Interactions of substrate with calreticulin, an endoplasmic reticulum chaperone

Abstract

Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc₁Man_7-9GlcNAc₂ present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc₁Man_7-9GlcNAc₂, determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (K_b ) and changes in enthalpy of binding (ΔH_b°) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19×10⁴ M⁻¹ at 279 K is obtained when glucose occurs in α-1,3 linkage to ManαMe as in Glcα1-3ManαMe. The binding constant increases by 25-fold from di- to trisaccharide and doubles from tri- to tetrasaccharide, demonstrating that the entire Glcα1-3Manα1-2Manα1-2ManαMe structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis à vis legume lectins, which have the same fold as this chaperone, are discussed.