Mannose for N-linked oligosaccharide synthesis is assumed to come from glucose via phosphomannose isomerase (PMI). Dr. Freeze's results challenge this key assumption by showing that mammalian cells have mannose-specific, glucose-tolerant transporters with a K uptake of ~60FM, similar to serum mannose concentrations. Fibroblasts presented with physiological concentrations of glucose (5.5mM) and mannose (50FM), use transported mannose for >70% of glycoprotein biosynthesis, showing that most mannose does not come from glucose in these cells. The principal investigator and his colleagues hypothesize that mannose, and not glucose, is the major source of mannose for glycosylation. Cells briefly deprived of glucose, actively abusing alcoholics, and patients with inherited Carbohydrate Deficient Glycoprotein Syndrome (CDGS) all underglycosylate a specific set of serum glycoproteins because they fail to add entire sugar chains. Adding mannose but not glucose to the culture medium of CDGS fibroblasts corrects underglycosylation and also prevents glucose depletion-induced underglycosylation. The mechanisms are unknown, but they probably result from deficiencies in mannose mobilization. To understand how underglycosylation occurs requires a better understanding of the role of mannose in glycoprotein biosynthesis. The investigators will study the mannose transporter and mannose mobilization in mouse cells and in the organism. Specifically they propose to: 1. Identify the mannose transporter(s) and develop transport assays and antibodies to study its biosynthesis and distribution. 2. Determine if mannose salvaged from normal oligosaccharide processing and exogenous glycoprotein catabolism is used for oligosaccharide biosynthesis. 3. Determine the preferred source of mannose in hepatoma cells under physiological conditions and how other sources compensate when cells are metabolically stressed. 4. Demonstrate glycoprotein digestion and alpha-mannosidases responsible for it. These studies will show the normal sources of mannose for glycoprotein biosynthesis, and may provide insight for other inherited or physiological defects in glycoprotein biosynthesis. In some cases, mannose may be a therapeutic dietary supplement. A mannose supplementation trial for CDGS patients is now underway.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Medical Biochemistry Study Section (MEDB)
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Marino, Pamela
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Sanford-Burnham Medical Research Institute
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Sharma, Vandana; Freeze, Hudson H (2011) Mannose efflux from the cells: a potential source of mannose in blood. J Biol Chem 286:10193-200
Freeze, Hudson H (2009) Towards a therapy for phosphomannomutase 2 deficiency, the defect in CDG-Ia patients. Biochim Biophys Acta 1792:835-40
DeRossi, Charles; Bode, Lars; Eklund, Erik A et al. (2006) Ablation of mouse phosphomannose isomerase (Mpi) causes mannose 6-phosphate accumulation, toxicity, and embryonic lethality. J Biol Chem 281:5916-27
Wu, Xiaohua; Freeze, Hudson H (2002) GLUT14, a duplicon of GLUT3, is specifically expressed in testis as alternative splice forms. Genomics 80:553-7
Wu, Xiaohua; Li, Weizhong; Sharma, Vandana et al. (2002) Cloning and characterization of glucose transporter 11, a novel sugar transporter that is alternatively spliced in various tissues. Mol Genet Metab 76:37-45
Davis, Joseph A; Wu, Xiao-Hua; Wang, Ling et al. (2002) Molecular cloning, gene organization, and expression of mouse Mpi encoding phosphomannose isomerase. Glycobiology 12:435-42
Davis, J A; Freeze, H H (2001) Studies of mannose metabolism and effects of long-term mannose ingestion in the mouse. Biochim Biophys Acta 1528:116-26
Freeze, H H; Westphal, V (2001) Balancing N-linked glycosylation to avoid disease. Biochimie 83:791-9
Freeze, H H (2001) Update and perspectives on congenital disorders of glycosylation. Glycobiology 11:129R-143R
Kim, S; Westphal, V; Srikrishna, G et al. (2000) Dolichol phosphate mannose synthase (DPM1) mutations define congenital disorder of glycosylation Ie (CDG-Ie) J Clin Invest 105:191-8

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