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N-Glycosylation Fingerprinting of Viral Glycoproteins by xCGE-LIF

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Carbohydrate-Based Vaccines

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1331))

Abstract

The ongoing threat of pathogens, increasing resistance against antibiotics, and the risk of fast spreading of infectious diseases in a global community resulted in an intensified development of vaccines. Antigens used for vaccination comprise a wide variety of macromolecules including glycoproteins, lipopolysaccharides, and complex carbohydrates. For all of these antigens the sugar composition plays a crucial role for immunogenicity and protective efficacy of the vaccine. Here, we provide a protocol for N-glycosylation fingerprinting utilizing high performance multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) technology. The method described, enables to analyze the N-glycosylation of specific proteins out of a complex sample or even the total of all N-glycans contained in such a sample. The protocol is exemplarily demonstrated for N-glycosylation fingerprinting of cell culture-derived influenza A and B viruses and their major antigens, the membrane glycoproteins hemagglutinin and neuraminidase.

*Both authors contributed equally

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References

  1. Aich U, Yarema KJ (2008) Glycobiology and immunology. In: Carbohydrate-based vaccines and immunotherapies. Wiley, Hoboken, NJ, pp 1–53

    Google Scholar 

  2. Pon RA, Jennings HJ (2008) Carbohydrate-based antibacterial vaccines. In: Carbohydrate-based vaccines and immunotherapies. Wiley, Hoboken, NJ, pp 117–166

    Google Scholar 

  3. Astronomo RD, Burton DR (2010) Carbohydrate vaccines: developing sweet solutions to sticky situations? Nat Rev Drug Discov 9(4):308–324

    Article  CAS  PubMed  Google Scholar 

  4. Ruhaak LR, Zauner G, Huhn C, Bruggink C, Deelder AM, Wuhrer M (2010) Glycan labeling strategies and their use in identification and quantification. Anal Bioanal Chem 397(8):3457–3481

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Vanderschaeghe D, Festjens N, Delanghe J et al (2010) Glycome profiling using modern glycomics technology: technical aspects and applications. Biol Chem 391(2-3):149–161

    CAS  PubMed  Google Scholar 

  6. Seeberger PH, Werz DB (2007) Synthesis and medical applications of oligosaccharides. Nature 446(7139):1046–1051

    Article  CAS  PubMed  Google Scholar 

  7. Wang P, Dong S, Shieh JH et al (2013) Erythropoietin derived by chemical synthesis. Science 342(6164):1357–1360

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Meuris L, Santens F, Elson G et al (2014) GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins. Nat Biotechnol 32(5):485–489

    Article  CAS  PubMed  Google Scholar 

  9. Kanda Y, Yamane-Ohnuki N, Sakai N et al (2006) Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC. Biotechnol Bioeng 94(4):680–688

    Article  CAS  PubMed  Google Scholar 

  10. Callewaert N, Laroy W, Cadirgi H et al (2001) Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-α-D-mannosidase for N-glycan engineering in Pichia pastoris. FEBS Lett 503(2–3):173–178

    Article  CAS  PubMed  Google Scholar 

  11. Hamilton SR, Davidson RC, Sethuraman N et al (2006) Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313(5792):1441–1443

    Article  CAS  PubMed  Google Scholar 

  12. Jacobs PP, Geysens S, Vervecken W et al (2008) Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology. Nat Protoc 4(1):58–70

    Article  Google Scholar 

  13. Aumiller JJ, Mabashi-Asazuma H, Hillar A et al (2012) A new glycoengineered insect cell line with an inducibly mammalianized protein N-glycosylation pathway. Glycobiology 22(3):417–428

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Cox MMJ (2012) Recombinant protein vaccines produced in insect cells. Vaccine 30(10):1759–1766

    Article  CAS  PubMed  Google Scholar 

  15. Abe Y, Takashita E, Sugawara K et al (2004) Effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza A/H3N2 virus hemagglutinin. J Virol 78(18):9605–9611

