3Department of Biochemistry, A.B.Chandler Medical Center, University of Kentucky College of Medicine, Lexington, KY and 4The Burnham Institute, La Jolla, CA 92037 USA
Received on November 29, 1999; revised on January 28, 2000; accepted on February 3, 2000.
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Abstract |
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Key words: GDP-mannose/mannose/Congenital Disorders of Glycosylation (CDG)/fibroblast/phosphomannose isomerase
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Introduction |
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Metabolic labeling of fibroblasts with [3H]mannose previously established that CDG-I patients abnormally assemble Glc3Man9GlcNAc2-P-P-Dol, the oligosaccharide donor in the protein N-glycosylation pathway (Figure 1; reviewed by Freeze and Aebi, 1999; Powell et al., 1994
; Krasnewich et al., 1995
). Furthermore, the defects in Glc0-3Man9GlcNAc2-P-P-Dol synthesis and reduced protein N-glycosylation could be reversed by the addition of mannose to the tissue culture medium (Panneerselvam and Freeze, 1996
a). It has been assumed that insufficient synthesis of GDP-Man occurs in several types of patients, but the cellular levels of GDP-Man have not been measured directly to confirm this tentative conclusion.
To test this hypothesis, we used a sensitive and reliable method (Rush and Waechter, 1995) to measure the levels of GDP-Man in normal and CDG-Ia and -Ib fibroblasts cultured in the presence and absence of exogenously supplied mannose. Metabolic labeling with [3H]mannose allowed us to calculate the specific activities of the GDP-[3H]Man pools and determine the amounts of Man-P-Dol and Glc0-3Man9GlcNAc2-P-P-Dol synthesized by the cells. These comparisons indicate that GDP-Man levels are dramatically lower in PMM- and PMI-deficient fibroblasts, and that adding 1 mM mannose to the tissue culture medium substantially corrects the GDP-Man deficiency and restores Man-P-Dol and Glc0-3Man9GlcNAc2-P-P-Dol synthesis to virtually normal levels. This study further shows that cellular GDP-Man levels are normal and unaffected by mannose supplementation in one Man-P-Dol synthase deficient fibroblast cell line (Kim et al., 2000
).
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Results and discussion |
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Exogenous mannose, but not glucose, restores normal GDP-Man levels in PMM-deficient fibroblasts
Previous studies have shown that inclusion of 1 mM mannose in the tissue culture medium corrected both the synthesis of truncated dolichol-linked intermediates and the under-N-glycosylation of glycoproteins observed in PMM-deficient fibroblasts (Panneerselvam and Freeze, 1996a). In order to determine if mannose supplementation also restored GDP-Man levels in PMM-deficient fibroblasts, cells from normal and affected individuals were cultured for 30 min in
-MEM containing 0.5 mM glucose in the presence and absence of 1 mM mannose. When fibroblast cultures from 4 unrelated PMM-deficient patients were incubated with 1 mM mannose, the GDP-Man levels increased from an average of 2.4 pmol/106 cells (<10% of normal fibroblasts) to an average of 15.5 pmol/106 cells (~75% of normal fibroblasts) (Table I). Mannose supplementation did not significantly affect the GDP-Man concentration in normal cells.
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To determine if the effect of mannose supplementation on cellular GDP-Man levels was specific for mannose, different amounts of glucose were added to control and PMM-deficient fibroblast cultures. As shown in Table II, GDP-Man in two PMM-deficient fibroblast cell lines was virtually undetectable when cultured in the presence of 0.5 mM glucose, whereas normal fibroblasts had 30.7 pmol/106 cells under these culture conditions. After incubation for 30 min with 2.5 mM glucose, the GDP-Man concentration in the PMM-deficient cells was 911 pmol/106 cells. However, when PMM-deficient fibroblasts were cultured in 0.5 mM glucose in the presence of 1 mM mannose, GDP-Man levels were restored to the normal range (2537 pmol/106 cells). This result indicates that mannose is more effective than glucose in correcting GDP-Man deficiency in PMM-deficient fibroblasts, although glucose, at a higher concentration, partially restores GDP-Man synthesis.
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The results in Table IV show that a large amount of radioactivity was incorporated into Man-P-Dol and Glc0-3Man9GlcNAc2-P-P-Dol under both labeling conditions. In the absence of added mannose, significantly more [3H]mannose is incorporated into the Man-P-Dol and Glc0-3Man9GlcNAc2-P-P-Dol fractions in PMI-deficient fibroblasts than in the normal cells. However, using the specific radioactivity of the GDP-Man pool in PMI-deficient fibroblasts cultured in the absence of added mannose (calculated from Table III), we estimate that the cellular levels of Man-P-Dol (~1.5% of normal) and Glc0-3Man9GlcNAc2-P-P-Dol (~6% of normal) are drastically reduced relative to normal fibroblasts (Table IV). When the PMI-deficient fibroblasts were cultured in the presence of 1 mM mannose, the synthesis of both Man-P-Dol (26% of normal fibroblasts) and Glc0-3Man9GlcNAc2-P-P-Dol (49% of normal fibroblasts) were significantly restored. Mannose supplementation did not affect the synthesis of Man-P-Dol or Glc0-3Man9GlcNAc2-P-P-Dol in normal cells.
