Functional analysis of plant free oligosaccharides through regulation of gene-expressions of oligosaccharide-related enzymes and application of the biological functions

• Project/Area Number: 17K08197
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Applied molecular and cellular biology
• Research Institution: Okayama University
• Principal Investigator: Kimura Yoshinobu 岡山大学, 環境生命科学研究科, 教授 (70195387)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2018: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2017: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 遊離N-グリカン / PNGase / ENGase / 発現制御 / 過剰発現 / ERAD / 遊離N-グリカン（FNG） / 過剰発現株 / 発現抑制株 / acidic PNGase / cytosolic PNGase / 発現抑制 / Arabidopsis thaliana / Micro Tom / 糖鎖生物学 / 糖鎖機能 / 糖鎖工学

Outline of Final Research Achievements

In developing or growing plants, we have found that the free N-glycans (FNGs) occur at a few micro molar concentrations and we have made a working hypothesis that these FNGs may carry important functions involved in plant physiology. The FNGs are classified into two categories; one produced from misfolded glycoproteins in ERAD system, and the other one produced from the turnover of function-lost proteins. In this project, through the constructions of mutant lines of A.thaliana or tomato, we have found the putative biological functions of FNGs involved in plant development or growth, and we have developed the basic research to apply the putative functions to regulate the plant growth or the fruit-development.

Academic Significance and Societal Importance of the Research Achievements

本課題は，植物に遍在する遊離型糖鎖の植物分化・成長に関わる生理機能の証明と利用に焦点を当てている点が学術的な特色であり，仮説が実証されれば，21世紀の食料生産を支える新たなアグリテクノロジーの確立につながる意義がある。本研究成果において，分化成長中の植物に遍在する遊離型糖鎖（遊離N-グリカン，FNGs）の生成に関わる３種酵素（cPNGase，aPNGase，ENGase）の発現制御株（発現抑制，過剰発現）を構築することで，PNGase/ENGase 非依存的な生成機構が存在することを明らかにするとともに，aPNGase過剰発現（T2株）により，FNGs増加と果実熟成促進傾向を確認した。

