Effects of glucose starvation and puromycin treatment on lipid-linked oligosaccharide precursors and biosynthetic enzymes in Chinese hamster ovary cells in vivo and in vitro

Abstract

Previous studies from several laboratories have demonstrated that glucose-starved Chinese hamster ovary (CHO) cells and other cells in culture switch from synthesis of the normal Glc3Man9GlcNAc2-P-P-Dol to Man5-GlcNAc2-P-P-Dol. In this study we have investigated this phenomenon in CHO cells in vitro and in vivo in order to determine the possible site of this block. Our results demonstrate that enzymatic activities responsible for Man9GlcNA2 synthesis in vitro are normal in glucose-starved cells. In vivo, however, the pool of GDP-[3H]Man is severely depleted, while [3H]mannose incorporation into lipid-linked and protein-bound Man5GlcNAc2 is increased. This result suggests that the available GDP-Man in starved cells is utilized to synthesize Man5GlcNAc2 preferentially, resulting in a reduction of Dol-P-Man and Man6-Man9 GlcNAc2 synthesis in vivo in glucose-starved cells. Conditions which prevent the depletion of GDP-[3H]Man in glucose-starved cells, such as puromycin or cycloheximide treatment, result in normal synthesis of Man9GlcNAc2 by glucose-starved cells. An unexpected finding in the course of this study is that puromycin or cycloheximide treatment of cells, which is known to inhibit lipid-linked oligosaccharide synthesis in glucose-fed cells, has no such inhibitory effect on glucose-starved cells.