CATABOLISM OF GLYCAN MOIETIES OF LIPID INTERMEDIATES LEADS TO A SINGLE MAN(5)GLCNAC OLIGOSACCHARIDE ISOMER - A STUDY WITH PERMEABILIZED CHOCELLS

This paper presents kinetic and structural analyses of oligosaccharide material released during glycosylation in permeabilized Chinese hamst er ovary cells incubated with sugar nucleotides. Permeabilized cells r eleased 30 times more oligosaccharide material than metabolically labe lled 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 ol igosaccharide-phosphate, depending on the nature and amount of the oli gosaccharide-lipids synthesized. The oligosaccharide-phosphates releas ed were recovered in the cytosol, and were exclusively Man(2)Glc-NAc2P and Man(5)GlcNAc(2)P, released from oligosaccharide-lipids thought to be facing the cytosol. In contrast, the structures found as neutral o ligosaccharide material were similar to those attached to newly synthe sized glycoproteins, indicating that the oligosaccharides were subject ed to the same processing enzymes whether or not they were protein bou nd. Importantly, the kinetics of the transfer to protein and the relea se of free neutral oligosaccharide were parallel, suggesting that the same enzyme was responsible for both processes. Structural analyses de monstrated that the same Man(5)GlcNAc(2) structure was transferred to protein and released as free oligosaccharide. Neutral oligosaccharides were found in both the cytosol and the pellet; however, oligosacchari des with one GlcNAc residue at the reducing end (OS-Gn(1)) were found exclusively in the supernate. The major neutral oligosaccharide produc ed after 2 h of metabolic labelling was Man(5)GlcNAc and it was found in the cytosol.