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 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.
Kmiécik
D
D
Laboratoire de Chimie Biologique, UMR N. 111 du CNRS, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Herman
V
V
Stroop
C J
CJ
Michalski
J C
JC
Mir
A M
AM
Labiau
O
O
Verbert
A
A
Cacan
R
R
D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002240:Carbohydrate Sequence; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D013237:Stereoisomerism
D009844:Oligosaccharides; D010710:Phosphates; D011134:Polysaccharides; C028569:oligomannoside