[Show abstract][Hide abstract] ABSTRACT: Lipid-linked oligosaccharides (LLOs) are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I congenital disorders of glycosylation. Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, simple and sensitive nonradioactive methods for LLO analysis are lacking. Thus, almost all studies of LLO synthesis have relied on metabolic labeling of the oligosaccharides with radioactive sugar precursors. We report that LLOs in cell cultures and tissues can be easily detected and quantified with a sensitivity of 1-2 pmol by fluorophore-assisted carbohydrate electrophoresis (FACE). These analyses required efficient removal of contaminants, most likely trace quantities of glycogen breakdown products, that interfered with FACE. Studies with CHO-K1 cells showed that LLOs detected by FACE and by metabolic labeling had similar turnover rates. Glc(3)Man(9)GlcNAc(2)-P-P-dolichol was the most prominent LLO detected by FACE in normal cultured cells and mouse tissues. However, the relative amounts of Glc(0-2)Man(5-9)GlcNAc(2)-P-P-dolichol intermediates in tissues, such as liver and kidney, were unexpectedly greater than for cultured cells. IV injection of D-mannose, raising the circulatory concentration by three- to fourfold, did not affect LLO composition. Thus, the relative accumulation of LLO intermediates in mouse liver and kidney is not likely due to inadequate D-mannose in the circulation. In summary, FACE is a facile, accurate, and sensitive method for LLO analysis, permitting investigations not feasible by metabolic labeling.
[Show abstract][Hide abstract] ABSTRACT: Lipid-linked oligosaccharides (LLOs) are the donors of glycans that modify newly synthesized proteins in the endoplasmic reticulum (ER) of eukaryotes, resulting in formation of N-linked glycoproteins. The vast majority of LLO analyses have relied on metabolic labeling with radioactive sugar precursors, but these approaches have technical limitations resulting in many important questions about LLO synthesis being left unanswered. Here we describe the application of a facile non-radioactive technique, fluorophore-assisted carbohydrate electrophoresis (FACE), which circumvents these limitations. With FACE, steady-state LLO compositions can be determined quantitatively from cell cultures and animal tissues. We also present FACE methods for analysis of phosphosugars and nucleotide sugars, which are metabolic precursors of LLOs.
Methods in Enzymology 02/2006; 415:3-20. · 2.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: GlcNAc-1-P transferase (GPT) transfers GlcNAc-1-P from UDP-GlcNAc to dolichol-P (Dol-P), forming GlcNAc-P-PDol to initiate synthesis of the lipid-linked oligosaccharide Glc3Man9GlcNAc2-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.
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