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

This paper presents kinetic and structural analyses of oligosaccharidematerial released during glycosylation in permeabilized Chinesehamster ovary cells incubated with sugar nucleotides. Permeabilizedcells released 30 times more oligosaccharide material than metabolicallylabelled cells, normalized to the amount of labelled glycoproteinacceptor, making this an amenable system for study. Fifteento forty per cent of the oligosaccharide material released bypermeabilized cells was oligosaccharide-phosphate, dependingon the nature and amount of the oligosaccharide-lipids synthesized.The oligosaccharide-phosphates released were recovered in thecytosol, and were exclusively Man²GlcNAc²P and Man⁵GlcNAc²P,released from oligosaccharide-lipids thought to be facing thecytosol. In contrast, the structures found as neutral oligosaccharidematerial were similar to those attached to newly synthesizedglycoproteins, indicating that the oligosaccharides were subjectedto the same processing enzymes whether or not they were proteinbound. Importantly, the kinetics of the transfer to proteinand 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 structurewas transferred to protein and released as free oligosaccharide.Neutral oligosaccharides were found in both the cytosol andthe pellet; however, oligosaccharides with one GlcNAc residueat the reducing end (OS-Gn¹) were found exclusively in the supemate.The major neutral oligosaccharide produced after 2 h of metaboliclabelling was Man⁵GlcNAc and it was found in the cytosol. lipid intermediates oligomannoside-phosphates permeabilized cells subcellular distribution of oligomannosides