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Klenk HD, Wagner R, Heuer D et al (2002) Importance of hemagglutinin glycosylation for the biological functions of influenza virus. Virus Res 82(1-2):73–75

    Article  CAS  PubMed  Google Scholar 

  17. Vigerust DJ, Shepherd VL (2007) Virus glycosylation: role in virulence and immune interactions. Trends Microbiol 15(5):211–218

    Article  CAS  PubMed  Google Scholar 

  18. Swarts BM, Guo Z (2008) Carbohydrate-based antiviral vaccines. In: Carbohydrate-based vaccines and immunotherapies. Wiley, Hoboken, NJ, pp 167–193

    Google Scholar 

  19. Wolfert MA, Boons G-J (2013) Adaptive immune activation: glycosylation does matter. Nat Chem Biol 9(12):776–784

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Avci FY, Li X, Tsuji M et al (2011) A mechanism for glycoconjugate vaccine activation of the adaptive immune system and its implications for vaccine design. Nat Med 17(12):1602–1609

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. de Vries RP, Smit CH, de Bruin E et al (2012) Glycan-dependent immunogenicity of recombinant soluble trimeric hemagglutinin. J Virol 86(21):11735–11744

    Article  PubMed Central  PubMed  Google Scholar 

  22. Hütter J, Rödig J, Höper D et al (2013) Toward animal cell culture-based influenza vaccine design: viral hemagglutinin N-glycosylation markedly impacts immunogenicity. J Immunol 190(1):220–230

    Article  PubMed  Google Scholar 

  23. Kailemia MJ, Ruhaak LR, Lebrilla CB et al (2014) Oligosaccharide analysis by mass spectrometry: a review of recent developments. Anal Chem 86(1):196–212

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Wuhrer M, de Boer AR, Deelder AM (2009) Structural glycomics using hydrophilic interaction chromatography (HILIC) with mass spectrometry. Mass Spectrom Rev 28(2):192–206

    Article  CAS  PubMed  Google Scholar 

  25. Harvey DJ (2005) Proteomic analysis of glycosylation: structural determination of N- and O-linked glycans by mass spectrometry. Expert Rev Proteomics 2(1):87–101

    Article  CAS  PubMed  Google Scholar 

  26. Roth Z, Yehezkel G, Khalaila I (2012) Identification and quantification of protein glycosylation. Int J Carbohydr Chem 2012(2012):10

    Google Scholar 

  27. Anumula KR (2006) Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem 350(1):1–23

    Article  CAS  PubMed  Google Scholar 

  28. Laroy W, Contreras R, Callewaert N (2006) Glycome mapping on DNA sequencing equipment. Nat Protoc 1(1):397–405

    Article  CAS  PubMed  Google Scholar 

  29. Raman R, Raguram S, Venkataraman G et al (2005) Glycomics: an integrated systems approach to structure-function relationships of glycans. Nat Methods 2(11):817–824

    Article  CAS  PubMed  Google Scholar 

  30. Huffman JE, Pučić-Baković M, Klarić L et al (2014) Comparative performance of four methods for high-throughput glycosylation analysis of immunoglobulin g in genetic and epidemiological research. Mol Cell Proteomics 13(6):1598–1610

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Lin G, Simmons G, Pohlmann S et al (2003) Differential N-linked glycosylation of human immunodeficiency virus and Ebola virus envelope glycoproteins modulates interactions with DC-SIGN and DC-SIGNR. J Virol 77(2):1337–1346

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Lin S-C, Jan J-T, Dionne B et al (2013) Different immunity elicited by recombinant H5N1 hemagglutinin proteins containing pauci-mannose, high-mannose, or complex type N-glycans. PLoS One 8(6):e66719

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Geyer R, Diabaté S, Geyer H et al (1987) Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus, strain Dutch. Glycoconj J 4(1):17–32

    Article  CAS  Google Scholar 

  34. Mir-Shekari SY, Ashford DA, Harvey DJ et al (1997) The glycosylation of the influenza A virus hemagglutinin by mammalian cells. A site-specific study. J Biol Chem 272(7):4027–4036

