Pivotal Role of Calnexin and Mannose Trimming in Regulating the Endoplasmic Reticulum-associated Degradation of Major Histocompatibility Complex Class I Heavy Chain

Abstract

We have established a mammalian semipermeabilized cell system that faithfully reconstitutes the proteasomemediated degradation of major histocompatibility complex Class I heavy chain. We show that degradation required unfolding of the protein and was cytosol-and ATP-dependent and that dislocation and degradation required proteasome activity. When the interaction of heavy chain with calnexin was prevented, the rate of degradation was accelerated, suggesting that an interaction with calnexin stabilized heavy chain. Stabilization of heavy chain to degradation was also achieved either by preventing mannose trimming or by removal of the N-linked glycosylation site. This demonstrates that glycosylation and mannose trimming are required to ensure degradation of heavy chain. When degradation or mannose trimming was inhibited, heavy chain formed a prolonged interaction with immunoglobulin heavy chain binding protein, ERp57, and protein disulfide isomerase. Taken together, these results indicate that calnexin association and mannose trimming provide a mechanism to regulate the folding, assembly, and degradation of glycoproteins entering the secretory pathway.

Multiple Endoplasmic Reticulum-associated Pathways Degrade Mutant Yeast Carboxypeptidase Y in Mammalian Cells

“…However, no consistent picture has emerged; in some cases accelerated degradation is observed when a glycoprotein is prevented from entering the cycle (26,51), whereas in other cases interaction with the cycle accelerated degradation (18,25). There are also cases in which glucose trimming has been shown to have no effect on ERAD (20,43).…”

“…Although some have suggested calnexin-substrate complexes as intermediates toward ERAD (18,25), other reports indicate that calnexin binding protects glycoproteins from degradation (26,51). To test the situation in our system, we used castanospermine to block glucosidase I and II and to prevent CPY* binding to calnexin.…”

“…A possible role for mannose trimming is also suggested by the effect on ERAD observed when a putative mannose lectin, named EDEM in mammalian cells and HtmI/ MnlI in yeast, is eliminated or overexpressed (21)(22)(23)(24)(25). For some glycoproteins, there is also evidence that trimming of glucose residues plays a role in regulating ERAD (11,26).…”

Transcription Factor NFIC Undergoes N-Glycosylation during Early Mammary Gland Involution

“…A general signal for retrotranslocation, suggested by Johnson and Haigh (67), is the prolonged exposure of a polypeptide sequence, surface, or glycosylation state that would elicit chaperone binding. Cytoplasmic proteins and ATP hydrolysis are also required (68,69). The retrotranslocated protein is probably pulled into the cytoplasm by a protein located in the cytoplasm (67).…”

Glycoprotein Folding in the Endoplasmic Reticulum

“…However, another group used the mannosidase inhibitors kifunensin and deoxymannojirimycin to conclude that secretion of the α 1 -antitrypsin Z mutant was increased when oligosaccharide trimming was prevented (Marcus and Perlmutter, 2000). Various other molecules have been shown to require mannose trimming in the ER prior to degradation, including CPY in yeast (Jakob et al, 1998) and the MHC class I molecule in man (Wilson et al, 2000).…”