Research Products

• [Int'l Joint Research] ゲント大学(ベルギー)
• [Int'l Joint Research] ウィーン大学(オーストリア)
• [Int'l Joint Research] ゲント大学/Prof. Els JM van Damme(ベルギー)
• [Int'l Joint Research] ウィーン大学/Prof. Richard Strasser(オーストリア)
• [Int'l Joint Research] ゲント大学(ベルギー)
• [Int'l Joint Research] ウィーン大学(オーストリア)
• [Journal Article] Synthesis and preliminary evaluation of neoglycopolymers carrying multivalent N-glycopeptide units. (2020)
  • Author(s): Takeda N, Maeda M, Itano S, Takase M, Kimura M, Kimura Y.
  • Journal Title: Int J Biol Macromol. Volume: 147 Pages: 1294-1300
  • DOI: 10.1016/j.ijbiomac.2019.09.255
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Convenient preparation of an antigenic oligosaccharide from white kidney bean powder: A useful plant oligosaccharide for synthesis of immunoactive glycopolymer. (2020)
  • Author(s): Kimura M, Ogura M, Akamatsu M, Sugimoto K, Maeda M, Nitoda T, Nagasawa-Fujimori H, Yamashita H, Kimura Y.
  • Journal Title: Int J Biol Macromol. Volume: 153 Pages: 1016-1023
  • DOI: 10.1016/j.ijbiomac.2019.10.231
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Plant complex type free N-glycans occur in tomato xylem sap (2019)
  • Author(s): Tusjimori, Y., Ogura, M., Rahman, MZ., Maeda, M., Kimura, Y.
  • Journal Title: Bioscience, Biotechnology, and Biochemistry Volume: 83 Issue: 7 Pages: 1310-1314
  • DOI: 10.1080/09168451.2019.1608803
  • NAID: 120006840498
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Analysis of aPNGase-overexpressed tomato line (T2 generation): phenotype, PNGase activity and structural features of FNGs (2019)
  • Author(s): Yamamoto, C., Ogura, M., Matsumaru, C., Maeda, M., Kimura, Y
  • Journal Title: Glycoconjugate Journal Volume: 36 Pages: 379-379
  • Peer Reviewed
• [Journal Article] Structural features of high-mannose type free N-glycans (FNGs) in A. thaliana mutant lines lacking in ERAD related proteins (2019)
  • Author(s): Furuta，K., Maeda，M., 1, Strasser, R., and Kimura, Y
  • Journal Title: Glycoconjugate Journal Volume: 36 Pages: 241-241
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Structural Features of FNGs and Salt Sensitivity of ENGase and ENGase/cPNGase knockout Arabidopsis thaliana (2019)
  • Author(s): Shirai, S., Uemura, R., Maeda, M., Misaki,R.,Fujiyama, K., and Kimura, Y.
  • Journal Title: Glycoconjugate Journal Volume: 36 Pages: 379-379
  • Peer Reviewed
• [Journal Article] Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract. (2018)
  • Author(s): Uemura R, Ogura M, Matsumaru C, Akiyama T, Maeda M, Kimura Y.
  • Journal Title: Biosci Biotechnol Biochem. Volume: 印刷中 Issue: 7 Pages: 1172-1175
  • DOI: 10.1080/09168451.2018.1459464
  • NAID: 120006619340
  • Peer Reviewed / Open Access
• [Journal Article] Molecular characterization of second tomato α1,3/4-fucosidase (α-Fuc'ase Sl-2), a member of glycosyl hydrolase family 29 active toward the core α1,3-fucosyl residue in plant N-glycans. (2018)
  • Author(s): Rahman Z, Tsujimori Y, Maeda M, Hossain A, Ishimizu T, Kimura Y.
  • Journal Title: J Biochem. Volume: 印刷中 Issue: 1 Pages: 53-63
  • DOI: 10.1093/jb/mvy029
  • NAID: 40021625325
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Occurrence of complex type free N-glycans with a single GlcNAc residue at the reducing termini in the fresh-water plant, Egeria densa (2017)
  • Author(s): Maeda Megumi、Ebara Natsuki、Tani Misato、Vavricka Christopher J.、Kimura Yoshinobu
  • Journal Title: Glycoconj. J. Volume: 34 Issue: 2 Pages: 1405-1408
  • DOI: 10.1007/s10719-016-9758-z
