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Molecular modeling of the effects of glycosylation on the structure and dynamics of human interferon-gamma

  • Elena LilkovaEmail author
  • Peicho Petkov
  • Nevena Ilieva
  • Elena Krachmarova
  • Genoveva Nacheva
  • Leandar Litov
Original Paper
  • 68 Downloads
Part of the following topical collections:
  1. 8th conference on Modeling & Design of Molecular Materials (MDMM 2018)

Abstract

Natural hIFNγ is a glycoprotein with two N-glycosylation sites in each monomer chain, which are independently and differentially glycosylated. Although glycosylation is not necessary for the activity of the cytokine, it was proposed that it protects the cytokine from proteolytic degradation and thus extends its circulatory half-life. Here, we report the development of model structures of glycosylated full-length native hIFNγ homodimers. Our aim is to shed light on the mechanism through which glycosylation preserves the integrity of the cytokine molecule. To this end, we employ molecular dynamics simulations to study the interaction of the carbohydrate chains with the receptor-binding sites in the cytokine and with its flexible highly positively charged C-termini. The glycans interact primarily with the globular part of the protein, but also occasionally form contacts with the solvent-exposed and sensitive to proteases C-terminal tails. We show that the glycans restrict the C-termini wagging motion into the solvent, limit their flexibility and keep them closer to the α-helical globule of hIFNγ, thus possibly protecting them from proteolytic processing.

Keywords

Human interferon-gamma (hIFNγGlycosylation Molecular dynamics simulations Glycan–protein interactions 

Notes

Acknowledgments

This work was supported in part under the Programme for young scientists’ career development at the Bulgarian Academy of Sciences (DFNP-17-146/2017) and under Grants DN-11/20/2017 and DNTS-Austia-01-2/2013 of the Bulgarian Science Fund.

Computational resources were provided by the HPC Cluster at the Faculty of Physics of Sofia University “St. Kl. Ohridski”.

Supplementary material

894_2019_4013_MOESM1_ESM.pdf (15.3 mb)
(PDF 15.3 MB)

