Advertisement

Measurement of Neutral and Sialylated IgG n-Glycome at Asn-297 by CE-LIF to Assess Hypogalactosylation in Rheumatoid Arthritis

  • Christian Schwedler
  • Véronique BlanchardEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1972)

Abstract

Modulations in immunoglobulin G (IgG) n-glycosylation have been observed in many human diseases including chronic inflammatory diseases such as rheumatoid arthritis and also cancer. In this chapter, we describe how to determine hypogalactosylation for clinical samples, namely the sample preparation of IgG n-glycans at Asn-297 as well as the measurement of neutral and sialylated n-glycans by capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF).

This semiautomated protocol describes the isolation polyclonal antibodies from serum, the separation of IgG-Fc glycopeptides from IgG antigen-binding fragment by pepsin digestion. Afterward, enzymatically released IgG-Fc n-glycans are cleaned up using a polyaromatic adsorbent resin followed by carbon purification. Sialic acids are then derivatized prior to glycan labeling. As a result, the agalactosylated n-glycan A2 does not co-migrate with sialylated n-glycans, which refines the measurement of hypogalactosylation by CE-LIF.

Key words

Immunoglobulin G n-Glycan analysis Asn297 Capillary electrophoresis Methylamidation Glycan biomarker 

Notes

Acknowledgements

Authors acknowledge Peggy Thiele for her technical assistance.

