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Investigating the Role of Artemin Glycosylation

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ABSTRACT

Purpose

Oligosaccharides play diverse and unpredictable functional roles when attached to proteins and are a largely unexplored scaffold for deconstructing and attributing novel functions to proteins during drug development. Here, the glycoprotein Artemin (ART) was carefully assessed by multiple analytical methods that allow us to provide a comprehensive understanding of how N-linked glycosylation impact the structural and functional properties of ART.

Methods

Modification of the N-linked glycan of ART was performed by incubation with various enzymes. Biological assays and systems were used to examine the relative activity and pharmacokinetic properties of ART as a function of glycosylation. In order to reveal the conformational impact of glycosylation on ART, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed in addition to differential scanning calorimetry. The colloidal stability of ART glycovariants was assessed by dynamic light scattering, viscometry, and solubility assays.

Results

No difference in pharmacokinetics or relative potency was revealed between glycosylated and nonglycosylated ART. Surprisingly, the HDX-MS data indicated that the glycan does not greatly influence the conformation and dynamics of the protein. In contrast, differences in thermal and colloidal stability clearly revealed a role of glycosylation in increasing the solubility and stability of ART.

Conclusions

Our findings demonstrate how careful analysis using multiple advanced techniques can be used to identify and dissect the multiple potential functions of protein glycosylation and form a prerequisite for glycoengineering and drug development of glycoproteins.

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Abbreviations

ART:

Artemin

CE-SDS:

Sodium-dodecyl sulfate capillary electrophoresis

CHO:

Chinese Hamster Ovary

DA:

Dopaminergic

DLS:

Dynamic light scattering

DSC:

Differential scanning calorimetry

GDNF:

Glial cell line-derived neurotrophic factor

GFLs:

GDNF family of ligands

GFRα:

GDNF family receptor-α

GlcNAc:

N-acetylglucosamine

GPI:

Glycosylphosphatidylinositol

HDX-MS:

Hydrogen/Deuterium exchange mass spectrometry

KIRA:

Kinase receptor activation

NeuAc:

Sialic acid

NRTN:

Neurturin

pI:

Isoelectric point

PSPN:

Persephin

RET:

REarranged during Transfection receptor tyrosine kinase

RMSD:

Root mean square deviation

SCX:

Strong cation-exchange

TCEP:

Tris(2-carboxyethyl)phosphine hydrochloride

TGF-β:

Transforming growth factor-β

UPLC:

Ultra performance liquid chromatography

REFERENCES

  1. Baloh RH, Tansey MG, Lampe PA, Fahrner TJ, Enomoto H, Simburger KS, et al. Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFRalpha3-RET receptor complex. Neuron. 1998;21(6):1291–302.

    Article  CAS  PubMed  Google Scholar 

  2. Wang X, Baloh RH, Milbrandt J, Garcia KC. Structure of artemin complexed with its receptor GFRalpha3: convergent recognition of glial cell line-derived neurotrophic factors. Structure. 2006;14(6):1083–92.

    Article  PubMed  Google Scholar 

  3. Masure S, Geerts H, Cik M, Hoefnagel E, Van Den Kieboom G, Tuytelaars A, et al. Enovin, a member of the glial cell-line-derived neurotrophic factor (GDNF) family with growth promoting activity on neuronal cells. Existence and tissue-specific expression of different splice variants. Eur J Biochem / FEBS. 1999;266(3):892–902.

    Article  CAS  Google Scholar 

  4. Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science. 1993;260(5111):1130–2.

    Article  CAS  PubMed  Google Scholar 

  5. Kotzbauer PT, Lampe PA, Heuckeroth RO, Golden JP, Creedon DJ, Johnson Jr EM, et al. Neurturin, a relative of glial-cell-line-derived neurotrophic factor. Nature. 1996;384(6608):467–70.

    Article  CAS  PubMed  Google Scholar 

  6. Milbrandt J, de Sauvage FJ, Fahrner TJ, Baloh RH, Leitner ML, Tansey MG, et al. Persephin, a novel neurotrophic factor related to GDNF and neurturin. Neuron. 1998;20(2):245–53.

    Article  CAS  PubMed  Google Scholar 

  7. Henderson CE, Phillips HS, Pollock RA, Davies AM, Lemeulle C, Armanini M, et al. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science. 1994;266(5187):1062–4.

