title: Mass isotopomer analysis of metabolically labeled nucleotide sugars and N- and O-glycans for tracing nucleotide sugar metabolisms.
abstract: Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using (13)C6-glucose and (13)C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS. Metabolic labeling of cultured cells with (13)C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a (13)C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells. Using (13)C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.
authors.affiliation: Disease Glycomics Team, Systems Glycobiology Research Group, Global Research Cluster, RIKEN Max Plank Joint Research Center, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Nakajima
authors.forename: Emi
authors.initials: E
authors.lastname: Ito
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Ohtsubo
authors.forename: Ken
authors.initials: K
authors.lastname: Shirato
authors.forename: Rina
authors.initials: R
authors.lastname: Takamiya
authors.forename: Shinobu
authors.initials: S
authors.lastname: Kitazume
authors.forename: Takashi
authors.initials: T
authors.lastname: Angata
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Taniguchi
mesh_terms: D000818:Animals; D002247:Carbon Isotopes; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D006595:Hexosamines / Q000378:metabolism; D006820:Hyaluronic Acid / Q000378:metabolism; D007340:Insulinoma / Q000378:metabolism; D007553:Isotope Labeling; D008113:Liver Neoplasms / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008954:Models, Biological; D008970:Molecular Weight; D009711:Nucleotides / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis / Q000378:metabolism*; D013402:Sugar Alcohols / Q000378:metabolism; D013997:Time Factors; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D006595:Hexosamines; D009711:Nucleotides; D011134:Polysaccharides; D013402:Sugar Alcohols; D014537:Uridine Diphosphate N-Acetylglucosamine; D006820:Hyaluronic Acid