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

Ningguo Gao, Jie Shang, Mark A. Lehrman

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)125-134
Number of pages10
JournalGlycobiology
Volume18
Issue number1
DOIs
StatePublished - Jan 1 2008

Keywords

  • Congenital disorder of glycosylation
  • Dolichol
  • GPT
  • Lec35
  • Lipid-linked oligosaccharide

ASJC Scopus subject areas

  • Biochemistry

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