Mannose supplementation corrects GDP-mannose deficiency in cultured fibroblasts from some patients with Congenital Disorders of Glycosylation (CDG).
Congenital Disorders of Glycosylation (CDG) are human deficiencies in glycoprotein biosynthesis. Previous studies showed that 1 mM mannose corrects defective protein N-glycosylation in cultured fibroblasts from some CDG patients. We hypothesized that these CDG cells have limited GDP-mannose (GDP-Man) and that exogenous mannose increases the GDP-Man levels. Using a well established method to measure GDP-Man, we found that normal fibroblasts had an average of 23.5 pmol GDP-Man/10(6) cells, whereas phosphomannomutase (PMM)-deficient fibroblasts had only 2.3-2.7 pmol/10(6) cells. Adding 1 mM mannose to the culture medium increased the GDP-Man level in PMM-deficient cells to approximately 15.5 pmol/10(6) cells, but had no significant effect on GDP-Man levels in normal fibroblasts. Similarly, mannose supplementation increased GDP-Man from 4.6 pmol/10(6) cells to 24.6 pmol/10(6) cells in phosphomannose isomerase (PMI)-deficient fibroblasts. Based on the specific activity of the GDP-[(3)H]Man pool present in [2-(3)H]mannose labeled cells, mannose supplementation also partially corrected the impaired synthesis of mannosylphosphoryldolichol (Man-P-Dol) and Glc(0)(-)(3)Man(9)GlcNAc(2)-P-P-Dol. These results confirm directly that deficiencies in PMM and PMI result in lowered cellular GDP-Man levels that are corrected by the addition of mannose. In contrast to these results, GDP-Man levels in fibroblasts from a CDG-Ie patient, who is deficient in Man-P-Dol synthase, were normal and unaffected by mannose supplementation even though mannose addition was found to correct abnormal lipid intermediate synthesis in another study (Kim et al. [2000] J. Clin. Invest., 105, 191-198). The mechanism by which mannose supplementation corrects abnormal protein N-glycosylation in Man-P-Dol synthase deficient cells is unknown, but this observation suggests that the regulation of Man-P-Dol synthesis and utilization may be more complex than is currently understood.
Department of Biochemistry, A.B.Chandler Medical Center, University of Kentucky College of Medicine, Lexington, KY, USA.
J S
JS
Rush
K
K
Panneerselvam
C J
CJ
Waechter
H H
HH
Freeze
D002239:Carbohydrate Metabolism, Inborn Errors / Q000188:drug therapy* / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D006031:Glycosylation; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism*; D006801:Humans; D008358:Mannose / Q000008:administration & dosage / Q000627:therapeutic use*; D008364:Mannosyltransferases / Q000378:metabolism
D006155:Guanosine Diphosphate Mannose; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D008358:Mannose