EFFECT OF CELL ATTACHMENT AND GROWTH ON THE SYNTHESIS AND FATE OF DOLICHOL-LINKED OLIGOSACCHARIDES IN CHINESE-HAMSTER OVARY CELLS

The inhibition of cellular processes in suspended anchorage-dependent Chinese hamster ovary (CHO) cell lines and their restoration upon atta chment to a solid substrate has been used as a model to study the rela tionship between oligosaccharide-diphospho-dolichols and their metabol ic products (glycoprotein and soluble oligosaccharide material, i.e. o ligosaccharide phosphates and neutral oligosaccharides). Using metabol ic labelling we demonstrated that suspended cells have a low incorpora tion rate into lipid intermediates and into glycoproteins. The oligosa ccharide-lipid populations are mainly glucosylated and the neutral oli gosaccharides have exclusively a chitobiosyl residue at their reducing end. In contrast, monolayer cells exhibit a high incorporation rate i nto lipid intermediates with a pattern dominated by two species contai ning either two or five mannose residues, and into glycoproteins with a pattern similar to the one observed for suspended cells (i.e. glucos ylated species). In monolayer cells the neutral oligosaccharides posse ss either one or two GlcNAc residues at their reducing end. The variat ions in the nature and in the quantity of soluble oligosaccharide mate rial as a function of the cell density reflects regulatory points in t he synthesis of N-glycosyl proteins. The first regulatory point could be the control of the quantity of non-glucosylated oligosaccharide-lip ids to be channelled toward the glucosylated lipid-donor pool. The lev el of this donor pool being constant, the oligosaccharide transferase could utilize oligosaccharide-lipid donors at a constant rate by two d ifferent reactions: either transfer onto protein when acceptors are av ailable, or transfer onto water generating neutral oligosaccharides po ssessing two GlcNAc residues at the reducing end. Another regulatory p oint would be the degradation of a part of neoglycoproteins leading to the release of neutral oligosaccharides possessing one GlcNAc residue at the reducing end.