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GDP-Man levels in Man-P-Dol synthase deficient fibroblasts
Fibroblasts from Man-P-Dol synthase-deficient CDG-Ie patients synthesize a truncated dolichol-linked intermediate (Imbach et al., 2000; Kim et al., 2000
) which was corrected when the cells were incubated with exogenous mannose (Kim et al., 2000
). To determine if mannose supplementation expanded the cellular GDP-Man pool, the GDP-Man levels of the Man-P-Dol synthase-deficient fibroblasts after metabolic labeling with [3H]mannose for 48 h in the presence and absence of 1 mM mannose were measured. As shown in Table V, the cellular GDP-Man level was not significantly different in Man-P-Dol-deficient cells compared to normal fibroblasts (see Table III) cultured in either the presence or absence of mannose. This surprising result indicates that mannose addition corrects truncated dolichol-linked oligosaccharide synthesis in Man-P-Dol synthase deficient fibroblasts without altering the total amount of cellular GDP-Man.
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In conclusion, deficiencies in PMM and PMI reduce the total GDP-Man pool in fibroblasts from these patients and supplementing the culture medium with mannose effectively restores this depleted pool to normal levels. Glucose is significantly less effective in restoring normal GDP-Man levels. The results of this study further emphasize the importance of considering the specific activity of GDP-Man when calculating the rates of mannolipid and protein N-glycosylation by metabolic labeling with [3H]mannose. It will be important to determine how cells select and utilize mannose or glucose for biosynthesis of GDP-Man in future studies. Finally, the observation that mannose supplementation apparently corrects a defect in Man-P-Dol synthase without affecting the size of the total GDP-Man pool suggests that there are aspects of the regulation of lipid intermediate biosynthesis that have yet to be elucidated.
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Materials and methods |
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Cell lines and tissue culture
PMM-deficient fibroblasts were described previously (Panneerselvam et al., 1997b). The PMI-deficient fibroblast was obtained from Dr. Marc Patterson, Mayo Clinic, Rochester, MN. The Man-P-Dol synthase deficient cells from the CDG-Ie patient were obtained from Dr. James Filiano, Dartmouth Hitchcock Medical Center. All cells were cultured in
-MEM medium (Gibco BRL) with 5 mM glucose and 10% fetal bovine serum, as described before (Panneerselvam et al., 1997
).
Metabolic labeling of cultured fibroblasts with [3H]mannose and extraction of nucleotide sugars
Primary fibroblast cultures (100 mm dishes) were metabolically labeled for 0.5 to 48 h at 37°C by incubation with [3H]mannose (2550 µCi/ml) in -MEM, 10% FBS supplemented with or without 1 mM mannose. Incorporation of [3H]mannose into GDP-Man, Man-P-Dol and Glc0-3Man9GlcNAc2-P-P-Dol was determined exactly as described previously except that the cells were disrupted in methanol instead of ethanol, and the aqueous phase following partitioning of the CHCl3/CH3OH (2:1) fraction was retained and analyzed for GDP-Man as described below (Waechter et al., 1983
; Rush and Waechter, 1995
). The only isotopically labeled compound in the Man-P-Dol fraction was chromatographically identical to authentic Man-P-Dol as determined by thin layer chromatography on silica gel G TLC plates in CHCl3/CH3OH/H2O/NH4OH (65:35:4:1). The radiolabeled compound exhibited the characteristic sensitivity of Man-P-Dol to hydrolysis in mild acid (0.1 N HCl, 50% isopropanol, 50°C, 1h) and was resistant to alkaline hydrolysis (0.1 M KOH, toluene/methanol[1:1], 0°C, 30 min) (Waechter and Scher, 1981
).
Assay of the incorporation of [3H]mannose into dolichol-linked oligosaccharides and N-linked glycoproteins
The amount of [3H]mannose incorporated into Glc0-3Man9GlcNAc2-P-P-Dol and glycoprotein in the delipidated residue, from the previous section, was determined by a multiple extraction procedure (Waechter et al., 1983). The radioactive products in the Glc0-3[3H]Man9GlcNAc2-P-P-Dol fraction from the PMI-deficient fibroblasts were sensitive to mild acid hydrolysis (10 mM HCl, 80% tetrahydrofuran, 10 min, 100°C) (Lucas et al., 1975
) which released a [3H]oligosaccharide that co-eluted with authentic Glc3Man9GlcNAc2 following gel filtration on Bio-Gel P-4 and by HPLC on an AX5 ion-exchange column. The delipidated residue was solubilized with 1ml of 1% SDS (3 min, 100°) and analyzed for protein (Rodriquez-Vico et al., 1989
) and for radioactivity. Incorporation of [3H]mannose into cellular products was normalized for cell number based on the amount of protein recovered in the delipidated membrane residue.
Partial purification of GDP-Man
The aqueous phases from the cellular extracts described above were dried under reduced pressure at 30°C, redissolved in 0.2 ml 15 mM ammonium phosphate (pH 3.5) containing 1 mM CaCl2, 1 mM MgCl2 and 1 mM MnCl2 (Con A buffer) and purified by chromatography on a Con ASepharose column (Rush and Waechter, 1995).
GDP-Man was purified further by anion exchange chromatography on a 0.5 x 20 cm column of Partisil-10 SAX silica gel using a Waters Baseline 810 Chromatography Workstation HPLC containing a Waters model U6K Universal Liquid Chromatograph Injector (equipped with a 10 ml injection loop), model 501 Solvent Delivery System and a Lambda-Max model 481 LC Spectrophotometer (Rush and Waechter, 1995). The UV absorbance peak associated with GDP-Man in the HPLC chromatogram was integrated using the Waters Baseline 810 peak detection and integration software. GDP-Man integrations were rigorously and carefully reviewed from each HPLC determination to insure that the software appropriately determined the baseline for peak detection. GDP-Man concentrations were normalized for each determination using the recovery of the GDP-[U-14C]Man as an internal standard and were calculated by comparison with standard injections of known amounts of GDP-Man.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed at: The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037
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