The CryIA(c) Receptor Purified from Manduca sexta Displays Multiple Specificities

Abstract

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.

Mapping the Epitope in Cadherin-like Receptors Involved inBacillus thuringiensis Cry1A Toxin Interaction Using Phage Display

“…GalNAc efficiently competes the binding of Cry1Ac to APN by binding to a pocket located in domain III (25,44,45). This suggests the Cry1Ac epitope that binds scFv73 is different than that known for initially binding to APN, and as our results suggest, is located on domain II.…”

“…The interaction between toxin and its receptor can be complex. For example, Cry1Ac binds to two sites on the APN purified from M. sexta, and only one of these sites is also recognized by Cry1Aa and Cry1Ab (25). Interestingly, binding of Cry1Ac to both receptor sites is inhibited by sugars, which do not inhibit the binding of Cry1Aa and Cry1Ab (25).…”

Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment

“…The affinity constant (K d ) obtained by SPR analysis of the Cry1Ac interaction with APN isolated from M. sexta was two orders of magnitude lower than that reported from equilibrium binding studies with Cry1Ac and intact M. sexta BBMV [4,19]. The SPR experiments used APN covalently attached to a carboxymethylated dextran matrix, in the absence of other membrane components.…”

“…The SPR experiments used APN covalently attached to a carboxymethylated dextran matrix, in the absence of other membrane components. It was suggested that the presence of other molecules in BBMV, particularly lipid, might be responsible for the observed differences [19]. The fact that Cry toxin binding to BBMV from susceptible insects is quickly irreversible [22], indicating toxin insertion into the membrane, could account for the higher affinity of Cry1Ac for intact BBMV.…”

“…Recently the technique of surface plasmon resonance (SPR) has been used to study Cry toxin binding to both insect midgut brush border membrane vesicles (BBMV) and a purified toxin receptor [19,20]. With this technique, ligands (receptor, BBMV or toxin) are immobilized on a sensor chip and the interaction of a specific analyte with the immobilized ligand can be followed in real time.…”