This paper presents kinetic and structural analyses of oligosaccharide material released during glycosylation in permeabilized Chinese hamster ovary cells incubated with sugar nucleotides. Permeabilized cells released 30 times more oligosaccharide material than metabolically labelled cells, normalized to the amount of labelled glycoprotein acceptor, making this an amenable system for study. Fifteen to forty per cent of the oligosaccharide material released by permeabilized cells was oligosaccharide-phosphate, depending on the nature and amount of the oligosaccharide-lipids synthesized. The oligosaccharide-phosphates released were recovered in the cytosol, and were exclusively Man2Glc-NAc2P and Man5GlcNAc2P, released from oligosaccharide-lipids thought to be facing the cytosol. In contrast, the structures found as neutral oligosaccharide material were similar to those attached to newly synthesized glycoproteins, indicating that the oligosaccharides were subjected to the same processing enzymes whether or not they were protein bound. Importantly, the kinetics of the transfer to protein and the release of free neutral oligosaccharide were parallel, suggesting that the same enzyme was responsible for both processes. Structural analyses demonstrated that the same Man5GlcNAc2 structure was transferred to protein and released as free oligosaccharide. Neutral oligosaccharides were found in both the cytosol and the pellet; however, oligosaccharides with one GlcNAc residue at the reducing end (OS-Gn1) were found exclusively in the supernate. The major neutral oligosaccharide produced after 2 h of metabolic labelling was Man5GlcNAc and it was found in the cytosol.
The N-glycosylation of proteins is accompanied by the release of soluble oligosaccharide material. Besides oligosaccharide phosphates originating from the cleavage of lipid intermediates, neutral free oligosaccharides represent the major part of this material and are heterogeneous depending on whether the reducing end has one or two N-acetylglucosamine residues. The present study focuses on the intracellular origin of neutral free oligosaccharides in a CHO cell line. Kinetic and pulse-chase experiments clearly indicate that oligosaccharides possessing a chitobiosyl unit are derived from oligosaccharide pyrophosphodolichol, whereas oligosaccharides possessing one N-acetyl-glucosamine residue are derived from newly synthesized glycoprotein. This relationship is confirmed by comparing the glycosylation pattern of lipid donors and glycoproteins with those of neutral free oligosaccharides under various incubation conditions (inhibition of protein synthesis, presence of processing inhibitors, presence or absence of glucose). Degradation of newly synthesized glycoprotein and formation of neutral oligosaccharides with one N-acetylglucosamine residue are inhibited at 16 degrees C but not affected by lysosomotropic agents such as leupeptin or NH4Cl. Together with the fact that the degradation of newly synthesized glycoproteins and the subsequent release of the glycan are recovered in permeabilized cells, these results suggest that this phenomenon occurs in the rough endoplasmic reticulum or in a closely related compartment.
Recent studies on the mechanism of degradation of newly synthesized glycoproteins suggest the involvement of a retrotranslocation of the glycoprotein from the lumen of the rough endoplasmic reticulum into the cytosol, where a deglycosylation process takes place. In the studies reported here, we used a glycosylation mutant of Chinese hamster ovary cells that does not synthesize mannosylphosphoryldolichol and has an increased level of soluble oligomannosides originating from glycoprotein degradation. In the presence of anisomycin, an inhibitor of protein synthesis, we observed an accumulation of glucosylated oligosaccharide-lipid donors (Glc3Man5GlcNAc2-PP-Dol), which are the precursors of the soluble neutral oligosaccharide material. Inhibition of rough endoplasmic reticulum glucosidase(s) by castanospermine led to the formation of Glc3Man5GlcNAc2(OSGn2) (in which OSGn2 is an oligomannoside possessing two GlcNAc residues at its reducing end), which was then retained in the lumen of intracellular vesicles. Thus they were protected during an 8 h chase period from the action of cytosolic chitobiase, which is responsible for the conversion of OSGn2 to oligomannosides possessing one GlcNAc residue at the reducing end (OSGn1). In contrast, when protein synthesis was maintained in the presence of castanospermine, glucosylated oligomannosides (Glc1-3Man5GlcNAc1) were recovered in cytosol. Except for monoglucosylated Man5 species, which are potential substrates for luminal calnexin and calreticulin, the pattern of oligomannosides was similar to that observed on glycoproteins. The occurrence in the cytosol of glucosylated species with one GlcNAc residue at the reducing end implies that the deglycosylation process that generates glucosylated OSGn1 from glycoproteins occurs in the cytosol.
Neutral oligomannosides possessing one GlcNAc (OS-Gn1) and two GlcNAc (Os-Gn2) at the reducing end have been reported to be released during the N-glycosylation process in various biological models. To investigate which enzyme is responsible for OS-Gn1 formation, we used the Madin-Darby bovine kidney (MDBK) cell line which exhibits neither lysosomal chitobiase nor endoglucosaminidase activities. However, these cells produced OS-Gn1 and we showed that a neutral chitobiase is responsible for the transformation of OS-Gn2 into OS-Gn1. Using streptolysin O-permeabilized MDBK cells, we demonstrated that this neutral chitobiase activity is located in the cytosolic compartment and is active on oligomannoside species released during the N-glycosylation process.
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