The Molecular Chaperone Calnexin Binds Glc1Man9GlcNAc2 Oligosaccharide as an Initial Step in Recognizing Unfolded Glycoproteins

Abstract

Calnexin is a molecular chaperone that resides in the membrane of the endoplasmic reticulum. Most proteins that calnexin binds are N-glycosylated, and treatment of cells with tunicamycin or inhibitors of initial glucose trimming steps interferes with calnexin binding. To test if calnexin is a lectin that binds early oligosaccharide processing intermediates, a recombinant soluble calnexin was created. Incubation of soluble calnexin with a mixture of Glc0-3Man9GlcNAc2 oligosaccharides resulted in specific binding of the Glc1Man9GlcNAc2 species. Furthermore, Glc1Man5-7GlcNAc2 oligosaccharides bound relatively poorly, suggesting that, in addition to a requirement for the single terminal glucose residue, at least one of the terminal mannose residues was important for binding. To assess the involvement of oligosaccharide-protein interactions in complexes of calnexin and newly synthesized glycoproteins, alpha 1-antitrypsin or the heavy chain of the class I histocompatibility molecule were purified as complexes with calnexin and digested with endoglycosidase H. All oligosaccharides on either glycoprotein were accessible to this probe and could be removed without disrupting the association with calnexin. Furthermore, the addition of 1 M alpha-methyl glucoside or alpha-methyl mannoside had no effect on complex stability. These findings suggest that once complexes between calnexin and glycoproteins are formed, oligosaccharide binding does not contribute significantly to the overall interaction. However, it is likely that the binding of Glc1Man9GlcNAc2 oligosaccharides is a crucial event during the initial recognition of newly synthesized glycoproteins by calnexin.

MHC Class I Quality Control

“…Inside the ER the HC instantly interacts with BiP, the Hsp90 protein, and soon also with calnexin, a general ER lectin chaperone. Calnexin binds to newly synthesized proteins with mono-glycosylated N-linked glycans, and in line with this also to early folding stages of the N-glycosylated HC (Ware, Vassilakos et al 1995). This initial chaperoning of the HC prevents it from collapse and aggregation, and also sets the HC in a conformation that allows mature  2 m to bind the HC forming the MHC-I heterodimer.…”

“…The two outer glucose residues are then removed by glycosidase I and II leaving the MHC-I HC mono-glucosylated (Glc 1 Man 9 GlcNAc 2 ) at this step in the maturation process (Elbein 1991). This enables subsequent interaction of HC with the lectin chaperones, calnexin and calreticulin (Hebert, Foellmer et al 1995;Ware, Vassilakos et al 1995).…”

Roles of calreticulin and calnexin during mucin synthesis in LS180 and HT29/A1 human colonic adenocarcinoma cells

“…They recognize glycan chains of the form Glc " Man * GlcNAc # and bind with similar affinities [25]. It has also been reported [23,25] that, after initial binding occurs via the glycan, CLN probably associates in a more stable fashion by peptide interaction with peptide moieties on the surface of the incompletely folded protein. The protein then remains associated with the chaperone until it has folded correctly and lost the conformational features responsible for the attachment.…”

“…CLN and CRT function as chaperones in a similar manner, although CLN is a type 1 membrane protein, whereas CRT is soluble, is present in the ER lumen, and contains a Cterminal KDEL (Lys-Asp-Glu-Leu) ER retrieval signal [21]. However, CRT shares extensive sequence identity with the luminal domain of CLN [21], and both proteins are unique as chaperones, since they preferentially behave as lectins, interacting specifically with partially trimmed monoglucosylated N-linked oligosaccharides on newly synthesized protein molecules [22][23][24]. They recognize glycan chains of the form Glc " Man * GlcNAc # and bind with similar affinities [25].…”

N‐glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum by ‘‘off‐line’’ liquid chromatography and mass spectrometry

“…Otherwise, the modA mutant displays some growth retardation and alterations in the levels of some intracellular and secreted glycosidases (16) . It is possible that the binding to ER lectins involved in quality control in the secretory pathway is affected by a lack of glucosidase II activity; certainly, calnexin binds Glc 1 Man 9 GlcNAc 2 (the intermediate product of glucosidase II during trimming or the product of UDP-glucose:glycoprotein glucosyltransferase), but not Glc 2-3 Man 9 GlcNAc 2 (17) and an absence of glucosidase II may in some cases prevent association with calnexin and so result in increased degradation of proteins in the endoplasmic reticulum (18) .…”