Analyses of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues by fluorophore-assisted carbohydrate electrophoresis

Ningguo Gao, Mark A. Lehrman

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37 Citations (Scopus)

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. Glc3Man9GlcNAc2-P-P-dolichol was the most prominent LLO detected by FACE in normal cultured cells and mouse tissues. However, the relative amounts of Glc0-2Man5-9GlcNAc2-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.

Original languageEnglish (US)
Pages (from-to)353-360
Number of pages8
JournalGlycobiology
Volume12
Issue number5
StatePublished - 2002

Fingerprint

Fluorophores
Electrophoresis
Oligosaccharides
Cell culture
Cell Culture Techniques
Carbohydrates
Tissue
Dolichol
Labeling
Mannose
Liver
Cultured Cells
dolichol pyrophosphate
lipid-linked oligosaccharides
Congenital Disorders of Glycosylation
Cells
Glycosylation
Kidney
CHO Cells
Asparagine

Keywords

  • Dolichol
  • Fluorophore-assisted carbohydrate electrophoresis
  • Glycosylation
  • Lipid-linked oligosaccharide

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Analyses of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues by fluorophore-assisted carbohydrate electrophoresis",
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. Glc3Man9GlcNAc2-P-P-dolichol was the most prominent LLO detected by FACE in normal cultured cells and mouse tissues. However, the relative amounts of Glc0-2Man5-9GlcNAc2-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.",
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T1 - Analyses of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues by fluorophore-assisted carbohydrate electrophoresis

AU - Gao, Ningguo

AU - Lehrman, Mark A.

PY - 2002

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N2 - 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. Glc3Man9GlcNAc2-P-P-dolichol was the most prominent LLO detected by FACE in normal cultured cells and mouse tissues. However, the relative amounts of Glc0-2Man5-9GlcNAc2-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.

AB - 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. Glc3Man9GlcNAc2-P-P-dolichol was the most prominent LLO detected by FACE in normal cultured cells and mouse tissues. However, the relative amounts of Glc0-2Man5-9GlcNAc2-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.

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