Kinetics and the Mechanism of Interaction of the Endoplasmic Reticulum Chaperone, Calreticulin, with Monoglucosylated (Glc1Man9GlcNAc2) Substrate

Abstract

Calreticulin is a lectin-like molecular chaperone of the endoplasmic reticulum in eukaryotes. Its interaction with N-glycosylated polypeptides is mediated by the glycan, Glc 1 Man 9 GlcNAc 2 , present on the target glycoproteins. In this work, binding of monoglucosyl IgG (chicken) substrate to calreticulin has been studied using real time association kinetics of the interaction with the biosensor based on surface plasmon resonance (SPR). By SPR, accurate association and dissociation rate constants were determined, and these yielded a micromolar association constant. The nature of reaction was unaffected by immobilization of either of the reactants. The Scatchard analysis values for K a agreed well with 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 interaction. The binding of calreticulin to IgG was used for the development of immunoassay and the relative affinity of the lectin-substrate association was indirectly measured. The values are in agreement with those obtained with SPR. Although the reactions are several orders of magnitude slower than the diffusion controlled processes, the data are qualitatively and quantitatively consistent with single-step bimolecular association and dissociation reaction. Analyses of the activation parameters indicate that reaction is enthalpically driven and does not involve a highly ordered transition state. Based on these data, the mechanism of its chaperone activity is briefly discussed.

Major Histocompatibility Complex Class I Molecules Expressed with Monoglucosylated N-Linked Glycans Bind Calreticulin Independently of Their Assembly Status

“…The K d values of the calreticulin interaction with free class I HC and refolded complexes were evaluated at 37°C and were determined to be 0.5/1.5 and 1.1 M, respectively. These values agree very well with the published surface plasmon resonance binding data for calreticulin and monoglucosylated chicken IgG (7). Because the affinities of free glycosylated HC and glycosylated native molecules are identical within the limits of the assays, we conclude that polypeptide-based interactions with calreticulin do not contribute to the binding to MHC class I molecules in vitro.…”

The Endoplasmic Reticulum Glucosyltransferase Recognizes Nearly Native Glycoprotein Folding Intermediates

“…As expected, GCI2-(1-53) triggered a much lower BiP/ Grp78 ATPase activity than GCI2-(1-40), although both neoglycoproteins shared the same Grp/BiP binding site centered at Leu-32 (8). It is doubtful that immediate partial deglucosylation of the transferred glycan mediated by glucosidases I and II would elicit a long-lived CNX/CRT-GCI2 interaction; because affinity of lectins for monoglucosylated glycans is rather low (16), it would be expected that an initial lectin binding would be followed by dissociation of the complex and glucosidases I-and II-mediated complete deglucosylation. No further glucosylation and, hence, CNX/CRT binding would occur until formation of molten globule-like conformations.…”

Beyond lectins: the calnexin/calreticulin chaperone system of the endoplasmic reticulum

“…The calnexin/calreticulin chaperone system glycosylated by oligosaccharyl transferase, the monoglucosylated structure is formed by the combined action of glucosidases I and II. In the lectin-only model, this then interacts with Cnx/Crt only through the lectin site, and dissociation is probably governed by the relatively weak affinity of this site (K d = 1-2 M) (Patil et al, 2000). Upon release from the chaperone, the glycoprotein is deglucosylated by the further action of glucosidase II.…”