Skip to main content
SpringerLink
Log in

Effects of N-Glycans on Glycoprotein Folding and Protein Dynamics

  • Chapter
  • First Online:
Glycobiophysics

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 1104))

Abstract

This chapter describes the folding of synthetic homogeneous glycosylpolypeptides into glycoproteins depending on the position and number of glycosylation sites and oligosaccharide structures. To evaluate the role of oligosaccharides in protein folding, we synthesized small glycoprotein models, homogeneous misfolded glycoproteins, and erythropoietins. In addition to these chemical syntheses, this chapter introduces a unique method for 15N-labeling of synthetic glycoproteins to enable structural analysis. Based on experimental results, it can be suggested that N-glycans stabilize the structure of glycoproteins.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
eBook
USD 16.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References and Note

  • Aebi M, Bernasconi R, Clerc S, Molinari M (2010) N-glycan structures: recognition and processing in the ER. Trends Biochem Sci 35(2):74–82. https://doi.org/10.1016/j.tibs.2009.10.001

    Article  CAS  PubMed  Google Scholar 

  • Anfinsen CB (1973) Principles that govern the folding of protein chains. Science 181:223–230

    Article  CAS  PubMed  Google Scholar 

  • Baldwin ET, Weber IT, St Charles R, Xuan JC, Appella E, Yamada M, Matsushima K, Edwards BF, Clore GM, Gronenborn AM (1991) Crystal structure of interleukin 8: symbiosis of NMR and crystallography. Proc Natl Acad Sci 88(2):502–506

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bazan JF, Bacon KB, Hardiman G, Wang W, Soo K, Rossi D, Greaves DR, Zlotnik A, Schall TJ (1997) CDF finding. Nature 385:640–644

    Article  CAS  PubMed  Google Scholar 

  • Culyba EK, Price JL, Hanson SR, Dhar A, Wong CH, Gruebele M, Powers ET, Kelly JW (2011) Protein native-state stabilization by placing aromatic side chains in N-glycosylated reverse turns. Science 331(6017):571–575. https://doi.org/10.1126/Science.1198461

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dawson PE, Muir TW, Clark-Lewis I, Kent SBH (1994) Synthesis of proteins by native chemical ligation. Science 266(5186):776–779

    Article  CAS  PubMed  Google Scholar 

  • Dedola S, Izumi M, Makimura Y, Seko A, Kanamori A, Sakono M, Ito Y, Kajihara Y (2014) Folding of synthetic homogeneous glycoproteins in the presence of a glycoprotein folding sensor enzyme. Angew Chem Int Ed 53(11):2883–2887. https://doi.org/10.1002/anie.201309665

    Article  CAS  Google Scholar 

  • Farrow NA, Muhandiram R, Singer AU, Pascal SM, Kay CM, Gish G, Shoelson SE, Pawson T, Forman-Kay JD, Kay LE (1994) Backbone dynamics of a free and a phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33(19):5984–6003. https://doi.org/10.1021/bi00185a040

    Article  CAS  PubMed  Google Scholar 

  • Hackenberger CPR, Friel CT, Radford SE, Imperiali B (2005) Semisynthesis of a glycosylated Im7 analogue for protein folding studies. J Am Chem Soc 127:12882–12889

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hebert DN, Bernasconi R, Molinari M (2010) ERAD substrates: which way out? Semin Cell Dev Biol 21(5):526–532

    Article  CAS  PubMed  Google Scholar 

  • Hien Minh N, Izumi M, Sato H, Okamoto R, Kajihara Y (2017) Chemical synthesis of glycoproteins with the specific installation of gradient-enriched 15N-labeled amino acids for getting insights into glycoprotein behavior. Chem Eur J 23(27):6579–6585. https://doi.org/10.1002/chem.201606049

    Article  PubMed  Google Scholar 

  • Hoover DM, Mizoue LS, Handel TM, Lubkowski J (2000) The crystal structure of the chemokine domain of fractalkine shows a novel quaternary arrangement. J Biol Chem 275:23187–23193

    Article  CAS  PubMed  Google Scholar 

  • Huang YW, Yang HI, Wu YT, Hsu TL, Lin TW, Kelly JW, Wong CH (2017) Residues comprising the enhanced aromatic sequon influence protein N-glycosylation efficiency. J Am Chem Soc 139(37):12947–12955. https://doi.org/10.1021/jacs.7b03868

