The CryIA(c) receptor purified from Manduca sexta displays multiple specificities.
The kinetic binding characteristics of four Bacillus thuringiensis CryI insecticidal crystal proteins to a Cry-binding protein, purified from Manduca sexta brush-border vesicles, were analyzed by an optical biosensor. This 120-kilodalton binding protein, previously determined to be aminopeptidase N, was converted to a 115-kilodalton water-soluble form by removing the attached glycosylphosphatidylinositol anchor with phospholipase C. The solubilized form recognized the three major subclasses of CryIA toxins but not CryIC even though all four CryI proteins are toxic to larvae of M. sexta. CryIA(a) and CryIA(b) toxins bound to a single site on the solubilized aminopeptidase N molecule whereas CryIA(c) bound to two distinct sites. Apparent kinetic rate constants were determined for each binding reaction. All three CryIA toxins exhibited moderately fast on rates (approximately 10(-5) M-1 s-1) and a slow reversible off rate (approximately 10(-3) s-1). Although the second CryIA(c)-binding site retained a moderately fast association rate, it was characterized by a rate of dissociation from the amino-peptidase an order of magnitude faster than observed for the other CryIA-binding sites. CryIA(c) binding to both sites was strongly inhibited in the presence of N-acetylgalactosamine (IC50 = 5 mM) but not N-acetylglucosamine, mannose, or glucose. CryIA(a) and CryIA(b) binding were unaffected in the presence of the same sugars. Our results serve to illustrate both the complexity and the diverse nature of toxin interactions with Cry-binding proteins.
Masson
L
L
National Research Council of Canada, Biotechnology Research Institute, Montreal, Quebec.
Lu
Y J
YJ
Mazza
A
A
Brousseau
R
R
Adang
M J
MJ
D000116:Acetylgalactosamine / Q000494:pharmacology; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001427:Bacterial Toxins; D001665:Binding Sites; D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000302:isolation & purification / Q000378:metabolism*; D006460:Hemolysin Proteins; D019476:Insect Proteins; D007700:Kinetics; D018402:Manduca; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C095416:Cry toxin receptors; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000116:Acetylgalactosamine