Semi-synthesis of bioactive large glycoproteins

• Project/Area Number: 26248040
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Bio-related chemistry
• Research Institution: Osaka University
• Principal Investigator: Kajihara Yasuhiro 大阪大学, 理学研究科, 教授 (50275020)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥36,270,000 (Direct Cost: ¥27,900,000、Indirect Cost: ¥8,370,000); Fiscal Year 2016: ¥7,020,000 (Direct Cost: ¥5,400,000、Indirect Cost: ¥1,620,000); Fiscal Year 2015: ¥7,020,000 (Direct Cost: ¥5,400,000、Indirect Cost: ¥1,620,000); Fiscal Year 2014: ¥22,230,000 (Direct Cost: ¥17,100,000、Indirect Cost: ¥5,130,000)
• Keywords: 糖鎖関連化学 / 糖鎖 / 糖タンパク質 / シアル酸転移酵素 / 糖 / タンパク質 / 抗体 / レクチン / 半化学合成 / 化学合成

Outline of Final Research Achievements

In order to synthesize large bioactive glycoproteins, we examined semisyntheses employing synthesis of glycopeptide by solid phase peptide synthesis (SPPS) and synthesis of long polypeptide in E. Coli expression. Glycopeptide-thioesters bearing human biantennary complex type or highmannose type oligosaccharides were synthesized by Boc-SPPS. Peptide expression in E. Coli was designed to yield either HisTag-Met-target-Cys-peptide or HisTag-ENLYFQ-target-Cys-peptide. These peptides prepared in E. Coli were converted into corresponding Cys-peptides by CNBr treatment or TEV-protease. Native chemical ligation of these peptides and glycopeptide-thioesters yielded Fc domain of human-antibody and sialyltransferase. Folding experiments successfully yielded enzymatically active sialyltransferase. These trials proved that large bioactive glycoprotein consisting over 300 amino acids can be prepared by chemical synthesis.

