Monoglucosylated Oligomannosides Are Released during the Degradation Process of Newly Synthesized Glycoproteins

Abstract

The Chinese hamster ovary mutant MI8 -5 is known to synthesize Man 9 GlcNAc 2 -P-P-dolichol rather than the fully glucosylated lipid intermediate Glc 3 Man 9 GlcNAc 2 -P-P-dolichol. This nonglucosylated oligosaccharide lipid precursor is used as donor for N-glycosylation. In this paper we demonstrate that a significant part of the glycans bound to the newly synthesized glycoproteins in MI8 -5 cells are monoglucosylated. The presence of monoglucosylated glycans on glycoproteins determines their binding to calnexin as part of the quality control machinery. Furthermore, we point out the presence of Glc 1 Man 5 GlcNAc 1 in the cytosol of MI8 -5 cells. This indicates that part of the monoglucosylated glycoproteins can be directed toward a deglycosylation process that occurs in the cytosol. Besides studies on glycoprotein degradation based on the disappearance of protein moieties, MI8 -5 cells can be used as a tool to elucidate the various step leading to glycoprotein degradation by studying the fate of the glycan moieties.A key reaction of N-glycosylation is the transfer en bloc of a Glc 3 Man 9 GlcNAc 2 oligosaccharide from a lipid intermediate to an Asn residue in the Asn-Xaa-Ser(Thr) consensus sequence of a nascent protein. This N-glycosylation process is immediately followed by sequential deglucosylation leading to Man 9 GlcNAc 2 protein. It is now clearly established that oligosaccharide trimming in the ER 1 is intimately linked to the quality control process, leading to the degradation of misfolded glycoproteins or unassembled multimeric proteins. Indeed the presence of monoglucosylated oligomannosides on glycoproteins retained in the ER is essential for their binding to the molecular chaperones calnexin and calreticulin (1, 2). The presence of a glucosyl residue is controlled by reglucosylation-deglucosylation cycles involving UDP-glucose : glycoprotein glucosyltransferase acting as a folding sensor (3) and glucosidase II, respectively. It has been demonstrated that glucose trimming and reglucosylation cycles determine the association of a glycoprotein with calnexin, as well as its folding (4,5).During this quality control, part of newly synthesized glycoproteins are degraded after translocation of the glycosylated polypeptide chains from the ER to the cytosol. As first demonstrated by Wiertz et al. (6), using labeled protein with an amino acid precursor, this retrotranslocation is accompanied by the removal of the glycans by a cytosolic peptide N-glycanase (PNGase), such as the one isolated by Suzuki et al. (7). The relationship between the location of the enzyme and the degradation process has been discussed by Karaivanova and Spiro (8). However, it is worth mentioning that few labs follow the fate of newly synthesized proteins by labeling with [2-3 H]mannose. In this case, the N-glycosylation process is accompanied by the release of glycans from either lipid intermediates or from newly synthesized glycoproteins (9 -11). The trafficking of the released oligomannosides from both origins leads to the formatio...

Brefeldin A promotes the appearance of oligosaccharyl phosphates derived from Glc3Man9GlcNAc2-PP-dolichol within the endomembrane system of HepG2 cells

“…Although BFA-provoked redistribution of the GA to the ER requires intact OSP (0.2-0.4% of all radioactive components recovered from the cells and medium). Misfolded glycoproteins also give rise to nfOS through ER-associated degradation processes (50). The changes in nfOS levels seen with the different glycosidase inhibitors are expected because, as previously reported (21), inhibition of the cytoplasmic and lysosomal mannosidases by SW leads to the accumulation of nfOSs that are produced during glycoprotein biosynthesis.…”

Getting In and Out from Calnexin/Calreticulin Cycles

“…2, residues b-g and l) was found to be a cytosolic by-product of the degradation of misfolded glycoproteins synthesized by mutant Chinese hamster ovary cells known to transfer Man 9 GlcNAc 2 in protein N-glycosylation (52). The glycan came from cytosolic degradation of Glc 1 Man 9 GlcNAc 2 and Glc 1 Man 8 GlcNAc 2 , the species that determined CNX/CRT recognition of folding intermediates in those cells.…”

Endoplasmic reticulum‐associated degradation of glycoproteins bearing Man5GlcNAc2 and Man9GlcNAc2 species in the MI8‐5 CHO cell line

“…The N‐glycosylation process is accompanied by the release of soluble oligomannosides. At least some of this soluble material has been shown to originate from glycoprotein degradation [16,17] after the quality control mechanism. Figure 4A shows the pattern of oligomannosides bound to glycoproteins after 20 min of preincubation at 0.175 m m Glc followed by 1 h of labeling and a 1 h chase in culture medium containing 5 m m Glc and 5 m m Man.…”