Unexpected basis for impaired Glc₃Man₉ GlcNAc₂-P-P-dolichol biosynthesis by elevated expression of GlcNAc-1-P transferase

GlcNAc-1-P transferase (GPT) transfers GlcNAc-1-P from UDP-GlcNAc to dolichol-P (Dol-P), forming GlcNAc-P-P-Dol to initiate synthesis of the lipid-linked oligosaccharide Glc₃Man₉ GlcNAc₂-P-P-dolichol (G₃M₉Gn₂ -P-P-Dol). Elevated expression of GPT in CHO-K1 cells is known to cause accumulation of the intermediate M₅Gn₂-P-P-Dol, presumably by excessively consuming Dol-P and thereby hindering Dol-P-dependent synthesis of Man-P-Dol (MPD) and Glc-P-Dol (GPD), which provide the residues for extending M₅Gn₂-P-P-Dol to G₃M₉Gn₂-P-P-Dol. If so, elevated GPT expression should increase oligosaccharide-P-P-Dol quantities and reduce monosaccharide-P-Dol quantities, while requiring GPT enzymatic activity. Here we report that elevated GPT expression failed to appreciably alter the quantities of the two classes of dolichol-linked saccharide, and that neither a GPT inhibitor nor introduction of an inactivating mutation into GPT prevented M₅Gn₂-P-P-Dol accumulation, arguing against excessive Dol-P consumption. Unexpectedly, we noticed similarities between the phenotypes of GPT overexpressers and of CHO-K1 cells lacking Lec35p (encoded by MPDU1, the congenital disorder of glycosylation (CDG)-If locus), which is required for utilization of MPD and GPD. By compensatory overexpression of Lec35p, G₃M₉Gn₂-P-P-Dol synthesis in GPT overexpressers could be restored. However, GPT overexpression did not affect the levels of Lec35 mRNA or protein. These results suggest that GPT may impair Lec35p function, and imply that upper as well as lower limits on GPT expression exist in normal cells. Since the mammalian GPT gene can undergo spontaneous amplification, the data also indicate a potential basis for forms of pseudo-CDG-If.