Mannose corrects altered N-glycosylation in carbohydrate-deficient glycoprotein syndrome fibroblasts.

Type I carbohydrate-deficient glycoprotein syndrome (CDGS) patients fail to add entire N-linked oligosaccharide chains to some serum glycoproteins.Here we show that four CDGS fibroblast cell lines have two related glycosylation abnormalities.First, they incorporate 3-10-fold less [ 3 H] mannose into proteins, and, second, the size of the lipid-linked oligosaccharide precursor (LLO) is much smaller than in controls.Addition of exogenous mannose, but not glucose, to these CDGS cells corrects both the lowered [ 3 H]mannose incorporation and the size of LLO.These corrections are not permanent, and the defects immediately reappear when mannose is removed.To explore further the basis of mannose correction, we analyzed the amount of 3 H-labeled LLO intermediates.Except for dolichol-P-mannose, other precursors, including mannose, mannose-6-phosphate, mannose-1-phosphate, and GDP-mannose, all showed a 3-10fold decrease in CDGS cells.Thus, there are no obvious lesions in the intracellular conversion of mannose into LLO, and, once inside the cell, [ 3 H]mannose appeared to be metabolized normally.Initial velocities of [ 3 H] mannose uptake were two-to threefold less in CDGS cells compared with controls, and this slower transport may partially explain the reduced [ 3 H]mannose incorporation in CDGS cells.Since we previously showed that the enzymes converting glucose to mannose-6-phosphate appear to be normal, our results suggest that cells may acquire or generate mannose in other ways.Although we have not identified the primary defect in CDGS, these studies show that intracellular mannose is limited and that some patients might benefit from including mannose in their regular diets.( J. Clin.Invest.1996.97:1478-1487.)Key words: glycosylation • lipidlinked oligosaccharide • glycoprotein • hexose transporter • CDG syndrome Methods Materials.Most of the materials were obtained from Sigma Chemical Co.(St.Louis, MO), except for the following: concanavalin A (Con A)-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ), ␣ -MEM (GIBCO BRL, Baltimore, MD), FBS (Hyclone Laboratories, Logan, UT), Micropak AX-5 HPLC column (Varian Instruments, Walnut Creek, CA), and microspin filters (Lida Manufacturing Corp., Kenosha, WI).Radiolabels.2-[ 3 H]Mannose (15 Ci/mmol), 2-deoxy[1,2-3 H(N)]glucose (40 Ci/mmol), and [ 35 S]methionine (1,217 Ci/mmol) were obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO).Cell lines.The CDGS and control cell lines were all obtained from Dr. Neil Buist (Department of Pediatrics, Oregon Health Sciences University, Portland, OR).CDGS 1 and 2, our designation, are patients from the same family (samples F27467 and F27468) described in our previous study (13).CDGS cell lines 3 and 4 are patients from another family (samples F20660 and F02661, coded by Dr. N. Buist).All these patients are diagnosed as type I CDGS based on the clinical and biochemical (transferrin pattern) analyses.