    Article  CAS  PubMed  Google Scholar 

  8. Baloh RH, Gorodinsky A, Golden JP, Tansey MG, Keck CL, Popescu NC, et al. GFRalpha3 is an orphan member of the GDNF/neurturin/persephin receptor family. Proc Natl Acad Sci U S A. 1998;95(10):5801–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Rosenblad C, Gronborg M, Hansen C, Blom N, Meyer M, Johansen J, et al. In vivo protection of nigral dopamine neurons by lentiviral gene transfer of the novel GDNF-family member neublastin/artemin. Mol Cell Neurosci. 2000;15(2):199–214.

    Article  CAS  PubMed  Google Scholar 

  10. Takahashi M. The GDNF/RET signaling pathway and human diseases. Cytokine Growth Factor Rev. 2001;12(4):361–73.

    Article  CAS  PubMed  Google Scholar 

  11. Airaksinen MS, Saarma M. The GDNF family: signalling, biological functions and therapeutic value. Nat Rev Neurosci. 2002;3(5):383–94.

    Article  CAS  PubMed  Google Scholar 

  12. Worby CA, Vega QC, Chao HH, Seasholtz AF, Thompson RC, Dixon JE. Identification and characterization of GFRalpha-3, a novel Co-receptor belonging to the glial cell line-derived neurotrophic receptor family. J Biol Chem. 1998;273(6):3502–8.

    Article  CAS  PubMed  Google Scholar 

  13. Silvian L, Jin P, Carmillo P, Boriack-Sjodin PA, Pelletier C, Rushe M, et al. Artemin crystal structure reveals insights into heparan sulfate binding. Biochemistry. 2006;45(22):6801–12.

    Article  CAS  PubMed  Google Scholar 

  14. Golden JP, Baloh RH, Kotzbauer PT, Lampe PA, Osborne PA, Milbrandt J, et al. Expression of neurturin, GDNF, and their receptors in the adult mouse CNS. J Comp Neurol. 1998;398(1):139–50.

    Article  CAS  PubMed  Google Scholar 

  15. Carmillo P, Dago L, Day ES, Worley DS, Rossomando A, Walus L, et al. Glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFR alpha 1) is highly selective for GDNF versus artemin. Biochemistry. 2005;44(7):2545–54.

    Article  CAS  PubMed  Google Scholar 

  16. Anglade P, Vyas S, Javoy-Agid F, Herrero MT, Michel PP, Marquez J, et al. Apoptosis and autophagy in nigral neurons of patients with Parkinson’s disease. Histol Histopathol. 1997;12(1):25–31.

    CAS  PubMed  Google Scholar 

  17. Orozco OE, Walus L, Sah DW, Pepinsky RB, Sanicola M. GFRalpha3 is expressed predominantly in nociceptive sensory neurons. Eur J Neurosci. 2001;13(11):2177–82.

    Article  CAS  PubMed  Google Scholar 

  18. Gardell LR, Wang R, Ehrenfels C, Ossipov MH, Rossomando AJ, Miller S, et al. Multiple actions of systemic artemin in experimental neuropathy. Nat Med. 2003;9(11):1383–9.

    Article  CAS  PubMed  Google Scholar 

  19. Connelly GP, Bai Y, Jeng MF, Englander SW. Isotope effects in peptide group hydrogen exchange. Proteins. 1993;17(1):87–92.

    Article  CAS  PubMed  Google Scholar 

  20. Eigenbrot C, Gerber N. X-ray structure of glial cell-derived neurotrophic factor at 1.9 A resolution and implications for receptor binding. Nat Struct Biol. 1997;4(6):435–8.

    Article  CAS  PubMed  Google Scholar 

  21. Ishino T, Economou NJ, McFadden K, Zaks-Zilberman M, Jost M, Baxter S, et al. A protein engineering approach differentiates the functional importance of carbohydrate moieties of interleukin-5 receptor alpha. Biochemistry. 2011;50(35):7546–56.

    Article  CAS  PubMed  Google Scholar 

  22. Houde D, Arndt J, Domeier W, Berkowitz S, Engen JR. Characterization of IgG1 Conformation and Conformational Dynamics by Hydrogen/Deuterium Exchange Mass Spectrometry. Anal Chem. 2009;81(14):5966.

