The Lectin Chaperone Calnexin Utilizes Polypeptide-based Interactions to Associate with Many of Its Substrates in Vivo

Abstract

Calnexin and calreticulin are molecular chaperones of the endoplasmic reticulum that bind to newly synthesized glycoproteins in part through a lectin site specific for monoglucosylated (Glc 1 Man 7-9 GlcNAc 2 ) oligosaccharides. In addition to this lectin-oligosaccharide interaction, in vitro studies have demonstrated that calnexin and calreticulin can bind to polypeptide segments of both glycosylated and nonglycosylated proteins. However, the in vivo relevance of this latter interaction has been questioned. We examined whether polypeptidebased interactions occur between calnexin and its substrates in vivo using the glucosidase inhibitor castanospermine or glucosidase-deficient cells to prevent the formation of monoglucosylated oligosaccharides. We show that if care is taken to preserve weak interactions, the block in lectin-oligosaccharide binding leads to the loss of some calnexin-substrate complexes, but many others remain readily detectable. Furthermore, we demonstrate that calnexin is capable of associating in vivo with a substrate that completely lacks Asn-linked oligosaccharides. The binding of calnexin to proteins that lack monoglucosylated oligosaccharides could not be attributed to nonspecific adsorption nor to its inclusion in protein aggregates. We conclude that both lectin-oligosaccharide and polypeptide-based interactions occur between calnexin and diverse proteins in vivo and that the strength of the latter interaction varies substantially between protein substrates. Glycoprotein folding within the endoplasmic reticulum (ER)1 is facilitated in part by the membrane-bound chaperone calnexin (CNX) and its soluble homolog calreticulin (CRT) (1). These proteins are unique among molecular chaperones in that they utilize a lectin site as a means to associate with unfolded glycoproteins (2-4). The lectin site is specific for Glc 1 Man 7-9 GlcNAc 2 oligosaccharides, which exist transiently as intermediates during the processing of Asn-linked glycoproteins. It is a widely held view that the removal and re-addition of the terminal glucose residue of these oligosaccharides, catalyzed, respectively, by the ER enzymes glucosidase II and UDPglucose:glycoprotein glucosyltransferase, regulate cycles of CNX and CRT binding to glycoproteins (5, 6). In this model, CNX and CRT do not function as classical molecular chaperones that prevent aggregation by binding to hydrophobic polypeptide segments. Rather, they are thought to promote folding by recruiting other chaperones and folding enzymes, such as the thiol oxidoreductase ERp57 (7,8), to the glycoprotein substrate.The concept that CNX and CRT associate with glycoproteins solely through lectin-oligosaccharide interactions is based primarily on experiments wherein cultured cells were treated with tunicamycin to block Asn-linked oligosaccharide addition or with the glucosidase I and II inhibitors castanospermine or deoxynojirimycin to prevent the conversion of the Glc 3 Man 9 GlcNAc 2 precursor to the monoglucosylated Glc 1 Man 9 GlcNAc 2 species. Subsequent immunoprec...

Missense Mutations in ABCG5 and ABCG8 Disrupt Heterodimerization and Trafficking

“…Glycan-independent binding of calnexin to other proteins has been documented (30 -32). Mutants of calnexin that lack a functional lectin domain associate with proteins via a polypeptide binding site and retain chaperone activity; however, the lectin site within calnexin enhances both the number and amount of substrates bound by calnexin (32)(33)(34)(35). The incomplete dissociation of calnexin with glycan-deficient G8 is consistent with the dual binding model of calnexin and suggests that the reduction in trafficking of G5/G8 dimers containing glycan-deficient G8 may reflect the reduced ability of calnexin to mediate folding of this dimer.…”

Productive Folding of Tyrosinase Ectodomain Is Controlled by the Transmembrane Anchor

“…Because calnexin may associate also with the polypeptide (2,3,16), an important point was to determine whether the TMD per se is required for the calnexin/tyrosinase interaction. We found that two different TMDs restored the interaction with calnexin, indicating that there is no direct interaction between the TMD of tyrosinase and calnexin.…”

Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells

“…These changes could either be due to a direct interaction between Crt-GFP and substrates (potentially in combination with other chaperones, such as ERp57), or indirectly through other substrate-bound chaperones that interact with Crt-GFP. To distinguish between these possibilities, we exploited the fact that most (if not all) substrate interactions with Crt are through its lectin activity (23,33,35). Crt(Y108F)-GFP is significantly reduced in its interactions with substrates ( Fig.…”