Analysis of glycosylation in CDG-Ia fibroblasts by fluorophore-assisted carbohydrate electrophoresis: Implications for extracellular glucose and intracellular mannose 6-phosphate

Ningguo Gao, Jie Shang, Mark A. Lehrman

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Abstract

Phosphomannomutase (PMM) deficiency causes congenital disorder of glycosylation (CDG)-Ia, a broad spectrum disorder with developmental and neurological abnormalities. PMM converts mannose 6-phosphate (M6P) to mannose-1-phosphate, a precursor of GDP-mannose used to make Glc 3Man9GlcNAc2-P-P-dolichol (lipid-linked oligosaccharide; LLO). LLO, in turn, is the donor substrate of oligosaccharyltransferase for protein N-linked glycosylation. Hepatically produced N-linked glycoproteins in CDG-Ia blood are hypoglycosylated. Upon labeling with [3H]mannose, CDG-Ia fibroblasts have been widely reported to accumulate [3H]LLO intermediates. Since these are thought to be poor oligosaccharyltransferase substrates, LLO intermediate accumulation has been the prevailing explanation for hypoglycosylation in patients. However, this is discordant with sporadic reports of specific glycoproteins (detected with antibodies) from CDG-Ia fibroblasts being fully glycosylated. Here, fluorophore-assisted carbohydrate electrophoresis (FACE, a nonradioactive technique) was used to analyze steady-state LLO compositions in CDG-Ia fibroblasts. FACE revealed that low glucose conditions accounted for previous observations of accumulated [3H]LLO intermediates. Additional FACE experiments demonstrated abundant Glc3Man9GlcNAc 2-P-P-dolichol, without hypoglycosylation, in CDG-Ia fibroblasts grown with physiological glucose. This suggested a "missing link" to explain hypoglycosylation in CDG-Ia patients. Because of the possibility of its accumulation, the effects of M6P on glycosylation were explored in vitro. Surprisingly, B16P was a specific activator for cleavage of Glc 3Man9GlcNAc2-P-P-dolichol. This led to futile cycling of the LLO pathway, exacerbated by GDP-mannose/ PMM deficiency. The possibilities that M6P may accumulate in hepatocytes and that M6P-stimulated LLO cleavage may account for both hypoglycosylation and the clinical failure of dietary mannose therapy with CDG-Ia patients are discussed.

Original languageEnglish (US)
Pages (from-to)17901-17909
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number18
DOIs
StatePublished - May 6 2005

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Congenital Disorders of Glycosylation
Glycosylation
Fluorophores
Fibroblasts
Electrophoresis
Carbohydrates
Glucose
Dolichol
Guanosine Diphosphate Mannose
Mannose
Glycoproteins
Substrate Cycling
mannose-6-phosphate
Hepatocytes
Substrates
Labeling
Tissue Donors
Blood

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Analysis of glycosylation in CDG-Ia fibroblasts by fluorophore-assisted carbohydrate electrophoresis: Implications for extracellular glucose and intracellular mannose 6-phosphate",
abstract = "Phosphomannomutase (PMM) deficiency causes congenital disorder of glycosylation (CDG)-Ia, a broad spectrum disorder with developmental and neurological abnormalities. PMM converts mannose 6-phosphate (M6P) to mannose-1-phosphate, a precursor of GDP-mannose used to make Glc 3Man9GlcNAc2-P-P-dolichol (lipid-linked oligosaccharide; LLO). LLO, in turn, is the donor substrate of oligosaccharyltransferase for protein N-linked glycosylation. Hepatically produced N-linked glycoproteins in CDG-Ia blood are hypoglycosylated. Upon labeling with [3H]mannose, CDG-Ia fibroblasts have been widely reported to accumulate [3H]LLO intermediates. Since these are thought to be poor oligosaccharyltransferase substrates, LLO intermediate accumulation has been the prevailing explanation for hypoglycosylation in patients. However, this is discordant with sporadic reports of specific glycoproteins (detected with antibodies) from CDG-Ia fibroblasts being fully glycosylated. Here, fluorophore-assisted carbohydrate electrophoresis (FACE, a nonradioactive technique) was used to analyze steady-state LLO compositions in CDG-Ia fibroblasts. FACE revealed that low glucose conditions accounted for previous observations of accumulated [3H]LLO intermediates. Additional FACE experiments demonstrated abundant Glc3Man9GlcNAc 2-P-P-dolichol, without hypoglycosylation, in CDG-Ia fibroblasts grown with physiological glucose. This suggested a {"}missing link{"} to explain hypoglycosylation in CDG-Ia patients. Because of the possibility of its accumulation, the effects of M6P on glycosylation were explored in vitro. Surprisingly, B16P was a specific activator for cleavage of Glc 3Man9GlcNAc2-P-P-dolichol. This led to futile cycling of the LLO pathway, exacerbated by GDP-mannose/ PMM deficiency. The possibilities that M6P may accumulate in hepatocytes and that M6P-stimulated LLO cleavage may account for both hypoglycosylation and the clinical failure of dietary mannose therapy with CDG-Ia patients are discussed.",
author = "Ningguo Gao and Jie Shang and Lehrman, {Mark A.}",
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T1 - Analysis of glycosylation in CDG-Ia fibroblasts by fluorophore-assisted carbohydrate electrophoresis

T2 - Implications for extracellular glucose and intracellular mannose 6-phosphate

AU - Gao, Ningguo

AU - Shang, Jie

AU - Lehrman, Mark A.

