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J. Biol. Chem., Vol. 280, Issue 18, 17901-17909, May 6, 2005

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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, and Mark A. Lehrman

From the Department of Pharmacology, University of Texas-Southwestern Medical Center, Dallas, Texas 75390

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 Glc₃Man₉GlcNAc₂-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 Glc₃Man₉GlcNAc₂-P-P-dolichol, without hypoglycosylation, 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, M6P was a specific activator for cleavage of Glc₃Man₉GlcNAc₂-P-P-dolichol. This led to futile cycling 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.

Received for publication, January 14, 2005

^* This work was funded by National Institutes of Health Grant GM38545 and Welch Foundation Grant I-1168 (to M. A. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains two additional figures.

To whom correspondence should be addressed: Dept. of Pharmacology, UT-Southwestern Medical Center, 6001 Forest Park Blvd., Dallas, TX 75390-9041. Tel.: 214-645-6172; Fax: 214-645-6131; E-mail: mark.lehrman{at}utsouthwestern.edu.

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