References

  1. 1.
    Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, Lindahl E (2015) GROMACS: high-performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2:19–25Google Scholar
  2. 2.
    Altobelli G, Nacheva G, Todorova K, Ivanov I, Karshikoff A (2001) Role of the C-terminal chain in human interferon-γ stability: an electrostatic study. Proteins: Struct Funct Bioinf 43(2):125–133Google Scholar
  3. 3.
    Arakawa T, Hsu Y, Chang D, Stebbing N, Altrock B (1986) Structure and activity of glycosylated human interferon-gamma. J Interf Res 6(6):687–695Google Scholar
  4. 4.
    Arakawa T, Hsu Y, Yphantis D (1987) Acid unfolding and self-association of recombinant Escherichia coli-derived human interferon γ. Biochemistry 26:5428–5432PubMedGoogle Scholar
  5. 5.
    Arnold JN, Radcliffe CM, Wormald MR, Royle L, Harvey DJ, Crispin M, Dwek RA, Sim RB, Rudd PM (2004) The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan-binding lectin. J Immunol 173:6831–6840PubMedGoogle Scholar
  6. 6.
    Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81:3684Google Scholar
  7. 7.
    Bohne-Lang A, von der Lieth CW (2005) GlyProt: in silico glycosylation of proteins. Nucleic Acids Res 33 (suppl_2):W214–W219PubMedPubMedCentralGoogle Scholar
  8. 8.
    Braude I (1984) Purification of human γ-interferon to essential homogeneity and its biochemical characterization. Biochemistry 23:5603PubMedGoogle Scholar
  9. 9.
    Bussi G, Donadio D, Parrinello M (2007) Canonical sampling through velocity rescaling. J Chem Phys 126(1):014101PubMedGoogle Scholar
  10. 10.
    Campbell MP, Peterson R, Mariethoz J, Gasteiger E, Akune Y, Aoki-Kinoshita KF, Lisacek F, Packer NH UniCarbKB: building a knowledge platform for glycoproteomics. Nucleic Acids Res. 42(Database issue)Google Scholar
  11. 11.
    Damerell D, Ceroni A, Maass K, Ranzinger R, Dell A, Haslam S (2012) The GlycanBuilder and GlycoWorkbench glycoinformatics tools: updates and new developments. Biol Chem 393(11):1357–62PubMedGoogle Scholar
  12. 12.
    Damerell D, Ceroni A, Maass K, Ranzinger R, Dell A, Haslam S (2015) Annotation of glycomics MS and MS/MS spectra using the GlycoWorkbench software tool. Methods Mol Biol 1273:3–15PubMedGoogle Scholar
  13. 13.
    Dotsenko AS, Gusakov AV, Volkov PV, Rozhkova AM, Sinitsyn AP (2016) N-linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verrucosum and its effect on the enzyme activity. Biotechnol Bioeng 113(2):283–291PubMedGoogle Scholar
  14. 14.
    Dumych T, Bridot C, Gouin SG, Lensink MF, Paryzhak S, Szunerits S, Blossey R, Bilyy R, Bouckaert J, Krammer EM (2018) A novel integrated way for deciphering the glycan code for the FimH lectin. Molecules 23(11):2794PubMedCentralGoogle Scholar
  15. 15.
    Ealick S, Cook WJ, Vijay-Kumar S, Carson M, Nagabhushan TL, Trotta PP, Bugg CE (1991) Three-dimensional structure of recombinant human interferon-γ. Science 252:698–702PubMedGoogle Scholar
  16. 16.
    Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG (1995) A smooth particle mesh Ewald method. J Chem Phys 103(19):8577–8593Google Scholar
  17. 17.
    Farrar MA, Schreiber RD (1993) The molecular cell biology of interferon-gamma and its receptor. Annu Rev Immunol 11(1):571–611PubMedGoogle Scholar
  18. 18.
    Ghanbari Z, Housaindokht M, Bozorgmehr M, Izadyar M (2016) The effect of glycosylation on the transferrin structure: a molecular dynamic simulation analysis. J Theor Biol 404:73–81PubMedGoogle Scholar
  19. 19.
    Gomi K, Morimoto M, Nakamizo N (1985) Characteristics of antiviral and anticellular activities of human recombinant interferon-gamma. Jpn J Cancer Res 76:224PubMedGoogle Scholar
  20. 20.
    Grzesiek S, Dobeli H, Gentz R, Garotta G, Labhardt A, Bax A (1992) H-1, C-13, and N-15 NMR backbone assignments and secondary structure of human interferon-gamma. Biochemistry 31(35):8180–8190PubMedGoogle Scholar
  21. 21.
    Guvench O, Mallajosyula SS, Raman EP, Hatcher E, Vanommeslaeghe K, Foster TJ, Jamison FW, MacKerell AD (2011) CHARMM additive all-atom force field for carbohydrate derivatives and its utility in polysaccharide and carbohydrate–protein modeling. J Chem Theory Comput 3162–3180PubMedPubMedCentralGoogle Scholar