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Molecular characterization of tomato α1,3/4-fucosidase, a member of glycosyl hydrolase family 29, involved in degradation of plant complex type N-glycans. (2017)
  • Author(s): Rahman, M.D., Maeda, M., Itano, S., Hossain, M.A., Ishimizu, T., Kimura, Y.
  • Journal Title: J. Biochem. Volume: 161 Pages: 421-432
  • DOI: 10.1093/jb/mvw089
  • NAID: 40021219805
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Glycoform of a newly identified pollen allergen, Cha o 3, from Chamaecyparis obtusa (Japanese cypress, Hinoki) (2017)
  • Author(s): Osada Toshihiro、Maeda Megumi、Tanabe Chinatsu、Furuta Kaori、Vavricka Christopher J.、Sasaki Eiji、Okano Mitsuhiro、Kimura Yoshinobu
  • Journal Title: Carbohydr. Res. Volume: 448 Pages: 18-23
  • DOI: 10.1016/j.carres.2017.05.005
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Structural feature of N-glycans of bamboo shoot glycoproteins: useful source of plant antigenic N-glycans (2017)
  • Author(s): Tanabe Chinatsu、Furuta Kaori、Maeda Megumi、Kimura Yoshinobu
  • Journal Title: Biosci. Biotechnol. Biochem. Volume: 81 Issue: 7 Pages: 1405-1408
  • DOI: 10.1080/09168451.2017.1320519
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Synthesis of Sulfo-Sialic Acid Analogues: Potent Neuraminidase Inhibitors in Regards to Anomeric Functionality (2017)
  • Author(s): Vavricka Christopher J.、Muto Chiaki、Hasunuma Tomohisa、Kimura Yoshinobu、Araki Michihiro、Wu Yan、Gao George F.、Ohrui Hiroshi、Izumi Minoru、Kiyota Hiromasa
  • Journal Title: Scientific Reports Volume: 7 Issue: 1 Pages: 8239-8239
  • DOI: 10.1038/s41598-017-07836-y
  • NAID: 120006352247
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] 新規人工糖鎖ポリマーによる植物複合型糖鎖結合タンパク質のスクリーニング (2020)
  • Author(s): 田中創平，前田恵，木村吉伸
  • Organizer: 日本農芸化学会中四国支部第５６回講演会
• [Presentation] 緑藻ミル Codium fragile 由来レクチン (CFL) のホモオリゴマー構造について (2020)
  • Author(s): 八塚真悟，前田恵，木村吉伸
  • Organizer: 日本農芸化学会中四国支部第５６回講演会
• [Presentation] 免疫活性測定を目的としたイモ類に含まれる貯蔵タンパク質の糖鎖構造解析 (2020)
  • Author(s): 蓮井泉保，木村吉伸，前田恵
  • Organizer: 日本農芸化学会中四国支部第５６回講演会
• [Presentation] 大腸菌 BL21(DE3)を用いたα-synuclein(AS)の多量発現と新たな精製法の確立 (2020)
  • Author(s): 小坂将太，前田恵，木村吉伸
  • Organizer: 日本農芸化学会中四国支部第５６回講演会
• [Presentation] Structural Features of FNGs and Salt Sensitivity of ENGase and ENGase/cPNGase knockout Arabidopsis thaliana (2019)
  • Author(s): Shirai, S., Uemura, R., Maeda, M., Misaki,R.,Fujiyama, K., and Kimura, Y.
  • Organizer: 25th Internatuonal Symposium on Glycoconjugates, Milano, Italia
  • Int'l Joint Research
• [Presentation] Analysis of aPNGase-overexpressed tomato line (T2 generation): phenotype, PNGase activity and structural features of FNGs (2019)
  • Author(s): Yamamoto, C., Ogura, M., Matsumaru, C., Maeda, M., Kimura, Y
  • Organizer: 25th Internatuonal Symposium on Glycoconjugates, Milano, Italia
  • Int'l Joint Research
• [Presentation] Structural features of high-mannose type free N-glycans (FNGs) in A. thaliana mutant lines lacking in ERAD related proteins (2019)
  • Author(s): Furuta，K., Maeda，M., 1, Strasser, R., and Kimura, Y
  • Organizer: 25th Internatuonal Symposium on Glycoconjugates, Milano, Italia
  • Int'l Joint Research
• [Presentation] Analysis of suppressive function of dendritic cells by glycopolymers carrying multivalent plant antigenic N-glycans beraing Lewis a epitope (2019)
  • Author(s): Maeda，M., Tanabe, C., Tani, M., and Kimura, Y
  • Organizer: 25th Internatuonal Symposium on Glycoconjugates, Milano, Italia
  • Int'l Joint Research
• [Presentation] Molcular characterization of peanut β-xylosidase active towards plant specific antigenic N-glycans (2019)