PY - 2005/5/6

Y1 - 2005/5/6

N2 - Phosphomannomutase (PMM) deficiency causes congenital disorder of glycosylation (CDG)-Ia, a broad spectrum disorder with developmental and neurological abnormalities. PMM converts mannose 6-phosphate (M6P) to mannose-1-phosphate, a precursor of GDP-mannose used to make Glc 3Man9GlcNAc2-P-P-dolichol (lipid-linked oligosaccharide; LLO). LLO, in turn, is the donor substrate of oligosaccharyltransferase for protein N-linked glycosylation. Hepatically produced N-linked glycoproteins in CDG-Ia blood are hypoglycosylated. Upon labeling with [3H]mannose, CDG-Ia fibroblasts have been widely reported to accumulate [3H]LLO intermediates. Since these are thought to be poor oligosaccharyltransferase substrates, LLO intermediate accumulation has been the prevailing explanation for hypoglycosylation in patients. However, this is discordant with sporadic reports of specific glycoproteins (detected with antibodies) from CDG-Ia fibroblasts being fully glycosylated. Here, fluorophore-assisted carbohydrate electrophoresis (FACE, a nonradioactive technique) was used to analyze steady-state LLO compositions in CDG-Ia fibroblasts. FACE revealed that low glucose conditions accounted for previous observations of accumulated [3H]LLO intermediates. Additional FACE experiments demonstrated abundant Glc3Man9GlcNAc 2-P-P-dolichol, without hypoglycosylation, in CDG-Ia fibroblasts grown with physiological glucose. This suggested a "missing link" to explain hypoglycosylation in CDG-Ia patients. Because of the possibility of its accumulation, the effects of M6P on glycosylation were explored in vitro. Surprisingly, B16P was a specific activator for cleavage of Glc 3Man9GlcNAc2-P-P-dolichol. This led to futile cycling of the LLO pathway, exacerbated by GDP-mannose/ PMM deficiency. The possibilities that M6P may accumulate in hepatocytes and that M6P-stimulated LLO cleavage may account for both hypoglycosylation and the clinical failure of dietary mannose therapy with CDG-Ia patients are discussed.

AB - Phosphomannomutase (PMM) deficiency causes congenital disorder of glycosylation (CDG)-Ia, a broad spectrum disorder with developmental and neurological abnormalities. PMM converts mannose 6-phosphate (M6P) to mannose-1-phosphate, a precursor of GDP-mannose used to make Glc 3Man9GlcNAc2-P-P-dolichol (lipid-linked oligosaccharide; LLO). LLO, in turn, is the donor substrate of oligosaccharyltransferase for protein N-linked glycosylation. Hepatically produced N-linked glycoproteins in CDG-Ia blood are hypoglycosylated. Upon labeling with [3H]mannose, CDG-Ia fibroblasts have been widely reported to accumulate [3H]LLO intermediates. Since these are thought to be poor oligosaccharyltransferase substrates, LLO intermediate accumulation has been the prevailing explanation for hypoglycosylation in patients. However, this is discordant with sporadic reports of specific glycoproteins (detected with antibodies) from CDG-Ia fibroblasts being fully glycosylated. Here, fluorophore-assisted carbohydrate electrophoresis (FACE, a nonradioactive technique) was used to analyze steady-state LLO compositions in CDG-Ia fibroblasts. FACE revealed that low glucose conditions accounted for previous observations of accumulated [3H]LLO intermediates. Additional FACE experiments demonstrated abundant Glc3Man9GlcNAc 2-P-P-dolichol, without hypoglycosylation, in CDG-Ia fibroblasts grown with physiological glucose. This suggested a "missing link" to explain hypoglycosylation in CDG-Ia patients. Because of the possibility of its accumulation, the effects of M6P on glycosylation were explored in vitro. Surprisingly, B16P was a specific activator for cleavage of Glc 3Man9GlcNAc2-P-P-dolichol. This led to futile cycling of the LLO pathway, exacerbated by GDP-mannose/ PMM deficiency. The possibilities that M6P may accumulate in hepatocytes and that M6P-stimulated LLO cleavage may account for both hypoglycosylation and the clinical failure of dietary mannose therapy with CDG-Ia patients are discussed.

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