    Article  CAS  PubMed  Google Scholar 

  23. Houde D, Peng Y, Berkowitz SA, Engen JR. Post-translational modifications differentially affect IgG1 conformation and receptor binding. Mol Cell Proteomics. 2010;9(8):1716–28.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Jensen PF, Larraillet V, Schlothauer T, Kettenberger H, Hilger M, Rand KD. Investigating the interaction between the neonatal Fc receptor and monoclonal antibody variants by hydrogen/deuterium exchange mass spectrometry. Mol Cellular Proteomics: MCP. 2014.

  25. Morell AG, Gregoriadis G, Scheinberg IH, Hickman J, Ashwell G. The role of sialic acid in determining the survival of glycoproteins in the circulation. J Biol Chem. 1971;246(5):1461–7.

    CAS  PubMed  Google Scholar 

  26. Kundra R, Kornfeld S. Asparagine-linked oligosaccharides protect Lamp-1 and Lamp-2 from intracellular proteolysis. J Biol Chem. 1999;274(43):31039–46.

    Article  CAS  PubMed  Google Scholar 

  27. van Veen HA, Geerts ME, van Berkel PH, Nuijens JH. The role of N-linked glycosylation in the protection of human and bovine lactoferrin against tryptic proteolysis. Eur J Biochem / FEBS. 2004;271(4):678–84.

    Article  Google Scholar 

  28. Shental-Bechor D, Levy Y. Effect of glycosylation on protein folding: a close look at thermodynamic stabilization. Proc Natl Acad Sci U S A. 2008;105(24):8256–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Hanson SR, Culyba EK, Hsu TL, Wong CH, Kelly JW, Powers ET. The core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability. Proc Natl Acad Sci U S A. 2009;106(9):3131–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Petrescu AJ, Milac AL, Petrescu SM, Dwek RA, Wormald MR. Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding. Glycobiology. 2004;14(2):103–14.

    Article  CAS  PubMed  Google Scholar 

  31. Raju TS, Briggs JB, Chamow SM, Winkler ME, Jones AJ. Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues. Biochemistry. 2001;40(30):8868–76.

    Article  CAS  PubMed  Google Scholar 

  32. Song K, Yoon IS, Kim NA, Kim DH, Lee J, Lee HJ, et al. Glycoengineering of interferon-beta 1a improves its biophysical and pharmacokinetic properties. PLoS One. 2014;9(5), e96967.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, et al. The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem. 2003;278(5):3466–73.

    Article  CAS  PubMed  Google Scholar 

  34. Gabius HJ, Siebert HC, Andre S, Jimenez-Barbero J, Rudiger H. Chemical biology of the sugar code. Chembiochem. 2004;5(6):740–64.

    Article  CAS  PubMed  Google Scholar 

  35. Tang L, Coales SJ, Morrow JA, Edmunds T, Hamuro Y. Characterization of the N370S mutant of glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry. Chembiochem. 2012;13(15):2243–50.

    Article  CAS  PubMed  Google Scholar 

  36. Schlee S, Carmillo P, Whitty A. Quantitative analysis of the activation mechanism of the multicomponent growth-factor receptor Ret. Nat Chem Biol. 2006;2(11):636–44.

    Article  CAS  PubMed  Google Scholar 

  37. Orozco OE, Walus L, Sah DW, Pepinsky RB, Sanicola M. GFRalpha3 is expressed predominantly in nociceptive sensory neurons. Eur J Neurosci. 2001;13(11):2177–82.

    Article  CAS  PubMed  Google Scholar 

  38. Sanicola M, Hession C, Worley D, Carmillo P, Ehrenfels C, Walus L, et al. Glial cell line-derived neurotrophic factor-dependent RET activation can be mediated by two different cell-surface accessory proteins. Proc Natl Acad Sci. 1997;94(12):6238–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Weis DD, Engen JR, Kass IJ. Semi-automated data processing of hydrogen exchange mass spectra using HX-Express. J Am Soc Mass Spectrom. 2006;17(12):1700–3.

    Article  CAS  PubMed  Google Scholar 

  40. Lehermayr C, Mahler HC, Mader K, Fischer S. Assessment of net charge and protein-protein interactions of different monoclonal antibodies. J Pharm Sci. 2011;100(7):2551–62.

