Contributions of the Lectin and Polypeptide Binding Sites of Calreticulin to Its Chaperone Functions in Vitro and in Cells

Abstract

Calreticulin is a lectin chaperone of the endoplasmic reticulum that interacts with newly synthesized glycoproteins by binding to Glc 1 Man 9 GlcNAc 2 oligosaccharides as well as to the polypeptide chain. In vitro, the latter interaction potently suppresses the aggregation of various non-glycosylated proteins. Although the lectin-oligosaccharide association is well understood, the polypeptide-based interaction is more controversial because the binding site on calreticulin has not been identified, and its significance in the biogenesis of glycoproteins in cells remains unknown. In this study, we identified the polypeptide binding site responsible for the in vitro aggregation suppression function by mutating four candidate hydrophobic surface patches. Mutations in only one patch, P19K/I21E and Y22K/ F84E, impaired the ability of calreticulin to suppress the thermally induced aggregation of non-glycosylated firefly luciferase. These mutants also failed to bind several hydrophobic peptides that act as substrate mimetics and compete in the luciferase aggregation suppression assay. To assess the relative contributions of the glycan-dependent and -independent interactions in living cells, we expressed lectin-deficient, polypeptide bindingdeficient, and doubly deficient calreticulin constructs in calreticulin-negative cells and monitored the effects on the biogenesis of MHC class I molecules, the solubility of mutant forms of ␣ 1 -antitrypsin, and interactions with newly synthesized glycoproteins. In all cases, we observed a profound impairment in calreticulin function when its lectin site was inactivated. Remarkably, inactivation of the polypeptide binding site had little impact. These findings indicate that the lectin-based mode of client interaction is the predominant contributor to the chaperone functions of calreticulin within the endoplasmic reticulum. Membrane-bound calnexin (Cnx)3 and its soluble paralog calreticulin (Crt) are glycoprotein-specific chaperones of the endoplasmic reticulum (ER). As components of the ER quality control system, they retain glycoprotein folding intermediates within this organelle and assist folding by preventing aggregation and by recruiting folding catalysts such as the thiol oxidoreductase ERp57 and peptidyl-prolyl isomerase cyclophilin B (for reviews, see Refs. 1 and 2). Both chaperones consist of a globular lectin domain and an extended arm or P domain (3-5) with the tip of the arm domain comprising the binding site for ERp57 (6) and cyclophilin B (7). The specificity for glycoproteins resides within their lectin domains, which recognize a monoglucosylated oligosaccharide-processing intermediate of composition Glc 1 Man 9 GlcNAc 2 (8 -10). Cycles of chaperone binding and release are regulated by the availability of the terminal glucose residue on this oligosaccharide with glucose removal catalyzed by glucosidase II and its readdition by UDPglucose:glycoprotein glucosyltransferase I (11). UDP-glucose: glycoprotein glucosyltransferase I acts as the folding sensor in the cycle, only...

Biosynthesis of human myeloperoxidase

“…Carbohydrate sidechains on MPO precursors interact with ER resident molecular chaperones, ERp57, calreticulin, and calnexin [70, 71] (Figure 1). Such proteins typically interact transiently with nascent glycoproteins during their transit in the ER and serve to prevent their aberrant and unproductive folding [72–74]. None interacts with mature MPO subunits or with non-glycosylated MPO precursors synthesized in the presence of tunicamycin [75, 76], an antibiotic that blocks N-linked glycosylation [77].…”

Dissecting physical structure of calreticulin, an intrinsically disordered Ca2+-buffering chaperone from endoplasmic reticulum

“…Being preferentially resided in the endoplasmic reticulum (ER), CALR is mostly known for its roles in Ca 2+ homeostasis (in ER lumen, more than 50% of Ca 2+ associates with CALR) (Nakamura et al, 2001) and acting as a lectin-like ER chaperon for many glycoproteins (Labriola, Cazzulo, & Parodi, 1999; Spiro, Zhu, Bhoyroo, & Soling, 1996; Vassilakos, Michalak, Lehrman, & Williams, 1998; Zapun et al, 1998). CALR binds monoglucosylated oligosaccharides (Helenius & Aebi, 2004; Helenius, Trombetta, Hebert, & Simons, 1997), as well as interacts with misfolded proteins that can be either glycosylated or non-glycosylated (Lum et al, 2016; Saito, Ihara, Leach, Cohen-Doyle, & Williams, 1999). As a matter of fact, together with another lectin chaperone (or carbohydrate-binding chaperone), calnexin (CNX), CALR is known to interact with practically all glycoproteins in the ER (Ellgaard & Frickel, 2003).…”

Calnexin cycle – structural features of the ER chaperone system

“…More recently, a surface distant from lectin site was identified as responsible for in vitro binding of nonglycosylated substrates [89]. In particular, two double mutants P19K/I21E and Y22K/F84E of calreticulin do not efficiently suppress aggregation of firefly luciferase and do not bind hydrophobic peptides.…”

“…In particular, two double mutants P19K/I21E and Y22K/F84E of calreticulin do not efficiently suppress aggregation of firefly luciferase and do not bind hydrophobic peptides. The use of these peptide‐binding‐deficient and lectin‐deficient mutants in calreticulin‐negative cells allowed accessing the relative contributions of glycan‐dependent and glycan‐independent in calreticulin function in biogenesis of MHC class I molecules [89]. The conclusion is that the lectin‐based interactions provide the major contribution, whereas the peptide‐binding site has little affect on calreticulin function in vivo .…”