Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms
Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms
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DOI：
10.1074/mcp.m112.027151
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发表时间：
2013-09-01
影响因子：
7
通讯作者：
Taniguchi, Naoyuki
中科院分区：
文献类型：
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作者：
Nakajima, Kazuki;Ito, Emi;Taniguchi, Naoyuki
核苷酸是各种糖基转移酶的供体底物，也是N-和O-糖链生物合成的重要组成部分。它们的细胞间浓度受细胞代谢状态的调节，包括癌症和糖尿病等疾病。为了研究UDP-GlcNAc的去向，我们建立了分别使用C-13(6)-葡萄糖和C-13(2)-氨基葡萄糖对UDP-GlcNAc的合成和利用进行示踪的方法，并用LC-MS分析了大量的同位素异构体。用C-13(6)-葡萄糖对培养细胞进行代谢标记，并对UDP-HexNAc(UDP-GlcNAc+UDP-GalNAc)和CMP-NeuAc进行分析，揭示了导致UDP-GlcNAc合成和使用的代谢途径的相对贡献。在胰腺胰岛素瘤细胞中，C-13(6)-葡萄糖基序在CMP-NeuAc中的标记效率低于在肝癌细胞中的标记效率。以C-13(2)-氨基葡萄糖为标记物，在同位素分析的基础上，观察了N-和O-葡聚糖在各糖基残渣中标记效率的差异。在胰岛素瘤细胞中，唾液酸以及三和四唾液酸N-聚糖的标记效率较低，而唾液酸N-聚糖的标记效率较高。肝癌细胞株和胰岛素瘤细胞株分泌的透明质酸基本上没有显著差异。这表明代谢流是胰岛素瘤细胞低唾液酸化的原因。我们的策略应该有助于系统地追踪细胞GlcNAc代谢的每个阶段。
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 C-13(6)-glucose and C-13(2)-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS. Metabolic labeling of cultured cells with C-13(6)-glucose and the analysis of isotopomers of UDP-HexNAc (UDPGlcNAc 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 C-13(6)-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells. Using C-13(2)-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.