    Article  CAS  PubMed  Google Scholar 

  • Izumi M, Makimura Y, Dedola S, Seko A, Kanamori A, Sakono M, Ito Y, Kajihara Y (2012) Chemical synthesis of intentionally misfolded homogeneous glycoprotein: a unique approach for the study of glycoprotein quality control. J Am Chem Soc 134(17):7238–7241. https://doi.org/10.1021/ja3013177

    Article  CAS  PubMed  Google Scholar 

  • Kajihara Y, Suzuki Y, Yamamoto N, Sasaki K, Sakakibara T, Juneja LR (2004) Prompt chemoenzymatic synthesis of diverse complex-type oligosaccharides and its application to the solid-phase synthesis of a glycopeptide with asn-linked sialyl-undeca- and asialo-nonasaccharides. Chem Eur J 10(4):971–985. https://doi.org/10.1002/chem.200305115

    Article  CAS  PubMed  Google Scholar 

  • Kajihara Y, Tanabe Y, Sasaoka S, Okamoto R (2012) Homogeneous Human Complex-Type Oligosaccharides in Correctly Folded Intact Glycoproteins: Evaluation of Oligosaccharide Influence On Protein Folding, Stability, and Conformational Properties. Chem Eur J 18(19):5944–5953

    Article  CAS  PubMed  Google Scholar 

  • Kasai T, Koshiba S, Yokoyama J, Kigawa T (2015) Stable isotope labeling strategy based on coding theory. J Biomol NMR 63(2):213–221

    Article  CAS  PubMed  Google Scholar 

  • Makimura Y, Kiuchi T, Izumi M, Dedola S, Ito Y, Kajihara Y (2012) Efficient synthesis of glycopeptide-α-thioesters with a high-mannose type oligosaccharide by means of tert-Boc-solid phase peptide synthesis. Carbohydr Res 364:41–48

    Article  CAS  PubMed  Google Scholar 

  • Martin G, Ulrich V, Manfred N, Roland W, Harald SC (1995) Characterization of changes in the glycosylation pattern of recombinant proteins from BHK-21 cells due to different culture conditions. J Biotechnol 42:117–131

    Article  Google Scholar 

  • Mizoue LS, Bazan JF, Johnson EC, Handel TM (1999) CDF NMR. Biochemistry 38:1402–1414

    Article  CAS  PubMed  Google Scholar 

  • Murakami M, Kiuchi T, Nishihara M, Tezuka K, Okamoto R, Izumi M, Kajihara Y (2016) Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity. Sci Adv 2:e1500678. https://doi.org/10.1126/sciadv.1500678

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Murakami M, Okamoto R, Izumi M, Kajihara Y (2012) Chemical Synthesis of an Erythropoietin Glycoform Containing a Complex-type Disialyloligosaccharide. Angew Chem Int Ed 51(15):3567–3572

    Article  CAS  Google Scholar 

  • Nguyen HM, Izumi M, Sato H, Okamoto R, Kajihara Y (2017) Chemical synthesis of glycoproteins with the specific installation of gradient-enriched 15N-labeled amino acids for getting insights into glycoprotein behavior. Chem Eur J 23(27):6579–6585. https://doi.org/10.1002/chem.201606049

    Article  PubMed  Google Scholar 

  • Okamoto R, Mandal K, Ling M, Luster AD, Kajihara Y, Kent SBH (2014a) Total chemical synthesis and biological activities of glycosylated and non-glycosylated forms of the chemokines CCL1 and Ser-CCL1. Angew Chem Int Ed 53(20):5188–5193. https://doi.org/10.1002/anie.201310574

    Article  CAS  Google Scholar 

  • Okamoto R, Mandal K, Sawaya MR, Kajihara Y, Yeates TO, Kent SBH (2014b) (Quasi-)racemic X-ray structures of glycosylated and non-glycosylated forms of the chemokine Ser-CCL1 prepared by total chemical synthesis. Angew Chem Int Ed 53(20):5194–5198. https://doi.org/10.1002/anie.201400679