  22. 22.
    Haelewyn J, Michiels L, Verhaert P, Hoylaerts MF, Witters R, De Ley M (1997) Interaction of truncated human interferon gamma variants with the interferon gamma receptor: crucial importance of Arg-129. Biochem J 324(2):591–595PubMedPubMedCentralGoogle Scholar
  23. 23.
    Hess B (2008) P-LINCS: a parallel linear constraint solver for molecular simulation. J Chem Theory Comput 4(1):116–122PubMedGoogle Scholar
  24. 24.
    Hockney R, Goel S, Eastwood J (1974) Quiet high-resolution computer models of a plasma. J Comput Phys 14(2):148–158Google Scholar
  25. 25.
    Huang J, Rauscher S, Nawrocki G, Ran T, Feig M, de Groot BL, Grubmüller H, MacKerell AD Jr (2016) CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nat Methods 14:71–73PubMedPubMedCentralGoogle Scholar
  26. 26.
    Jo S, Song KC, Desaire H, MacKerell AD, Im W (2011) Glycan reader: automated sugar identification and simulation preparation for carbohydrates and glycoproteins. J Comput Chem 32(14):3135–3141PubMedPubMedCentralGoogle Scholar
  27. 27.
    Kelker H, Yip Y, Anderson P, Vilcek J (1983) Effects of glycosidase treatment on the physicochemical properties and biological activity of human interferon-gamma. J Biol Chem 258(13):8010–3PubMedGoogle Scholar
  28. 28.
    Kelker HC, Le J, Rubin BY, Yip YK, Nagler C, Vilćek J (1984) Three molecular weight forms of natural human interferon-γ revealed by immunoprecipitation with monoclonal antibody. J Biol Chem 259(7):4301–4304Google Scholar
  29. 29.
    Kirschner KN, Yongye AB, Tschampel SM, González-Outeirin̄o J, Daniels CR, Foley BL, Woods RJ (2015) GLYCAM06: a generalizable biomolecular force field. Carbohydrates. J Comput Chem 29(4):622–655Google Scholar
  30. 30.
    Knott BC, Crowley MF, Himmel ME, Ståhlberg J, Beckham GT (2014) Carbohydrate–protein interactions that drive processive polysaccharide translocation in enzymes revealed from a computational study of cellobiohydrolase processivity. J Am Chem Soc 136(24):8810–8819PubMedGoogle Scholar
  31. 31.
    Kognole AA, Payne CM (2017) Inhibition of mammalian glycoprotein YKL-40: identification of the physiological ligand. J Biol Chem 292(7):2624–2636PubMedPubMedCentralGoogle Scholar
  32. 32.
    Kony D, Damm W, Stoll S, Van Gunsteren W (2002) An improved OPLS-AA force field for carbohydrates. J Comput Chem 23(15):1416–29PubMedGoogle Scholar
  33. 33.
    Liu Y, Pan D, Bellis SL, Song Y (2008) Effect of altered glycosylation on the structure of the i-like domain of β1 integrin: a molecular dynamics study. Proteins 73(4):989–1000PubMedGoogle Scholar
  34. 34.
    Lopez C, Rzepiela A, de Vries A, Dijkhuizen L, Huenenbergera P, Marrink S (2009) The MARTINI coarse grained force field: extension to carbohydrates. J Chem Theory Comput 5:3195–3210PubMedGoogle Scholar
  35. 35.
    Loss A, Lütteke T (2015) Using NMR data on GLYCOSIENCES.de. Methods Mol Biol 1273:87–95PubMedGoogle Scholar
  36. 36.
    Lundell D, Lunn C, Dalgarno D, Fossetta J, Greenberg R, Reim R, Grace M, Narula S (1991) The carboxyl-terminal region of human interferon gamma is important for biological activity: mutagenic and NMR analysis. Protein Eng 4(3):335–341PubMedGoogle Scholar
  37. 37.
    Mortz E, Sareneva T, Haebel S, Julkunen I, Roepstorff P (1996) Mass-spectrometric characterization of glycosylated interferon-gamma variants separated by gel-electrophoresis. Electrophoresis 17(5):925–931PubMedGoogle Scholar
  38. 38.
    Nacheva G, Todorova K, Boyanova M, Berzal-Herranz A, Karshikoff A, Ivanov I (2003) Human interferon gamma: significance of the C-terminal flexible domain for its biological activity. Arch Biochem Biophys 413(1):91–98PubMedGoogle Scholar
  39. 39.
    Pan YCE, Stern AS, Familletti PC, Chizzonite R, Khan FR (1987) Structural characterization of human interferon γ heterogeneity of the carboxyl terminus. Eur J Biochem 166(1):145–149PubMedGoogle Scholar
  40. 40.
    Parrinello M, Rahman A (1980) Crystal structure and pair potentials: a molecular-dynamics study. Phys Rev Lett 45:1196Google Scholar
  41. 41.
    Parrinello M, Rahman A (1981) Polymorphic transitions in single crystals: a new molecular dynamics method. J Appl Phys 52:7182Google Scholar
  42. 42.
