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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(3)Man(9)GlcNAc(2)-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 [(3)H]mannose, CDG-Ia fibroblasts have been widely reported to accumulate [(3)H]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 [(3)H]LLO intermediates. Additional FACE experiments demonstrated abundant Glc(3)Man(9)GlcNAc(2)-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 Glc(3)Man(9)GlcNAc(2)-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.
Highlights
The congenital disorders of glycosylation (CDG) are inherited disorders in which the synthesis of LLO (Type I) or the processing of the N-linked oligosaccharide (Type II) is defective
Mannose 6-Phosphate Stimulates Selective Cleavage of G3M9Gn2-P-P-Dol, with Futile Cycling of the LLO Pathway—In the absence of a clear explanation for hypoglycosylation of serum proteins in congenital disorder of glycosylation (CDG)-Ia patients, we considered that mannose 6-phosphate (M6P) could be a “missing link,” because it might accumulate in PMMdeficient hepatocytes
Choice of Analytical Method for LLO Analysis in Fibroblasts—In contrast to previous analyses of [3H]mannose-labeled CDG-Ia fibroblasts, which involved culture in low glucose and indicated robust LLO abnormalities and hypoglycosylation of protein, fluorophore-assisted carbohydrate electrophoresis (FACE) analyses of CDG-Ia fibroblasts showed only moderate LLO abnormalities in 5 mM glucose (ϳ50% accumulation of M5Gn2-P-P-Dol), which did not lead to obvious hypoglycosylation
Summary
The congenital disorders of glycosylation (CDG) are inherited disorders in which the synthesis of LLO (Type I) or the processing of the N-linked oligosaccharide (Type II) is defective. Depending upon the CDG-I subtype, this hypoglycosylation can be due to either inadequate production of mature LLO (limiting the amount of donor substrate for the oligosaccharyltransferase (OT), which transfers oligosaccharide to protein) or production of structurally immature LLOs that are poor donor substrates for OT (4, 5) and can be caused by defects in mannose metabolism or the LLO assembly pathway. Since 1994 (6, 8 –12), studies on [3H]mannose-labeled LLOs in CDG-Ia dermal fibroblasts have reported reduced quantities of G3M9Gn2-P-P-Dol, and all but one (12) reported accumulation of immature LLOs (typically M3–6Gn2-P-P-Dol). 7-amino-1,3-napthalenedisulfonic acid; CDG, congenital disorder(s) of glycosylation; Dol, dolichol; FACE, fluorophore-assisted carbohydrate electrophoresis; F6P, fructose 6-phosphate; GDP-M, GDP-mannose; G3M9Gn2, Glc3Man9GlcNAc2; G6P, glucose 6-phosphate; LLO, lipidlinked oligosaccharide; M6P, mannose 6-phosphate; M1P, mannose 1-phosphate; SLO, streptolysin-O; PMM, phosphomannomutase; PMI, phosphomannose isomerase; HPLC, high pressure liquid chromatography; OT, oligosaccharyltransferase; FBS, fetal bovine serum. It is a paradox that dietary mannose therapy has failed to improve protein glycosylation or symptoms in CDG-Ia patients (4), LLO defects in cultured CDG-Ia fibroblasts were corrected by supplemental mannose (10), and dietary mannose therapy is highly effective for CDG-Ib (13)
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