References

  1. 1.
    Ohtsubo K, Marth JD (2006) Glycosylation in cellular mechanisms of health and disease. Cell 126(5):855–867CrossRefGoogle Scholar
  2. 2.
    Kim YJ, Varki A (1997) Perspectives on the significance of altered glycosylation of glycoproteins in cancer. Glycoconj J 14(5):569–576CrossRefGoogle Scholar
  3. 3.
    Lau KS, Dennis JW (2008) N-Glycans in cancer progression. Glycobiology 18(10):750–760CrossRefGoogle Scholar
  4. 4.
    Bondt A, Selman MH, Deelder AM, Hazes JM, Willemsen SP, Wuhrer M, Dolhain RJ (2013) Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation. J Proteome Res 12(10):4522–4531CrossRefGoogle Scholar
  5. 5.
    Van Beneden K, Coppieters K, Laroy W, De Keyser F, Hoffman IE, Van den Bosch F, Vander Cruyssen B, Drennan M, Jacques P, Rottiers P et al (2009) Reversible changes in serum immunoglobulin galactosylation during the immune response and treatment of inflammatory autoimmune arthritis. Ann Rheum Dis 68(8):1360–1365CrossRefGoogle Scholar
  6. 6.
    Nimmerjahn F, Ravetch JV (2007) Fc-receptors as regulators of immunity. Adv Immunol 96:179–204CrossRefGoogle Scholar
  7. 7.
    Deisenhofer J (1981) Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry 20(9):2361–2370CrossRefGoogle Scholar
  8. 8.
    Rudd PM, Leatherbarrow RJ, Rademacher TW, Dwek RA (1991) Diversification of the IgG molecule by oligosaccharides. Mol Immunol 28(12):1369–1378CrossRefGoogle Scholar
  9. 9.
    Stanley P, Schachter H, Taniguchi N (2009) N-Glycans. In: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME (eds) Essentials of glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  10. 10.
    Parekh RB, Dwek RA, Sutton BJ, Fernandes DL, Leung A, Stanworth D, Rademacher TW, Mizuochi T, Taniguchi T, Matsuta K et al (1985) Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature 316(6027):452–457CrossRefGoogle Scholar
  11. 11.
    Arnold JN, Wormald MR, Sim RB, Rudd PM, Dwek RA (2007) The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu Rev Immunol 25:21–50CrossRefGoogle Scholar
  12. 12.
    Kobata A (2008) The N-linked sugar chains of human immunoglobulin G: their unique pattern, and their functional roles. Biochim Biophys Acta 1780(3):472–478CrossRefGoogle Scholar
  13. 13.
    Albrecht S, Unwin L, Muniyappa M, Rudd PM (2014) Glycosylation as a marker for inflammatory arthritis. Cancer Biomark 14(1):17–28CrossRefGoogle Scholar
  14. 14.
    Shields RL, Lai J, Keck R, O'Connell LY, Hong K, Meng YG, Weikert SH, Presta LG (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J Biol Chem 277(30):26733–26740CrossRefGoogle Scholar
  15. 15.
    Niwa R, Natsume A, Uehara A, Wakitani M, Iida S, Uchida K, Satoh M, Shitara K (2005) IgG subclass-independent improvement of antibody-dependent cellular cytotoxicity by fucose removal from Asn297-linked oligosaccharides. J Immunol Methods 306(1–2):151–160CrossRefGoogle Scholar
  16. 16.
    Nimmerjahn F, Anthony RM, Ravetch JV (2007) Agalactosylated IgG antibodies depend on cellular Fc receptors for in vivo activity. Proc Natl Acad Sci U S A 104(20):8433–8437CrossRefGoogle Scholar
  17. 17.
    Rook GA, Steele J, Brealey R, Whyte A, Isenberg D, Sumar N, Nelson JL, Bodman KB, Young A, Roitt IM et al (1991) Changes in IgG glycoform levels are associated with remission of arthritis during pregnancy. J Autoimmun 4(5):779–794CrossRefGoogle Scholar
  18. 18.
    Pekelharing JM, Hepp E, Kamerling JP, Gerwig GJ, Leijnse B (1988) Alterations in carbohydrate composition of serum IgG from patients with rheumatoid arthritis and from pregnant women. Ann Rheum Dis 47(2):91–95CrossRefGoogle Scholar
  19. 19.
    Parekh R, Roitt I, Isenberg D, Dwek R, Rademacher T (1988) Age-related galactosylation of the N-linked oligosaccharides of human serum IgG. J Exp Med 167(5):1731–1736CrossRefGoogle Scholar
  20. 20.
    Schwedler C, Haupl T, Kalus U, Blanchard V, Burmester GR, Poddubnyy D, Hoppe B (2018) Hypogalactosylation of immunoglobulin G in rheumatoid arthritis: relationship to HLA-DRB1 shared epitope, anticitrullinated protein antibodies, rheumatoid factor, and correlation with inflammatory activity. Arthritis Res Ther 20(1):44CrossRefGoogle Scholar
  21. 21.
    Matsumoto A, Shikata K, Takeuchi F, Kojima N, Mizuochi T (2000) Autoantibody activity of IgG rheumatoid factor increases with decreasing levels of galactosylation and sialylation. J Biochem 128(4):621–628CrossRefGoogle Scholar
  22. 22.
    Wuhrer M, Stavenhagen K, Koeleman CA, Selman MH, Harper L, Jacobs BC, Savage CO, Jefferis R, Deelder AM, Morgan M (2015) Skewed Fc glycosylation profiles of anti-proteinase 3 immunoglobulin G1 autoantibodies from granulomatosis with polyangiitis patients show low levels of bisection, galactosylation, and sialylation. J Proteome Res 14(4):1657–1665CrossRefGoogle Scholar