Research Products

• [Journal Article] Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity (2016)
  • Author(s): Masumi Murakami, Tatsuto Kiuchi, Mika Nishihara, Katsunari Tezuka, Ryo Okamoto, Masayuki Izumi, Yasuhiro Kajihara
  • Journal Title: Science Advances Volume: 2
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Semi-synthesis of intact complex-type triantennary oligosaccharides from a biantennary oligosaccha-ride isolated from a natural source by selective chemical and enzymatic glycosylation (2016)
  • Author(s): Yuta Maki,Ryo Okamoto, Masayuki Izumi, Takefumi Murase, Yasuhiro Kajihara
  • Journal Title: J. Am. Chem. Soc Volume: 138 Issue: 10 Pages: 3461-3468
  • DOI: 10.1021/jacs.5b13098
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Synthesis of Glc1Man9-glycoprotein probes by a unique misfolding-enzymatic glucosylation-intentional misfolding approach (2016)
  • Author(s): Masayuki Izumi, Yukiho Oka, Ryo Okamoto, Akira Seko, Yoichi Takeda, Yukishige Ito, Yasuhiro Kajihara
  • Journal Title: Angew. Chem. Int. Ed. Volume: 128 Issue: 12 Pages: 4036-4039
  • DOI: 10.1002/ange.201511491
  • Peer Reviewed
• [Journal Article] Chemical Synthesis of a Synthetic Analogue of the Sialic Acid-Binding Lectin Siglec-7 (2014)
  • Author(s): M. Izumi, A. Otsuki, M. Nishihara, R. Okamoto, Y. Kajihara.
  • Journal Title: ChemBioChem Volume: 15 Issue: 17 Pages: 2503-2507
  • DOI: 10.1002/cbic.201402494
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Semisynthesis of a Post-Translationally Modified Protein by Using Chemical Cleavage and Activation of an Expressed Fusion Polypeptide (2014)
  • Author(s): R. Okamoto, M. Kimura, T. Ishimizu, M. Izumi, Y. Kajihara
  • Journal Title: Chemistry A European Journal Volume: 20 Issue: 33 Pages: 10425-10430
  • DOI: 10.1002/chem.201403035
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Decoration of proteins with sugar chains: recent advances in glycoprotein synthesis (2014)
  • Author(s): R. Okamoto, M. Izumi, Y. Kajihara
  • Journal Title: Current Opinion in Chemical Biology Volume: 22 Pages: 92-99
  • DOI: 10.1016/j.cbpa.2014.09.029
  • Peer Reviewed
• [Journal Article] Efficient synthesis of polypeptide-α-thioester by the method combining polypeptide expression and chemical activation for the semi-synthesis of interferon-γ having oligosaccharides (2014)
  • Author(s): Y. Kajihara, Y. Kanemitsu, M. Nishihara, R. Okamoto, M Izumi
  • Journal Title: J. Peptide Science Volume: 20 Issue: 12 Pages: 958-963
  • DOI: 10.1002/psc.2709
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] ハイマンノース型糖鎖を有するシアル酸転移酵素の半化学合成研究 (2017)
  • Author(s): 島田 有彩、岡本 亮、和泉 雅之、梶原 康宏
  • Organizer: 日本化学会 第97春季年会
  • Place of Presentation: 横浜 慶応大学日吉校
  • Year and Date: 2017-03-16
• [Presentation] Semi-synthesis of α2,6-sialyltransferase bearing high-mannose type oligosaccharide (2016)
  • Author(s): Arisa Shimada、Ryo Okamoto、Masayuki Izumi、Yasuhiro Kajihara
  • Organizer: International Carbohydrate Symposium 2016
  • Place of Presentation: New Orleans、USA
  • Year and Date: 2016-07-17
• [Presentation] Chemical Synthesis of homogeneous glycoprotein and insight into the relationship between glycosylation pattern and biological activity (2015)
  • Author(s): Yasuhiro Kajihara
  • Organizer: Pacifichem 2015, session: Strategies for Coupling and Decoupling Diverse Molecular Units in the Glycosciences
  • Place of Presentation: ホノルル ハワイ アメリカ
  • Year and Date: 2015-12-16
  • Int'l Joint Research / Invited
• [Presentation] Chemical synthesis of correctly folded and misfolded glycoproteins for understanding of glycoprotein quality control system (2015)
  • Author(s): Yasuhiro Kajihara
  • Organizer: Pacifichem 2015, session: Carbohydrate Recognition in Health and Disease
  • Place of Presentation: ホノルル ハワイ アメリカ
  • Year and Date: 2015-12-16
  • Int'l Joint Research / Invited
• [Presentation] Chemical Synthesis of homogeneous glycoprotein and insight into the relationship between glycosylation pattern and biological activity (2015)
  • Author(s): Yasuhiro Kajihara
  • Organizer: Pacifichem, session: Advances in Peptide and Protein Chemistry
  • Place of Presentation: ホノルル ハワイ アメリカ
  • Year and Date: 2015-12-16
  • Int'l Joint Research / Invited
• [Presentation] Chemical Synthesis of homogeneous glycoprotein and insight into the relationship between glycosylation pattern and biological activity (2015)
  • Author(s): Yasuhiro Kajihara
  • Organizer: 5 th Modern solid Peptide Symposium
  • Place of Presentation: Ramada Couran Cove Island Resort, Australia
  • Year and Date: 2015-10-22
  • Invited
• [Presentation] Chemical synthesis of homogeneous erythropoietins (2015)
  • Author(s): Yasuhiro Kajihara
  • Organizer: Bachem Spring symposium
  • Place of Presentation: バーゼル スイス
  • Year and Date: 2015-04-23
  • Int'l Joint Research / Invited
• [Presentation] A new approach for the semisynthesis of N-glycosylated proteins using chemical thioesterification reaction via peptide-α-guanidide (2014)
  • Author(s): Ryo Okamoto, Motoharu, Kimura, Masayuki Izumi, Yasuhiro Kajihara
  • Organizer: Asian Community of glycoscience and glycotechnology
  • Place of Presentation: Hyderabad, India
  • Year and Date: 2014-12-09 – 2014-12-12
• [Presentation] A synthetic study of a homogeneous hematopoietic glycoprotein bearing three di-branched sialyloligosaccharides (2014)
  • Author(s): Masumi Murakami, Ryo Okamoto, Masayuki Izumi, Yasuhiro Kajihara
  • Organizer: US & Japan joint Glycobiology meeting
  • Place of Presentation: Honolulu, Hawaii
  • Year and Date: 2014-11-16 – 2014-11-18
• [Presentation] 修飾可能なシステイン残基を有するSiglec-7の化学合成とその糖鎖結合活性 (2014)
  • Author(s): 和泉雅之、大槻晃久、西原三佳、岡本 亮、梶原康宏
  • Organizer: 33回日本糖質学会
  • Place of Presentation: 名古屋大学
  • Year and Date: 2014-08-10 – 2014-08-12
• [Presentation] Chemical Synthesis of Homogeneous Glycoproteins: Elucidation of Oligosaccharide Functions on Protein Surface (2014)
  • Author(s): 梶原康宏
  • Organizer: 13th Chinese International peptide symposium, China Datong
  • Place of Presentation: Datong, China
  • Year and Date: 2014-06-30 – 2014-07-04
  • Invited
• [Presentation] Chemical synthesis of homogeneous glycoproteins: Elucidation of oligosaccharide functions on protein surface (2014)
  • Author(s): 梶原康宏
  • Organizer: Asia-Canada Glycoscience Satellite Meeting
  • Place of Presentation: Vancouver, Canada
  • Year and Date: 2014-05-31 – 2014-06-01
  • Invited
• [Book] Glycochemical synthesis, chapter 11 (2016)
  • Author(s): Yasuhiro Kajihara, Masumi Murakami, Carlo Unverzagt
  • Total Pages: 550
  • Publisher: Wiley
• [Book] 「糖鎖の新機能開発・応用ハンドブック」第４編第２章第４節「糖タンパク質合成」 (2015)
  • Author(s): 和泉雅之、伊藤幸成、梶原康宏
  • Total Pages: 2
  • Publisher: 株式会社エヌティーエス
• [Book] 糖タンパク質の品質管理の仕組み (2015)
  • Author(s): 和泉雅之、伊藤幸成、梶原康宏
  • Total Pages: 5
  • Publisher: 東京化学同人 現代化学