Kinetics and the mechanism of interaction of the endoplasmic reticulum chaperone, calreticulin, with monoglucosylated (Glc(1)Man(9)GlcNAc(2)) substrate

Calreticulin is a lectin-like molecular chaperone of the endoplasmic reticu lum in eukaryotes. Its interaction with N-glycosylated polypeptides is medi ated by the glycan, Glc(1)Man(9)GlcNAc(2), present on the target glycoprote ins. In this work, binding of monoglucosyl IgG (chicken) substrate to calre ticulin has been studied using real time association kinetics of the intera ction with the biosensor based on surface plasmon resonance (SPR). By SPR, accurate association and dissociation rate constants were determined, and t hese yielded a micromolar association constant. The nature of reaction was unaffected by immobilization of either of the reactants. The Scatchard anal ysis values for K-a agreed web crith the one obtained by the ratio k(1)/k(- 1). The interaction was completely inhibited by free oligosaccharide, Glc(1 )Man(9)GlcNAc(2), whereas Man(9)GlcNAc(2) did not bind to the calreticulin- substrate complex, attesting to the exquisite specificity of this interacti on. The binding of calreticulin to IgG was used for the development of immu noassay and the relative affinity of the lectin-substrate association was i ndirectly measured. The values are in agreement with those obtained with SP R. Although the reactions are several orders of magnitude slower than the d iffusion controlled processes, the data are qualitatively and quantitativel y consistent with single-step bimolecular association and dissociation reac tion. Analyses of the activation parameters indicate that reaction is entha lpically driven and does not involve a highly ordered transition state. Bas ed on these data, the mechanism of its chaperone activity is briefly discus sed.