    Patel DS, Re S, Wu EL, Qi Y, Klebba PE, Widmalm G, Yeom MS, Sugita Y, Im W (2016) Dynamics and interactions of OmpF and LPS: influence on pore accessibility and ion permeability. Biophys J 110 (4):930–938PubMedPubMedCentralGoogle Scholar
  43. 43.
    Petkov P, Lilkova E, Ilieva N, Nacheva G, Ivanov I, Litov L (2018) Computational modelling of the full length hIFN-γ homodimer. In: Lecture notes in computer science (including subseries lecture notes in artificial intelligence and lecture notes in bioinformatics) 10665 LNCS, pp 544–551Google Scholar
  44. 44.
    Petrescu AJ, Milac AL, Petrescu SM, Dwek RA, Wormald MR (2004) Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding. Glycobiology 14 (2):103–114PubMedGoogle Scholar
  45. 45.
    Rabbani S, Krammer EM, Roos G, Zalewski A, Preston R, Eid S, Zihlmann P, Prévost M, Lensink MF, Thompson A, Ernst B, Bouckaert J (2017) Mutation of Tyr137 of the universal Escherichia coli fimbrial adhesin FimH relaxes the tyrosine gate prior to mannose binding. IUCrJ 4(1):7–23PubMedPubMedCentralGoogle Scholar
  46. 46.
    Rinderknecht E, O’Connor B, Rodriguez H (1984) Natural human interferon-γ. Complete amino acid sequence and determination of sites of glycosylation. J Biol Chem 259(11):6790–6797PubMedGoogle Scholar
  47. 47.
    Sareneva T, Cantell K, Pyhälä L, Pirhonen J, Julkunen I (1993) Effect of carbohydrates on the pharmacokinetics of human interferon-gamma. J Interf Res 13(4):267–9Google Scholar
  48. 48.
    Sareneva T, Mørtz E, Tölö H, Roepstorff P, Julkunen I (1996) Biosynthesis and N-glycosylation of human interferon-γ. Asn25 and Asn97 differ markedly in how efficiently they are glycosylated and in their oligosaccharide composition. Eur J Biochem 242(2):191–200PubMedGoogle Scholar
  49. 49.
    Sareneva T, Pirhonen J, Cantell K, Julkunen L (1995) N-glycosylation of human interferon-γ: glycans at Asn-25 are critical for protease resistance. Biochem J 308(1):9–14PubMedPubMedCentralGoogle Scholar
  50. 50.
    Sareneva T, Pirhonen J, Cantell K, Kalkkinent N, Julkunen L (1994) Role of N-glycosylation in the synthesis, dimerization and secretion of human interferon-γ. Biochem J 303:831– 840PubMedPubMedCentralGoogle Scholar
  51. 51.
    Schein CH, Haugg M (1995) Deletions at the C-terminus of interferon gamma reduce RNA binding and activation of double-stranded-RNA cleavage by bovine seminal ribonuclease. Biochem J 307(1):123–127PubMedPubMedCentralGoogle Scholar
  52. 52.
    Shental-Bechor D, Levy Y (2008) Effect of glycosylation on protein folding: a close look at thermodynamic stabilization. Proc Natl Acad Sci 105(24):8256–8261PubMedGoogle Scholar
  53. 53.
    Thiel D, le Du MH, Walter R, D’Arcy A, Chène C, Fountoulakis M, Garotta G, Winkler F, Ealick S (2000) Observation of an unexpected third receptor molecule in the crystal structure of human interferon-γ receptor complex. Structure 8:927–936PubMedGoogle Scholar
  54. 54.
    Varesio L, Blasi E, Thurman G, Talmadge J, Wiltrout R, Herberman R (1984) Potent activation of mouse macrophages by recombinant interferon-gamma. Cancer Res 44(10):4465–4469PubMedGoogle Scholar
  55. 55.
    Yamamoto S, Hase S, Fukuda S, Sano O, Ikenaka T (1989) Structures of the sugar chains of interferon-γ produced by human myelomonocyte cell line HBL-38. Biochem J 105:547–555Google Scholar
  56. 56.
    Yamamoto S, Hase S, Yamauchi H, Tanimoto T, Ikenaka T (1989) Studies on the sugar chains of interferon-γ from human peripheral-blood lymphocytes. Biochem J 105:1034–1039Google Scholar
  57. 57.
    Yip YK, Barrowclough BS, Urban C, Vilcek J (1982) Purification of two subspecies of human γ (immune) interferon. Proc Natl Acad Sci USA 79(6):1820–1824PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Information and Communication Technologies at the Bulgarian Academy of SciencesSofiaBulgaria
  2. 2.Faculty of Physics, Atomic Physics DepartmentSofia University “St. Kliment Ohridski”SofiaBulgaria
  3. 3.Institute of Mathematics and Informatics at the Bulgarian Academy of SciencesSofiaBulgaria
  4. 4.Institute of Molecular Biology “Roumen Tsanev” at the Bulgarian Academy of SciencesSofiaBulgaria

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