  23. 23.
    Tomana M, Schrohenloher RE, Koopman WJ, Alarcon GS, Paul WA (1988) Abnormal glycosylation of serum IgG from patients with chronic inflammatory diseases. Arthritis Rheum 31(3):333–338CrossRefGoogle Scholar
  24. 24.
    Saldova R, Royle L, Radcliffe CM, Abd Hamid UM, Evans R, Arnold JN, Banks RE, Hutson R, Harvey DJ, Antrobus R et al (2007) Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG. Glycobiology 17(12):1344–1356CrossRefGoogle Scholar
  25. 25.
    Kanoh Y, Mashiko T, Danbara M, Takayama Y, Ohtani S, Egawa S, Baba S, Akahoshi T (2004) Changes in serum IgG oligosaccharide chains with prostate cancer progression. Anticancer Res 24(5B):3135–3139PubMedGoogle Scholar
  26. 26.
    Schwedler C, Kaup M, Petzold D, Hoppe B, Braicu EI, Sehouli J, Ehlers M, Berger M, Tauber R, Blanchard V (2014) Sialic acid methylation refines capillary electrophoresis laser-induced fluorescence analyses of immunoglobulin G N-glycans of ovarian cancer patients. Electrophoresis 35(7):1025–1031CrossRefGoogle Scholar
  27. 27.
    Ruhaak LR, Barkauskas DA, Torres J, Cooke CL, Wu LD, Stroble C, Ozcan S, Williams CC, Camorlinga M, Rocke DM et al (2015) The serum immunoglobulin G glycosylation signature of gastric cancer. EuPA Open Proteom 6:1–9CrossRefGoogle Scholar
  28. 28.
    Mehta AS, Long RE, Comunale MA, Wang M, Rodemich L, Krakover J, Philip R, Marrero JA, Dwek RA, Block TM (2008) Increased levels of galactose-deficient anti-Gal immunoglobulin G in the sera of hepatitis C virus-infected individuals with fibrosis and cirrhosis. J Virol 82(3):1259–1270CrossRefGoogle Scholar
  29. 29.
    Moore JS, Wu X, Kulhavy R, Tomana M, Novak J, Moldoveanu Z, Brown R, Goepfert PA, Mestecky J (2005) Increased levels of galactose-deficient IgG in sera of HIV-1-infected individuals. AIDS 19(4):381–389CrossRefGoogle Scholar
  30. 30.
    de Jong SE, Selman MH, Adegnika AA, Amoah AS, van Riet E, Kruize YC, Raynes JG, Rodriguez A, Boakye D, von Mutius E et al (2016) IgG1 Fc N-glycan galactosylation as a biomarker for immune activation. Sci Rep 6:28207CrossRefGoogle Scholar
  31. 31.
    Anumula KR (2012) Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc. J Immunol Methods 382(1–2):167–176CrossRefGoogle Scholar
  32. 32.
    Mimura Y, Ashton PR, Takahashi N, Harvey DJ, Jefferis R (2007) Contrasting glycosylation profiles between Fab and Fc of a human IgG protein studied by electrospray ionization mass spectrometry. J Immunol Methods 326(1–2):116–126CrossRefGoogle Scholar
  33. 33.
    Wright A, Tao MH, Kabat EA, Morrison SL (1991) Antibody variable region glycosylation: position effects on antigen binding and carbohydrate structure. EMBO J 10(10):2717–2723CrossRefGoogle Scholar
  34. 34.
    Bondt A, Wuhrer M, Kuijper TM, Hazes JM, Dolhain RJ (2016) Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy. Arthritis Res Ther 18(1):274CrossRefGoogle Scholar
  35. 35.
    Berger M, Kaup M, Blanchard V (2012) Protein glycosylation and its impact on biotechnology. Adv Biochem Eng Biotechnol 127:165–185PubMedGoogle Scholar
  36. 36.
    Mittermayr S, Bones J, Doherty M, Guttman A, Rudd PM (2011) Multiplexed analytical glycomics: rapid and confident IgG N-glycan structural elucidation. J Proteome Res 10(8):3820–3829CrossRefGoogle Scholar
  37. 37.
    Schwedler C, Kaup M, Weiz S, Hoppe M, Braicu EI, Sehouli J, Hoppe B, Tauber R, Berger M, Blanchard V (2014) Identification of 34 N-glycan isomers in human serum by capillary electrophoresis coupled with laser-induced fluorescence allows improving glycan biomarker discovery. Anal Bioanal Chem 406(28):7185–7193CrossRefGoogle Scholar
  38. 38.
    Zhuang Z, Starkey JA, Mechref Y, Novotny MV, Jacobson SC (2007) Electrophoretic analysis of N-glycans on microfluidic devices. Anal Chem 79(18):7170–7175CrossRefGoogle Scholar
  39. 39.
    Mitra I, Snyder CM, Zhou X, Campos MI, Alley WR Jr, Novotny MV, Jacobson SC (2016) Structural characterization of serum N-glycans by methylamidation, fluorescent labeling, and analysis by microchip electrophoresis. Anal Chem 88(18):8965–8971CrossRefGoogle Scholar
  40. 40.
    Frisch E, Schwedler C, Kaup M, Iona Braicu E, Grone J, Lauscher JC, Sehouli J, Zimmermann M, Tauber R, Berger M et al (2013) Endo-beta-N-acetylglucosaminidase H de-N-glycosylation in a domestic microwave oven: application to biomarker discovery. Anal Biochem 433(1):65–69CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Charité—Universitätsmedizin Berlin, Campus Virchow KlinikumInstitut für Laboratoriumsmedizin, Klinische Chemie und PathobiochemieBerlinGermany

Personalised recommendations