Application of fluorophore-assisted carbohydrate electrophoresis for the study of the dolichol pyrophosphate-linked oligosaccharides pathway in cell cultures and animal tissues.
Defects in the synthesis of dolichol-linked oligosaccharide (or lipid-linked oligosaccharide [LLO]) cause severe, multisystem human diseases called type 1 congenital disorders of glycosylation (CDG type 1). LLOs are also involved in another disease, neuronal ceroid lipofuscinosis. Because of the low abundance of LLOs, almost all studies of LLO synthesis have relied upon metabolic labeling of the oligosaccharides with radioactive sugar precursors such as [3H]mannose or [14C]glucosamine, and therefore have been limited almost entirely to cell cultures and tissue slices. A procedure is presented for a facile, accurate, and sensitive non-radioactive method for LLO pathway analysis based on fluorophore-assisted carbohydrate electrophoresis (FACE). It is feasible to analyze almost any component in the LLO pathway with the application FACE, from sugar precursors to mature LLO (Glc3Man9GlcNAc2-P-P-dolichol).
Gao
Ningguo
N
Department of Pharmacology, University of Texas, Southern Medical Center, Dallas, TX, USA.
D000818:Animals; D001000:Ants / Q000737:chemistry; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004288:Dolichol Phosphates / Q000737:chemistry*; D004286:Dolichols / Q000737:chemistry; D004586:Electrophoresis / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000737:chemistry; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D011105:Polyisoprenyl Phosphate Sugars / Q000032:analysis / Q000378:metabolism; D012680:Sensitivity and Specificity; D015398:Signal Transduction
D002241:Carbohydrates; D004288:Dolichol Phosphates; D004286:Dolichols; D005456:Fluorescent Dyes; C066723:Glc(3)Man(9)(GlcNAc)(2)-diphosphate-dolichol; D009282:Naphthalenesulfonates; D009844:Oligosaccharides; D011105:Polyisoprenyl Phosphate Sugars; C026406:dolichol pyrophosphate; C019152:3-azido-2,7-naphthalene disulfonate