Abstract
BackgroundDuring mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini. After sequential trimming by cytosolic endo β-N-acetylglucosaminidase (ENGase) and Man2c1 mannosidase, cytosolic fOS are transported into lysosomes. Why mammalian cells generate such large quantities of fOS remains unexplored, but fOSGN2 could be liberated from LLO by oligosaccharyltransferase, or from glycoproteins by NGLY1-encoded Peptide-N-Glycanase (PNGase). Also, in addition to converting fOSGN2 to fOSGN, the ENGASE-encoded cytosolic ENGase of poorly defined function could potentially deglycosylate glycoproteins. Here, the roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells.Methods/Principal FindingsDuring metabolic radiolabeling and chase incubations, RNAi-mediated Engase1p down regulation delays fOSGN2-to-fOSGN conversion, and it is shown that Engase1p and Man2c1p are necessary for efficient clearance of cytosolic fOS into lysosomes. Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Δ deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation. In metabolically radiolabeled HepG2 cells evidence was obtained for a small but significant Engase1p-mediated generation of fOS in 1 h chase but not 30 min pulse incubations. Ngly1p down regulation revealed an Ngly1p-independent fOSGN2 pool comprising mainly Man8GlcNAc2, corresponding to ∼70% of total fOS, and an Ngly1p-dependent fOSGN2 pool enriched in Glc1Man9GlcNAc2 and Man9GlcNAc2 that corresponds to ∼30% of total fOS.Conclusions/SignificanceAs the generation of the bulk of fOS is unaffected by co-down regulation of Ngly1p and Engase1p, alternative quantitatively important mechanisms must underlie the liberation of these fOS from either LLO or glycoproteins during protein N-glycosylation. The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming.
Highlights
During protein N-glycosylation, oligosaccharyltransferase (OST) transfers the oligosaccharide Glc3Man9GlcNAc2 from the mature lipid linked oligosaccharide precursor (LLO, Glc3Man9GlcNAc2PP-dolichol) onto asparagine residues in the consensus sequence Asn-X-Ser/Thr of nascent proteins [1]
Experimental strategy Potentially both Ngly1p and Engase1p could give rise to free oligosaccharides (fOS) in mammalian cells and could have overlapping functions
Engase1p down regulation slows down fOSGN2-tofOSGN conversion in HepG2 cells
Summary
During protein N-glycosylation, oligosaccharyltransferase (OST) transfers the oligosaccharide Glc3Man9GlcNAc2 from the mature lipid linked oligosaccharide precursor (LLO, Glc3Man9GlcNAc2PP-dolichol) onto asparagine residues in the consensus sequence Asn-X-Ser/Thr of nascent proteins [1]. N-linked oligosaccharides play crucial roles in the quality control, folding, ER-associated degradation (ERAD) and subcellular trafficking of glycoproteins [2] During both mammalian LLO biosynthesis and ERAD, free oligosaccharides (fOS) possessing the di-N-acetylchitobiose moiety at their reducing end (fOSGN2), are generated [3,4]. Cytosolic trimming of fOSGN2 is accomplished by an endo-b-N-acetylglucosaminidase (ENGase, [13]) or chitobiase [14] to yield fOS bearing a single N-acetylglucosamine (GlcNAc) residue at their reducing termini (fOSGN) These structures are the preferred substrates for the cytosolic mannosidase [15], encoded by the MAN2C1 gene [16,17], that trims Man9GlcNAc to generate (Mana1-2Mana1-2Mana1-3(Mana1-6)Manb1-4GlcNAc: linear isomer of Man5GlcNAc) that is transported into lysosomes [12]. The roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells
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