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NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic

Abstract

The N-glycan at Asn297 of the immunoglobulin G Fc fragment modulates cellular responses of the adaptive immune system. However, the underlying mechanism remains undefined, as existing structural data suggest the glycan does not directly engage cell surface receptors. Here we characterize the dynamics of the glycan termini using solution NMR spectroscopy. Contrary to previous conclusions based on X-ray crystallography and limited NMR data, our spin relaxation studies indicate that the termini of both glycan branches are highly dynamic and experience considerable motion in addition to tumbling of the Fc molecule. Relaxation dispersion and temperature-dependent chemical shift perturbations demonstrate exchange of the α1-6Man-linked branch between a protein-bound and a previously unobserved unbound state, suggesting the glycan samples conformational states that can be accessed by glycan-modifying enzymes and possibly glycan recognition domains. These findings suggest a role for Fc-glycan dynamics in Fc-receptor interactions and enzymatic glycan remodeling.

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Figure 1: Immunoglobulin G contains an N-linked glycan at Asn297.
Figure 2: A two-dimensional 13C-1H HMQC correlation spectrum of 13C-galactose-labeled IgG1 and IgG1 Fab fragment.
Figure 3: Two-dimensional 13C-HMQC spectra and assignments of 13C-galactose-labeled IgG Fc.
Figure 4: 13C spin relaxation measurements of galactose resonances.
Figure 5: Relaxation dispersion and temperature-dependent chemical shift measurements show evidence of two states.
Figure 6: Models for Fc glycan dynamics and accessibility showing that exposed glycan conformations are possible.

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References

  1. Roitt, I.M., Brostoff, J. & Male, D.K. Immunology 6th edn. (Mosby, 2001).

  2. Goronzy, J.J. & Weyand, C.M. Developments in the scientific understanding of rheumatoid arthritis. Arthritis Res. Ther. 11, 249 (2009).

    Article  Google Scholar 

  3. Arnold, J.N. et al. The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu. Rev. Immunol. 25, 21–50 (2007).

    Article  CAS  Google Scholar 

  4. Alavi, A. & Axford, J.S. Sweet and sour: the impact of sugars on disease. Rheumatology 47, 760–770 (2008).

    Article  CAS  Google Scholar 

  5. Parekh, R.B. et al. Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature 316, 452–457 (1985).

    Article  CAS  Google Scholar 

  6. Kaneko, Y., Nimmerjahn, F., Madaio, M.P. & Ravetch, J.V. Pathology and protection in nephrotoxic nephritis is determined by selective engagement of specific Fc receptors. J. Exp. Med. 203, 789–797 (2006).

    Article  CAS  Google Scholar 

  7. Anthony, R.M. et al. Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 320, 373–376 (2008).

    Article  CAS  Google Scholar 

  8. Malhotra, R. et al. Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nat. Med. 1, 237–243 (1995).

    Article  CAS  Google Scholar 

  9. Yamaguchi, Y. et al. Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy. Biochim. Biophys. Acta 1760, 693–700 (2006).

    Article  CAS  Google Scholar 

  10. Mimura, Y. et al. Role of oligosaccharide residues of IgG1-Fc in Fc γ RIIb binding. J. Biol. Chem. 276, 45539–45547 (2001).

    Article  CAS  Google Scholar 

  11. Scallon, B.J. et al. Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality. Mol. Immunol. 44, 1524–1534 (2007).

    Article  CAS  Google Scholar 

  12. Anthony, R.M., Wermeling, F., Karlsson, M.C. & Ravetch, J.V. Identification of a receptor required for the anti-inflammatory activity of IVIG. Proc. Natl. Acad. Sci. USA 105, 19571–19578 (2008).

    Article  CAS  Google Scholar 

  13. Burmeister, W.P., Huber, A.H. & Bjorkman, P.J. Crystal structure of the complex of rat neonatal Fc receptor with Fc. Nature 372, 379–383 (1994).

    Article  CAS  Google Scholar 

  14. Sondermann, P., Huber, R., Oosthuizen, V. & Jacob, U. The 3.2-A crystal structure of the human IgG1 Fc fragment-Fc γRIII complex. Nature 406, 267–273 (2000).

    Article  CAS  Google Scholar 

  15. Radaev, S. et al. The structure of a human type III Fcγ receptor in complex with Fc. J. Biol. Chem. 276, 16469–16477 (2001).

    Article  CAS  Google Scholar 

  16. Idusogie, E.E. et al. Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc. J. Immunol. 164, 4178–4184 (2000).

    Article  CAS  Google Scholar 

  17. Deisenhofer, J. 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-Å resolution. Biochemistry 20, 2361–2370 (1981).

    Article  CAS  Google Scholar 

  18. Yamaguchi, Y. et al. Dynamics of the carbohydrate chains attached to the Fc portion of immunoglobulin G as studied by NMR spectroscopy assisted by selective C-13 labeling of the glycans. J. Biomol. NMR 12, 385–394 (1998).

    Article  CAS  Google Scholar 

  19. Wormald, M.R. et al. Variations in oligosaccharide-protein interactions in immunoglobulin G determine the site-specific glycosylation profiles and modulate the dynamic motion of the Fc oligosaccharides. Biochemistry 36, 1370–1380 (1997).

