596:Amino Acids; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002241:Carbohydrates; C095416:Cry toxin receptors; D004731:Endotoxins; D006001:Glycoconjugates; D006460:Hemolysin Proteins; D019476:Insect Proteins; D010636:Phenols; D011956:Receptors, Cell Surface; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010450:Endopeptidases