Studies on the regulation of the lipid-linked oligosaccharide pathway in cultured cells

Although the reactions involved in the formation of Glc3Man9(GlcNAc)2-PP-dolichol are reasonably well understood, little is known about the regulation of these reactions. Previous studies in the authors lab and the labs of Lennarz and Robbins showed that when protein synthesis was inhibited with puromycin (P) or cycloheximide (C), mannose (man) incorporation into lipid-linked oligosaccharides (LLO) was also inhibited. This inhibition of LLO could be due to limitations in the amount of dol-P as a result of absence of oligosaccharide acceptor, or to accumulation of a metabolic inhibitor. They have examined the effect of P and C on incorporation of 3H-man and 3H-GlcN into LLO and protein in MDCK cells. Both P and C inhibited man incorporation into LLO and protein, but formation of dol-P-man was not affected. On the other hand, GlcN incorporation into dol-PP-GlcNAc and dol-PP-(GlcNAc)2 was inhibited by P and C, as were incorporation into LLO and protein. Man incorporation into LLO was also inhibited by hydroxynorvaline, a thr analog that inhibits glycosylation. Inhibition of man incorporation into LLO and protein by P and C could not be overcome by adding dol-P or dol to the cells, although both lipids greatly stimulated man incorporation into lipids and protein in normal cells. Enzyme measurements of control or inhibited cells did not show any difference. These results suggest that inhibition of LLO is due to inhibition of dol-PP-GlcNAc formation, perhaps by accumulation of a metabolic inhibitor