    Article  CAS  Google Scholar 

  20. Kobata, A. The N-linked sugar chains of human immunoglobulin G: their unique pattern, and their functional roles. Biochim. Biophys. Acta 1780, 472–478 (2008).

    Article  CAS  Google Scholar 

  21. Barb, A.W., Brady, E.K. & Prestegard, J.H. Branch-specific sialylation of IgG-Fc glycans by ST6Gal-I. Biochemistry 48, 9705–9707 (2009).

    Article  CAS  Google Scholar 

  22. Mittermaier, A. & Kay, L.E. New tools provide new insights in NMR studies of protein dynamics. Science 312, 224–228 (2006).

    Article  CAS  Google Scholar 

  23. Chang, V.T. et al. Glycoprotein structural genomics: solving the glycosylation problem. Structure 15, 267–273 (2007).

    Article  CAS  Google Scholar 

  24. Barb, A.W. et al. Intramolecular glycan-protein interactions in glycoproteins. Methods Enzymol. 477, 365–388 (2010).

    Article  Google Scholar 

  25. Cavanagh, J. Protein NMR Spectroscopy: Principles and Practice 2nd edn. (Academic Press, 2007).

  26. Palmer, A.G. III, Kroenke, C.D. & Loria, J.P. Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Methods Enzymol. 339, 204–238 (2001).

    Article  CAS  Google Scholar 

  27. Hansen, D.F. et al. Probing chemical shifts of invisible states of proteins with relaxation dispersion NMR spectroscopy: how well can we do? J. Am. Chem. Soc. 130, 2667–2675 (2008).

    Article  CAS  Google Scholar 

  28. Raju, T.S. Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr. Opin. Immunol. 20, 471–478 (2008).

    Article  CAS  Google Scholar 

  29. Bock, K., Pedersen, C. & Pedersen, H. Carbon-13 nuclear magnetic resonance data for oligosaccharides. Adv. Carbohydr. Chem. Biochem. 42, 193–225 (1984).

    Article  CAS  Google Scholar 

  30. Wieruszeski, J.M., Michalski, J.C., Montreuil, J. & Strecker, G. Sequential H-1 and C-13 resonance assignments for an octasaccharide and decasaccharide of the N-acetyllactosamine type by multiple-step relayed correlation and hetero-nuclear correlation nuclear magnetic-resonance. Glycoconj. J. 6, 183–194 (1989).

    Article  CAS  Google Scholar 

  31. Vliegenthart, J.F.G., Dorland, L. & van Halbeek, H. High-resolution, 1H-nuclear magnetic resonance spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins. Adv. Carbohydr. Chem. Biochem. 41, 209–374 (1983).

    Article  CAS  Google Scholar 

  32. Voynov, V. et al. Dynamic fluctuations of protein-carbohydrate interactions promote protein aggregation. PLoS ONE 4, e8425 (2009).

    Article  Google Scholar 

  33. Lipari, G. & Szabo, A. Model-free approach to the interpretation of nuclear magnetic-resonance relaxation in macromolecules. 1. Theory and range of validity. J. Am. Chem. Soc. 104, 4546–4559 (1982).

    Article  CAS  Google Scholar 

  34. Lipari, G. & Szabo, A. Model-free approach to the interpretation of nuclear magnetic-resonance relaxation in macromolecules. 2. Analysis of experimental results. J. Am. Chem. Soc. 104, 4559–4570 (1982).

    Article  CAS  Google Scholar 

  35. Raju, T.S. et al. Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues. Biochemistry 40, 8868–8876 (2001).

    Article  CAS  Google Scholar 

  36. Lund, J. et al. Multiple interactions of IgG with its core oligosaccharide can modulate recognition by complement and human Fc γ receptor I and influence the synthesis of its oligosaccharide chains. J. Immunol. 157, 4963–4969 (1996).

    CAS  PubMed  Google Scholar 

  37. DeLano, W.L., Ultsch, M.H., de Vos, A.M. & Wells, J.A. Convergent solutions to binding at a protein-protein interface. Science 287, 1279–1283 (2000).

    Article  CAS  Google Scholar 

  38. Hirotsu, K. & Shimada, A. Crystal and molecular-structure of β-lactose. Bull. Chem. Soc. Jpn. 47, 1872–1879 (1974).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank T. Yang and M. Bar-Peled for the galactokinase, Y. Liu for discussions of dynamics and pulse sequences and E. Brady for preparing the UDP-13C-galactose, all of the University of Georgia. This research was funded by grants from the US National Institutes of Health (R01GM033225 and P41RR005351). A.W.B. was supported by a Kirschstein National Research Service Award fellowship (F32AR058084).

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A.W.B. and J.H.P. designed experiments, analyzed data and wrote the manuscript. A.W.B. carried out experiments.

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Correspondence to James H Prestegard.

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Supplementary Methods, Supplementary Figures 1–3 and Supplementary Table 1 (PDF 232 kb)

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Barb, A., Prestegard, J. NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic. Nat Chem Biol 7, 147–153 (2011). https://doi.org/10.1038/nchembio.511

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