Analyses of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues by fluorophore-assisted carbohydrate electrophoresis.
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.
Gao
Ningguo
N
Department of Pharmacology, University of Texas-Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas 75390-9041, USA.
Lehrman
Mark A
MA
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004288:Dolichol Phosphates / Q000737:chemistry*; D004586:Electrophoresis / Q000379:methods*; D005456:Fluorescent Dyes; D006031:Glycosylation; D008099:Liver / Q000737:chemistry; D009844:Oligosaccharides / Q000737:chemistry*
D004288:Dolichol Phosphates; D005456:Fluorescent Dyes; D009844:Oligosaccharides; C026406:dolichol pyrophosphate