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The CryIA(c) receptor purified from Manduca sexta displays multiple specificities
1National Research Council of Canada, Biotechnology Research Institute, Montreal, Quebec.
The Journal of Biological Chemistry
|September 1, 1995
Summary
Bacillus thuringiensis CryI toxins interact differently with the Manduca sexta aminopeptidase N. CryIA(a) and CryIA(b) bind to one site, while CryIA(c) binds to two sites, with varying affinities and sugar inhibition.
Area of Science:
- Biochemistry
- Molecular Biology
- Insect Toxicology
Background:
- Bacillus thuringiensis (Bt) produces Cry insecticidal crystal proteins toxic to various insect larvae.
- Cry proteins interact with specific receptors on the insect midgut brush-border membrane.
- Aminopeptidase N (APN) from Manduca sexta is a known Cry-binding protein crucial for Bt toxin activity.
Purpose of the Study:
- To characterize the kinetic binding interactions between four Bacillus thuringiensis CryI insecticidal proteins and a purified Cry-binding protein (aminopeptidase N) from Manduca sexta.
- To elucidate the binding sites and affinities of different CryIA toxin subclasses to aminopeptidase N.
- To investigate the role of specific sugars in modulating these toxin-APN interactions.
Main Methods:
- Purification of a soluble form of Manduca sexta aminopeptidase N using phospholipase C treatment.
- Analysis of kinetic binding characteristics using an optical biosensor.
- Determination of apparent kinetic rate constants (on-rates and off-rates) for toxin-APN interactions.
- Inhibition assays using various sugars (N-acetylgalactosamine, N-acetylglucosamine, mannose, glucose) to study binding specificity.
Main Results:
- The soluble aminopeptidase N recognized CryIA(a), CryIA(b), and CryIA(c) toxins, but not CryIC.
- CryIA(a) and CryIA(b) bound to a single site on APN, while CryIA(c) bound to two distinct sites.
- All CryIA toxins showed moderately fast association rates, but CryIA(c) exhibited a significantly faster dissociation rate from its second binding site.
- CryIA(c) binding was inhibited by N-acetylgalactosamine, but not other tested sugars, while CryIA(a) and CryIA(b) binding were unaffected.
Conclusions:
- Toxin interactions with the Cry-binding protein aminopeptidase N are complex and diverse.
- Differential binding kinetics and specific sugar interactions highlight the distinct nature of CryIA toxin recognition.
- These findings contribute to understanding the molecular basis of Cry protein toxicity and insect resistance mechanisms.