REDUCED UTILIZATION OF MAN(5)GLCNAC(2)-P-P-LIPID IN A LEC9 MUTANT OF CHINESE-HAMSTER OVARY CELLS - ANALYSIS OF THE STEPS IN OLIGOSACCHARIDE-LIPID ASSEMBLY

Recently we reported that CHB11-1-3, a Chinese hamster ovary cell muta nt defective in glycosylation of asparagine-linked proteins, is defect ive in the synthesis of dolichol [Quellhorst et al., 343:19-26, 1997: Arch Biochem Biophys]. CHB11-1-3 was found to be in the Lec9 complemen tation group, which synthesizes polyprenol rather than dolichol. In th is paper, levels of various polyprenyl derivatives in CHB11-1-3 are co mpared to levels of the corresponding dolichyl derivatives in parental cells. CHB11-1-3 was found to maintain near normal levels of Man(5)Gl cNAc(2)-P-P-polyprenol and mannosylphosphorylpolyprenol, despite reduc ed rates of synthesis, by utilizing those intermediates at a reduced r ate. The Man(5)GlcNAc(2) oligosaccharide attached to prenol in CHB11-1 -3 cells and to dolichol in parental cells is the same structure, as d etermined by acetolysis. Man(5)GlcNAc(2)-P-P-polyprenol and Man(5)GlcN Ac(5)-P-P-dolichol both appeared to be translocated efficiently in an in vitro reaction. Glycosylation of G protein was compared in vesicula r stomatitus virus (VSV)-infected parent and mutant; although a portio n of G protein was normally glycosylated in CHB11-1-3 cells, a large p ortion of G was underglycosylated, resulting in the addition of either one or no oligosaccharide to C. Addition of a single oligosaccharide occurred randomly rather than preferentially at one of the two sites. (C) 1997 Wiley-Liss, Inc.