Catabolism of glycan moieties of lipid intermediates leads to a single Man₅GlcNAc oligosaccharide isomer: a study with permeabilized CHO cells

Abstract

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.

Mannose corrects altered N-glycosylation in carbohydrate-deficient glycoprotein syndrome fibroblasts.

“…The size of these oligosaccharides also closely resembles that of those derived from protein-bound oligosaccharides (shown in Fig. 1), and they are most likely cleaved from protein or LLO as reported by others (34)(35)(36)(37)(38)(39)(40).…”

Endo-β- N -acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol

“…Although the sequential processing events of free OSs have been well characterized (15)(16)(17)(18)(19), the molecular nature of the enzyme͞transporters involved in this process has not been identified, with the exception of cytosolic PNGase (35). Because free OSs in the lumen of the ER can potentially interfere with the glycan-dependent protein quality control system in the ER (2-4), the rapid clearance of them from the lumen of the ER may be of great importance.…”

“…In recent years, it has been found that, during the N-glycosylation of proteins in the ER, a significant number of unconjugated, free oligosaccharides (free OSs) are formed (7)(8)(9). The occurrence of free OSs has been reported in a wide variety of cells (10)(11)(12)(13)(14), and their sequential processing event of free OSs in mammalian cells has also been well characterized (15)(16)(17)(18)(19). When the free OSs are formed in the lumen of the ER, they are transported out of the ER to the cytosol (15).…”

Study of free oligosaccharides derived from the bacterial N -glycosylation pathway

“…Free oligosaccharides (fOS) have also been described in mammalian cells, yeast, plants, and fish (11). In these cases, fOS are released from N-linked glycoproteins by peptide-N-glycanase (PNGase) within the endoplasmic reticulum associated protein degradation pathway (12) or from dolichol-pyrophosphate-linked oligosaccharides (Dol-PP-OS) by a pyrophosphatase that generates phosphorylated free oligosaccharides (fOS-P) (13,14). Other studies have suggested that in the absence of a sufficient acceptor, OS are transferred from their Dol-PP carrier to water within the ER (15,16).…”