    Article  CAS  Google Scholar 

  • Park SS, Park J, Ko J, Chen L, Meriage D, Crouse-Zeineddini J, Wong W, Kerwin BA (2009) Biochemical assessment of erythropoietin products from Asia versus US Epoetin alfa manufactured by Amgen. J Pharm Sci 98(5):1688–1699. https://doi.org/10.1002/jps.21546

    Article  CAS  PubMed  Google Scholar 

  • Phan AT, Patel DJ (2002) A site-specific low-enrichment 15N,13C isotope-labeling approach to unambiguous NMR spectral assignments in nucleic acids. J Am Chem Soc 124(7):1160–1161

    Article  CAS  PubMed  Google Scholar 

  • Price JL, Powers DL, Powers ET, Kelly JW (2011) Glycosylation of the enhanced aromatic sequon is similarly stabilizing in three distinct reverse turn contexts. Proc Natl Acad Sci U S A 108(34):14127–14132., S14127/14121-S14127/14127. https://doi.org/10.1073/pnas.1105880108

    Article  PubMed  PubMed Central  Google Scholar 

  • Sinclair AM, Elliott S (2005) Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins. J Pharm Sci 94(8):1626–1635. https://doi.org/10.1002/jps.20319

    Article  CAS  PubMed  Google Scholar 

  • Teeter MM, Mazer JA, L’Italien JJ (1981) Primary structure of the hydrophobic plant protein crambin. Biochemistry 20(19):5437–5443. https://doi.org/10.1021/bi00522a013

    Article  CAS  PubMed  Google Scholar 

  • Tirado-Rives J, Jorgensen WL (1990) Molecular dynamics of proteins with the OPLS potential functions. Simulation of the third domain of silver pheasant ovomucoid in water. J Am Chem Soc 112(7):2773–2781. https://doi.org/10.1021/ja00163a046

    Article  CAS  Google Scholar 

  • Unverzagt C, Kajihara Y (2013) Chemical assembly of N-glycoproteins: a refined toolbox to address a ubiquitous posttranslational modification. Chem Soc Rev 42(10):4408–4420

    Article  CAS  PubMed  Google Scholar 

  • Varki A (2017) Biological roles of glycans. Glycobiology 27(1):3–49. https://doi.org/10.1093/glycob/cww086

    Article  CAS  PubMed  Google Scholar 

  • Wuthrich K (1986) NMR of proteins and nucleic acids. Wiley, New York

    Book  Google Scholar 

  • Yabuki T, Kigawa T, Dohmae N, Takio K, Terada T, Ito Y, Laue ED, Cooper JA, Kainosho M, Yokoyama S (1998) Dual amino acid-selective and site-directed stable isotope labeling of the human c-Ha-Ras protein by cell-free synthesis. J Biomol NMR 11(3):295–306

    Article  CAS  PubMed  Google Scholar 

  • Yamashita K, Kamerling JP, Kobata A (1983) Structural studies of the sugar chains of hen ovomucoid. Evidence indicating that they are formed mainly by the alternate biosynthetic pathway of asparagine-linked sugar chains. J Biol Chem 258(5):3099–3106

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry, Osaka University, Toyonaka, Japan

    Yoko Amazaki, Ryo Okamoto, Yuta Maki & Yasuhiro Kajihara

  2. School of Medicine, Vietnam National University, Ho Chi Minh City, Vietnam

    Hien Minh Nguyen

Authors
  1. Yoko Amazaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hien Minh Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ryo Okamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuta Maki
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yasuhiro Kajihara
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yasuhiro Kajihara .

Editor information

Editors and Affiliations

  1. Synthetic Cellular Chemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama, Japan

    Yoshiki Yamaguchi

  2. Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Okazaki, Aichi, Japan

    Koichi Kato

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Nature Singapore Pte Ltd.

About this chapter

Check for updates. Verify currency and authenticity via CrossMark

Cite this chapter

Amazaki, Y., Nguyen, H.M., Okamoto, R., Maki, Y., Kajihara, Y. (2018). Effects of N-Glycans on Glycoprotein Folding and Protein Dynamics. In: Yamaguchi, Y., Kato, K. (eds) Glycobiophysics. Advances in Experimental Medicine and Biology, vol 1104. Springer, Singapore. https://doi.org/10.1007/978-981-13-2158-0_1

Download citation

Publish with us

Policies and ethics

Search

Navigation