  • Author(s): Md Asaduzzaman, Maeda, M., and Kimura, Y
  • Organizer: 第92回日本生化学会大会
• [Presentation] Protein-folding promoting activity of free N-glycans occurring in plant and animal cells (2018)
  • Author(s): Katsube, M., Abe, Y., Maeda, M., Kimura, M., Ueda, T., Kimura, Y
  • Organizer: 29th Joint Glycobiology Meeting 2018
  • Int'l Joint Research
• [Presentation] Screening of nuclear/cytoplasmic soybean lectins with glycopolymers bearing multivalent N-glycans (2018)
  • Author(s): Maeda, M., Takase, M., Els J.M. van Damme., Kimura, Y
  • Organizer: 29th Joint Glycobiology Meeting 2018
  • Int'l Joint Research
• [Presentation] ルイスa抗原含有N-グリカンを多価に結合した糖鎖ポリマーの作製と細胞性免疫活性解析 (2018)
  • Author(s): 田邉 千夏, 前田 恵, 木村 吉伸
  • Organizer: 日本生化学会大会
• [Presentation] 植物 ERAD 関連タンパク質を欠損した A. thaliana が産生する遊離N-グリカン（FNGs）の構造特性 (2018)
  • Author(s): 古田 佳織1, 前田 恵1, Strasser Richard2, 木村 吉伸
  • Organizer: 日本生化学会大会
• [Presentation] 遊離N-グリカン及びキトオリゴ糖のタンパク質フォールディング促進活性及びアミロイド形成阻害活性 (2018)
  • Author(s): 勝部諒，阿部義人，平石綾香，前田恵，植田正，木村吉伸
  • Organizer: 日本糖質学会年会
• [Presentation] 新たな酸性PNGase 活性測定系の開発と応用利用 (2018)
  • Author(s): 小椋美夏子，山本千晴，上村亮太，前田恵，木村吉伸
  • Organizer: 日本農芸化学会中四国支部講演会
• [Presentation] 酸性PNGase過剰発現トマト（T4世代）の PNGase活性と遊離糖鎖構造解析 (2018)
  • Author(s): 小椋美夏子，前田恵，木村吉伸
  • Organizer: 日本農芸化学会中四国第50回講演会（支部例会）
• [Presentation] Enzyme assay and free N-glycans structural analysis of acidic peptide:N-glycanase(aPNGase) candidate genes knockout plant. (2017)
  • Author(s): Uemura Ryota 1, Maeda Megumi 1, Akiyama Tsuyoshi 1, Misaki Ryo 2, Fujiyama Kazuhito 2, Kimura Yoshinobu
  • Organizer: 19th European Carbohydrate Symposium
  • Int'l Joint Research
• [Presentation] Protein-folding promoting activity and amyloid-formation inhibitory activity of free N-glycans (2017)
  • Author(s): Makoto Katsube1, Yoshito Abe2, Maeda Megumi1, Tatsuya Tanaka1, Tadashi Ueda2, Yoshinobu Kimura1
  • Organizer: 19th European Carbohydrate Symposium
  • Int'l Joint Research
• [Presentation] Molecular characterization Solanum lycopersicum α1,3/4-fucosidase II ( -Fuc'ase Sl-II) involved in degradation of plant complex type N-glycan (2017)
  • Author(s): Yuta Tsujimori,1 Md. Ziaur Rahman, 1,2 Megumi Maeda,1 Md. Anowar Hossain,3 Yoshinobu Kimura1
  • Organizer: 19th European Carbohydrate Symposium
  • Int'l Joint Research
• [Presentation] Significant decrease in the amount of GN1-type free N-glycans in ER- -mannosidase knocked-out A. thaliana (2017)
  • Author(s): Kaori Furuta, Megumi Maeda,Richard Strasser, Yoshinobu Kimura
  • Organizer: 12th Carbohydrate Bioengineering Meeting
  • Int'l Joint Research
• [Presentation] Glycoform of a newly found pollen allergen, Chao3, from Chamaecyparis obtusa (Japanese cypress, Hinoki) (2017)
  • Author(s): Chinatsu Tanabe 1, Megumi Maeda 1, Toshihiko Osada2, Eiji Sasaki2, Mitsuhiro Okano3, Yoshinobu Kimura1
  • Organizer: 12th Carbohydrate Bioengineering Meeting
  • Int'l Joint Research
• [Presentation] Accumulation of GN1-type plant complex type free N-glycans in α1,3/4-fucosidase knockout mutant of A. thaliana (2017)
  • Author(s): Megumi Maeda, Shun Takata, Tsuyoshi Ishimizu, Els JM Van Damme, Yoshinobu Kimura
  • Organizer: 12th Carbohydrate Bioengineering Meeting
  • Int'l Joint Research
• [Presentation] 遊離N-グリカンのタンパク質フォールディング促進活性とアミロイド形成阻害活性 (2017)
  • Author(s): 勝部諒，阿部義人，前田恵， 田中達也，植田正，木村吉伸
  • Organizer: 日本農芸化学会2017年度大会
• [Presentation] 酸性ペプチド:N-グリカナーゼ遺伝子を完全にノックアウトしたArabidopsis thaliana 中のPNGase 活性と遊離N-グリカンの行動特性 (2017)
  • Author(s): 上村亮太，前田恵，秋山剛，三崎亮，藤山和仁，木村吉伸
  • Organizer: 日本農芸化学会2017年度大会