High-speed and high-sensitive methods of carbohydrates based on mass isotopomer analysis with the aim of monitoring glycan dynamics

• Project/Area Number: 24790301
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: General medical chemistry
• Research Institution: The Institute of Physical and Chemical Research
• Principal Investigator: NAKAJIMA Kazuki 独立行政法人理化学研究所, 脳科学総合研究センター, 研究員 (10442998)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2014: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2013: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2012: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
• Keywords: 糖鎖修飾 / 糖ヌクレオチド / 質量分析 / クロマトグラフィー / グルコース代謝 / シアル酸 / 糖鎖 / 糖タンパク質

Outline of Final Research Achievements

In order to clarify biological significance of glycan dynamics, we have developed a tracing method for UDP-GlcNAc synthesis and utilization, and GlcNAc utilization using 13C6-glucose and 13C2-glucosamine, respectively, followed by the analysis of mass isotopomers. Because nucleotide sugars an important building block in glycan biosynthesis. Analysis of isotopomers of UDP-HexNAc and CMP-NeuAc, labeled with 13C6-glucose, revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and utilization. Using 13C2-glucosamine, the diversity of glycan turnover was observed in each glycan. In pancreatic insulinoma cells, the labeling efficiency of CMP-NeuAc was lower compared with that in hepatoma cells, indicating that metabolic flows are responsible for the low sialylation in the insulinoma cells. Thus, our strategy should be useful for systematically tracing each stage of cellular glycan dynamics.

Research Products

• [Journal Article] Simultaneous Quantification of Nucleotide Sugar Metabolism by LC and LC-MS (2014)
  • Author(s): Kazuki Nakajima, Naoyuki Taniguchi
  • Journal Title: Glycoscience: Biology and Medicine Volume: 1 Pages: 103-110
  • Acknowledgement Compliant
• [Journal Article] The absence of core fucose upregulates GnT-III and Wnt target genes: a possible mechanism for an adaptive response in terms of glycan function (2014)
  • Author(s): Kurimoto A, Kitazume S, Kizuka Y, Nakajima K, Oka R, Fujinawa R, Korekane H, Yamaguchi Y, Wada Y, Taniguchi N
  • Journal Title: J Biol Chem. (Epub 2014 Mar10.) Volume: 289(17) Issue: 17 Pages: 11704-14
  • DOI: 10.1074/jbc.m113.502542
  • Peer Reviewed
• [Journal Article] Ceramide galactosyltransferase expression is regulated positively by Nkx2.2 and negatively by OLIG2. (2014)
  • Author(s): Okahara K, Kizuka Y, Kitazume S, Ota F, Nakajima K, Hirabayashi Y, Maekawa M, Yoshikawa T, Taniguchi N.
  • Journal Title: Glycobiology Volume: 10 Issue: 10 Pages: 926-34
  • DOI: 10.1093/glycob/cwu042
  • Peer Reviewed
• [Journal Article] The Absence of Core Fucose Up-regulates GnT-III and Wnt Target Genes: A POSSIBLE MECHANISM FOR AN ADAPTIVE RESPONSE IN TERMS OF GLYCAN FUNCTION. (2014)
  • Author(s): Kurimoto A, Kitazume S, Kizuka Y, Nakajima K, Oka R, Fujinawa R, Korekane H, Yamaguchi Y, Wada Y, Taniguchi N
  • Journal Title: J Biol Chem Volume: 289 Pages: 11704-11714
  • Peer Reviewed
• [Journal Article] Mass isotopomer analysis of metabolically labeled nucleotide sugars and N- and O-glycans for tracing nucleotide sugar metabolisms. (2013)
  • Author(s): Nakajima, K., Ito, E., Ohtsubo, K., Shirato, K., Takamiya, R., Kitazume, S., Angata, T., Taniguchi, N.
  • Journal Title: Mol. & Cell Proteomics. Volume: 12 Pages: 2468-2080
  • Peer Reviewed
• [Journal Article] Metabolically programmed quality control system for dolichol-linked oligosaccharides. (2013)
  • Author(s): Harada, Y., Nakajima, K., Masahara-Negishi, Y., Freeze, H.H., Angata, T., Taniguchi, N., Suzuki, T.
  • Journal Title: Proc. Natl. Acad. Sci. U.S.A Volume: 110 Pages: 19366-19371
  • Peer Reviewed
• [Journal Article] Identification of ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) as a regulator of N-acetylglucosaminyltransferase GnT-IX (GnT-Vb). (2013)
  • Author(s): Korekane, H., Park, J.Y, Matsumoto, A., Nakajima, K., Takamatsu, S., Ohtsubo, K., Miyamoto, Y., Hanashima, S., Kanekiyo, K., Kitazume, S., Yamaguchi, Y., Matsuo, I., Taniguchi, N.
  • Journal Title: J. Biol. Chem. Volume: 288 Pages: 27912-27926
  • Peer Reviewed
• [Presentation] 双性イオイ性カラム(ZIC)-HILIC-ESI-MSによる脳内グルコース化分子の異性体定量 (2014)
  • Author(s): 8. 中嶋 和紀、秋山 央子、田中 香織、谷口 直之、平林 義雄
  • Organizer: 第33回日本糖質学会年会
  • Place of Presentation: 名古屋
  • Year and Date: 2014-08-10 – 2014-08-12
• [Presentation] Mass isotopomer analysis of metabolically labeled nucleotide sugars and N- and O-glycans for tracing nucleotide sugar metabolisms. (2013)
  • Author(s): Nakajima, K., Ito, E., Ohtsubo, K., Shirato, K., Takamiya, R., Kitazume, S., Angata, T., Taniguchi, N.
  • Organizer: HUPO 12th Annual World Congress
  • Place of Presentation: yokohama
• [Presentation] 質量同位体測定によるグルコサミンの代謝動態の追跡 (2013)
  • Author(s): 中嶋和紀、伊藤恵実、大坪和明、白土健、高宮里奈、北爪しのぶ、安形高志、谷口直之
  • Organizer: 第32回糖質学会年会
  • Place of Presentation: 大阪
• [Presentation] 質量同位体測定によるグルコサミンの代謝動態の追跡 (2013)
  • Author(s): 中嶋和紀、伊藤恵実、大坪和明、白土健、高宮里奈、北爪しのぶ、安形高志、谷口直之
  • Organizer: 第86回日本生化学会年会
  • Place of Presentation: 横浜
• [Presentation] Tracing Strategy of GlcNAc Cycle by Mass Isotopomers Analysis (2012)
  • Author(s): Nakajima, K., Ito, E., Ohtsubo, K., Shirato, K., Takamiya, R., Kitazume, S., Angata, T., and Taniguchi, N.
  • Organizer: HUPO 2012
  • Place of Presentation: Boston
• [Presentation] 質量同位体測定に基づくN-アセチルグルコサミンサイクルの追跡戦略 (2012)
  • Author(s): 中嶋和紀、伊藤恵実、大坪和明、白土健、高宮里奈、北爪しのぶ、安形高志、谷口直之
  • Organizer: 第85回日本生化学会年会
  • Place of Presentation: 福岡