    Article  CAS  PubMed  Google Scholar 

  35. Yagi H, Watanabe S, Suzuki T et al (2012) Comparative analyses of N-glycosylation profiles of influenza A viruses grown in different host cells. Open Glycosci 5(1):2–12

    Article  CAS  Google Scholar 

  36. Bateman AC, Karamanska R, Busch MG et al (2010) Glycan analysis and influenza A virus infection of primary swine respiratory epithelial cells: the importance of NeuAc 2-6 glycans. J Biol Chem 285(44):34016–34026

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Doores KJ, Bonomelli C, Harvey DJ et al (2010) Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens. Proc Natl Acad Sci U S A 107(31):13800–13805

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Schwarzer J, Rapp E, Reichl U (2008) N-glycan analysis by CGE–LIF: profiling influenza A virus hemagglutinin N-glycosylation during vaccine production. Electrophoresis 29(20):4203–4214

    Article  CAS  PubMed  Google Scholar 

  39. Schwarzer J, Rapp E, Hennig R et al (2009) Glycan analysis in cell culture-based influenza vaccine production: influence of host cell line and virus strain on the glycosylation pattern of viral hemagglutinin. Vaccine 27(32):4325–4336

    Article  CAS  PubMed  Google Scholar 

  40. Rödig J, Rapp E, Höper D et al (2011) Impact of host cell line adaptation on quasispecies composition and glycosylation of influenza A virus hemagglutinin. PLoS One 6(12):e27989

    Article  Google Scholar 

  41. Rödig J, Rapp E, Bohne J et al (2013) Impact of cultivation conditions on N-glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture. Biotechnol Bioeng 110(6):1691–1703

    Article  PubMed  Google Scholar 

  42. Genzel Y, Rödig J, Rapp E et al (2014) Vaccine production: upstream processing with adherent or suspension cell lines. Methods Mol Biol 1104:371–393

    Article  CAS  PubMed  Google Scholar 

  43. Ruhaak LR, Hennig R, Huhn C et al (2010) Optimized workflow for preparation of APTS-labeled N-glycans allowing high-throughput analysis of human plasma glycomes using 48-channel multiplexed CGE-LIF. J Proteome Res 9(12):6655–6664

    Article  CAS  PubMed  Google Scholar 

  44. Hennig R, Reichl U, Rapp E (2011) A software tool for automated high-throughput processing of CGE-LIF based glycoanalysis data, generated by a multiplexing capillary DNA sequencer. Glycoconj J 28:331

    Google Scholar 

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Acknowledgements

This work was supported by the Max Planck Society and by the European Union’s Seventh Framework Programme (FP7-Health-F5-2011) under grant agreement no. 278535 “HighGlycan.”

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    Authors and Affiliations

    1. Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse1, 39106, Magdeburg, Germany

      René Hennig, Erdmann Rapp, Robert Kottler, Samanta Cajic & Udo Reichl

    2. glyXera GmbH, Leipziger Straße 44, 39120, Magdeburg, Germany

      René Hennig, Erdmann Rapp, Samanta Cajic & Matthias Borowiak

    3. Otto-von-Guericke University, Universitätsplatz 2, 39106, Magdeburg, Germany

      Udo Reichl

    Authors
    1. René Hennig
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    2. Erdmann Rapp
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    3. Robert Kottler
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    4. Samanta Cajic
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    5. Matthias Borowiak
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    6. Udo Reichl
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    Correspondence to Erdmann Rapp .

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    1. Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine, Hannover, Germany

      Bernd Lepenies

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    Hennig, R., Rapp, E., Kottler, R., Cajic, S., Borowiak, M., Reichl, U. (2015). N-Glycosylation Fingerprinting of Viral Glycoproteins by xCGE-LIF. In: Lepenies, B. (eds) Carbohydrate-Based Vaccines. Methods in Molecular Biology, vol 1331. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2874-3_8

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    • DOI: https://doi.org/10.1007/978-1-4939-2874-3_8

    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-2873-6

    • Online ISBN: 978-1-4939-2874-3

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