Endoplasmic Reticulum-associated Degradation of Mammalian Glycoproteins Involves Sugar Chain Trimming to Man6–5GlcNAc2

Abstract

Endoplasmic reticulum-associated degradation of misfolded or misprocessed glycoproteins in mammalian cells is prevented by inhibitors of class I ␣-mannosidases implicating mannose trimming from the precursor oligosaccharide Glc 3 Man 9 GlcNAc 2 as an essential step in this pathway. However, the extent of mannose removal has not been determined. We show here that glycoproteins subject to endoplasmic reticulum-associated degradation undergo reglucosylation, deglucosylation, and mannose trimming to yield Man 6 GlcNAc 2 and Man 5 GlcNAc 2 . These structures lack the mannose residue that is the acceptor of glucose transferred by UDPGlc:glycoprotein glucosyltransferase. This could serve as a mechanism for removal of the glycoproteins from folding attempts catalyzed by cycles of reglucosylation and calnexin/calreticulin binding and result in targeting of these molecules for proteasomal degradation.Trimming of the asparagine-linked precursor oligosaccharide Glc 3 Man 9 GlcNAc 2 in the endoplasmic reticulum (ER) 1 yields Glc 1 Man 9 -7 GlcNAc 2 (G1M9 -7) that associates with the ER chaperones/lectins calnexin (CNX) and calreticulin (CRT) (1). A folding and quality control process is then initiated that consists of cycles of deglucosylation by glucosidase II, release from the chaperone, reglucosylation by the folding sensor enzyme UDP-Glc:glycoprotein glucosyltransferase (GT), and reassociation with CNX and CRT (2). Glycoproteins that achieve proper folding are no longer recognized by GT and leave this cycle.Trimming of mannose (Man) residues is involved in the disposal of ER-retained glycoproteins that cannot be driven to their native forms during the cycles of binding to CNX/CRT (3). It was proposed that Man trimming may provide a timer that allows these folding attempts before degradation starts (3, 4). Thus, inhibitors that prevent Man trimming were found to inhibit the ERAD of many defective glycoproteins (4 -8). In the absence of these inhibitors, the glycoproteins are ubiquitinated and delivered to the cytosolic proteasomes for degradation (9). Prior to dislocation to the cytosol, the substrates are transported to a specialized compartment termed the quality control (QC) compartment together with CNX and CRT (10). This compartment is pericentriolar, located near (but not overlapping) the Golgi and the ER-Golgi intermediate compartment (ERGIC), and is dependent on microtubules.Whereas the inhibitor studies provide strong evidence that Man trimming is involved in ERAD, the structures of the N-linked oligosaccharides that result from trimming have not been determined in mammalian cells. The inhibitors that prevent Man trimming leading to ERAD (such as 1-deoxymannojirimycin (dMNJ) and kifunensine) are blockers of class I mannosidases. Therefore, the major ER class I enzyme, ER mannosidase I, has been proposed to be responsible for this pathway. Consequently, the product of cleavage by this enzyme, an M8 isomer lacking the middle branch terminal Man (M8B), has been suggested as the signal that marks the glycoprotein for...

Deletion of the Glucosidase II Gene in Trypanosoma brucei Reveals Novel N-Glycosylation Mechanisms in the Biosynthesis of Variant Surface Glycoprotein

“…Furthermore, the results suggest that T. brucei UGGT glucosylates Man 5 GlcNAc 2 , and not Man 9 GlcNAc 2 , on VSG, whereas UGGT from other organisms has been reported to have a marked preference for Man 9 -7 GlcNAc 2 over Man 5 GlcNAc 2 (13). These unexpected results prompted us to re-evaluate our assumptions, based on examples from mammalian cells (41), that Man 9 GlcNAc 2 structures attached to protein in the ER of the parasite could not be processed by ER mannosidases to anything other than triantennary endo H-sensitive Man 5 GlcNAc 2 structures. Consequently, we analyzed the effects of a mixture of three potent cell-permeable ␣-mannosidase inhibitors (kifunensin, swainsonine, and 1-deoxymannojirimycin), which between them can inhibit all known classes of ER and Golgi ␣-mannosidases on the processing of wild-type VSG221 N-glycans.…”

Endoplasmic reticulum–retained podocin mutants are massively degraded by the proteasome

“…Cnx recognizes a complex code of glucose and mannose trimming in the N-glycan groups of its substrate proteins (21,(24)(25)(26). The fact that Pod R138Q topology and degradation is sensitive to annose trimming implies that most probably there is an interaction with Cnx through its N-glycan groups.…”

Genesis of ER stress in Huntington’s Disease

“…Extensive trimming of mannose residues of the precursor oligosaccharide is an essential requirement for glycoprotein ERAD [57] and α1,2 ER mannosidase I was proposed to function as an ERAD timer in the quality control of glycoproteins [58][59][60], together with a family of ER degradation enhancing α-mannosidase-like proteins (EDEMs), [61][62][63][64][65].…”