Regulation of nuclear protein functions by N-acetylglucosamine modification

• Project/Area Number: 24390015
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Biological pharmacy
• Research Institution: The University of Tokyo
• Principal Investigator: YAMAMOTO Kazuo 東京大学, 新領域創成科学研究科, 教授 (20174782)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥18,590,000 (Direct Cost: ¥14,300,000、Indirect Cost: ¥4,290,000); Fiscal Year 2014: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000); Fiscal Year 2013: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000); Fiscal Year 2012: ¥6,890,000 (Direct Cost: ¥5,300,000、Indirect Cost: ¥1,590,000)
• Keywords: Nーアセチルグルコサミン修飾 / 核内タンパク質 / レクチン / イメージング / 糖修飾 / N-アセチルグルコサミン / 糖鎖 / 蛋白質

Outline of Final Research Achievements

O-GlcNAcylation of nuclear proteins is a unique reaction in several points of view. O-GlcNAcyation levels are modulated in response to cellular signaling and affect protein localization, activity and stability. We screened engineered lectins to monitor O-GlcNAc modification using mammalian cell surface display from randomized lectin library. Furthermore, we expressed a soluble form of β-N-acetylgalactosaminyltransferase in the nucleus to change O-GlcNAc to GalNAc-GlcNAc. A novel lectin specific for GalNAc-GlcNAc was purified from Wisteria japonica seeds. Using these lectins as a probe, we identified more than 80 kinds of O-GlcNAc-modified proteins including 10 kinds of novel proteins from the nuclear and cytosolic fractions. O-GlcNAc-moidifed peptides were purified by lectin affinity chromatography and identified O-GlcNAc-modified residues by mass spectrometry. A lectin-GFP was expressed in the cytoplasm for monitoring O-GlcNAcylation in a live cell during cell activation.

Research Products

• [Journal Article] Association of malectin with ribophorin I is crucial for attenuation of misfolded glycoprotein secretion (2014)
  • Author(s): Takeda, K., Qin, S., Matsumoto, N., Yamamoto, K.
  • Journal Title: Biochem. Biophys. Res. Commun. Volume: 454 Issue: 3 Pages: 436-440
  • DOI: 10.1016/j.bbrc.2014.10.102
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Assessment of weak sugar-binding ability using lectin tetramer and membrane-based glycans (2014)
  • Author(s): Yamamoto, K.
  • Journal Title: Methods Mol. Biol. Volume: 1200 Pages: 413-418
  • DOI: 10.1007/978-1-4939-1292-6_36
  • ISBN: 9781493912919, 9781493912926
• [Journal Article] Carbohydrate-binding specificity of lectins using multiplexed glyco-bead array (2014)
  • Author(s): Yamamoto, K.
  • Journal Title: Methods Mol. Biol. Volume: 1200 Pages: 319-326
  • DOI: 10.1007/978-1-4939-1292-6_27
  • ISBN: 9781493912919, 9781493912926
• [Journal Article] Comprehensive list of lectins: origins, natures, and carbohydrate specificities (2014)
  • Author(s): Kobayashi, Y, Tateno, H, Ogawa, H, Yamamoto, K, Hirabayashi, J.
  • Journal Title: Methods Mol. Biol. Volume: 1200 Pages: 555-577
  • DOI: 10.1007/978-1-4939-1292-6_45
  • ISBN: 9781493912919, 9781493912926
• [Journal Article] Intracellular lectins are involved in quality control of glycoproteins (2014)
  • Author(s): Yamamoto, K.
  • Journal Title: Proceedings of the Japan Academy, Series B Volume: 90 Issue: 2 Pages: 67-82
  • DOI: 10.2183/pjab.90.67
  • NAID: 130003392354
  • ISSN: 0386-2208, 1349-2896
  • Peer Reviewed
• [Journal Article] Terminal N-acetylgalactosamine-specific leguminous lectin from Wisteria japonica as a probe for human lung squamous cell carcinoma. (2013)
  • Author(s): Soga et al.
  • Journal Title: PLoS ONE Volume: 8(12) Issue: 12 Pages: e83886-e83886
  • DOI: 10.1371/journal.pone.0083886
  • Peer Reviewed
• [Journal Article] Regulation of Mac-2BP secretion is mediated by its N-glycan binding to ERGIC-5 (2013)
  • Author(s): Chen, et al.
  • Journal Title: Glycobiology Volume: 23 Issue: 7 Pages: 904-916
  • DOI: 10.1093/glycob/cwt027
  • Peer Reviewed
• [Journal Article] Malectin forms a complex with r i bophor in I for enhanced association with misfolded glycoproteins. (2012)
  • Author(s): Qin, et al.
  • Journal Title: J. Biol. Chem. Volume: 287 Pages: 38080-38089
  • Peer Reviewed
• [Presentation] A novel method to identify O-GlcNAcylated proteins using a soluble N-acetylgalactosaminyltransferase (2015)
  • Author(s): Yamamoto, K.
  • Organizer: 2nd International symposium on protein modifications in pathogenic dysregulation of signaling
  • Place of Presentation: 東京大学医科学研究所大講堂（東京都、港区）
  • Year and Date: 2015-01-23 – 2015-01-24
• [Presentation] 核移行糖転移酵素を用いた核内N-アセチルグルコサミン修飾タンパク質同定法の確立 (2014)
  • Author(s): 山本一夫
  • Organizer: 第65回日本電気泳動学会総会
  • Place of Presentation: 横浜情報文化センター（神奈川県、横浜市）
  • Year and Date: 2014-10-24 – 2014-10-25
  • Invited
• [Presentation] マメ科レクチンのスキャフォールド解析 (2014)
  • Author(s): 山本一夫
  • Organizer: 第87回日本生化学会大会
  • Place of Presentation: 国立京都国際会館（京都府、京都市）
  • Year and Date: 2014-10-15 – 2014-10-18
  • Invited
• [Presentation] 糖鎖認識のための分子基盤：スキャフォールド解析から見えたこと (2014)
  • Author(s): 曽我慶介、山本一夫
  • Organizer: 第33回日本糖質学会年会
  • Place of Presentation: 名古屋大学豊田講堂（愛知県、名古屋市）
  • Year and Date: 2014-08-10 – 2014-08-12
  • Invited
• [Presentation] がん細胞表面糖鎖抗原を標的とした改変レクチンの探索 (2013)
  • Author(s): 田中敦洋 他
  • Organizer: 第32回日本糖質学会年会
  • Place of Presentation: 大阪国際交流センター
• [Presentation] 細胞提示法による改変レクチン創出 (2013)
  • Author(s): 曽我慶介 他
  • Organizer: 第32回日本糖質学会年会
  • Place of Presentation: 大阪国際交流センター
• [Remarks]
  • URL: http://park,itc.u-tokyo.ac.jp/iyaku/