    Article  CAS  PubMed  Google Scholar 

  41. Hanlon AD, Larkin MI, Reddick RM. Free-solution, label-free protein-protein interactions characterized by dynamic light scattering. Biophys J. 2010;98(2):297–304.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Pirofsky B. The determination of blood viscosity in man by a method based on Poiseuille’s law. J Clin Investig. 1953;32(4):292.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Allmendinger A, Dieu L-H, Fischer S, Mueller R, Mahler H-C, Huwyler J. High-throughput viscosity measurement using capillary electrophoresis instrumentation and its application to protein formulation. J Pharm Biomed Anal. 2014;99:51–8.

    Article  CAS  PubMed  Google Scholar 

  44. Lavoisier A, Schlaeppi J-M, editors. Early developability screen of therapeutic antibody candidates using Taylor dispersion analysis and UV area imaging detection. MAbs; 2015: Taylor & Francis.

  45. Sadick MD, Intintoli A, Quarmby V, McCoy A, Canova-Davis E, Ling V. Kinase receptor activation (KIRA): a rapid and accurate alternative to end-point bioassays. J Pharm Biomed Anal. 1999;19(6):883–91.

    Article  CAS  PubMed  Google Scholar 

  46. Piccinini E, Kalkkinen N, Saarma M, Runeberg-Roos P. Glial cell line-derived neurotrophic factor: characterization of mammalian posttranslational modifications. Ann Med. 2013;45(1):66–73.

    Article  CAS  PubMed  Google Scholar 

  47. Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 1993;3(2):97–130.

    Article  CAS  PubMed  Google Scholar 

  48. Arnold JN, Wormald MR, Sim RB, Rudd PM, Dwek RA. The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu Rev Immunol. 2007;25:21–50.

    Article  CAS  PubMed  Google Scholar 

  49. Hoofnagle AN, Resing KA, Ahn NG. Protein analysis by hydrogen exchange mass spectrometry. Annu Rev Biophys Biomol Struct. 2003;32(1):1–25.

    Article  CAS  PubMed  Google Scholar 

  50. Englander JJ, Del Mar C, Li W, Englander SW, Kim JS, Stranz DD, et al. Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry. Proc Natl Acad Sci. 2003;100(12):7057–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Konermann L, Pan J, Liu Y-H. Hydrogen exchange mass spectrometry for studying protein structure and dynamics. Chem Soc Rev. 2011;40(3):1224–34.

    Article  CAS  PubMed  Google Scholar 

  52. Wales TE, Engen JR. Hydrogen exchange mass spectrometry for the analysis of protein dynamics. Mass Spectrom Rev. 2006;25(1):158–70.

    Article  CAS  PubMed  Google Scholar 

  53. Guttman M, Scian M, Lee KK. Tracking hydrogen/deuterium exchange at glycan sites in glycoproteins by mass spectrometry. Anal Chem. 2011;83(19):7492–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Bai Y, Milne JS, Mayne L, Englander SW. Primary structure effects on peptide group hydrogen exchange. Proteins. 1993;17(1):75–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Bruinzeel W, Masure S. Exceptional stability of artemin neurotrophic factor dimers: effects of temperature, pH, buffer and storage conditions on protein integrity and activity. Appl Biochem Biotechnol. 2011;165(5–6):1379–90.

    Article  CAS  PubMed  Google Scholar 

  56. Gallagher PJ, Henneberry JM, Sambrook JF, Gething MJ. Glycosylation requirements for intracellular transport and function of the hemagglutinin of influenza virus. J Virol. 1992;66(12):7136–45.

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Narhi LO, Arakawa T, Aoki K, Wen J, Elliott S, Boone T, et al. Asn to Lys mutations at three sites which are N-glycosylated in the mammalian protein decrease the aggregation of Escherichia coli-derived erythropoietin. Protein Eng. 2001;14(2):135–40.

    Article  CAS  PubMed  Google Scholar 

  58. Wang C, Eufemi M, Turano C, Giartosio A. Influence of the carbohydrate moiety on the stability of glycoproteins. Biochemistry. 1996;35(23):7299–307.

    Article  CAS  PubMed  Google Scholar 

  59. Liu J, Nguyen MD, Andya JD, Shire SJ. Reversible self‐association increases the viscosity of a concentrated monoclonal antibody in aqueous solution. J Pharm Sci. 2005;94(9):1928–40.

    Article  CAS  PubMed  Google Scholar 

  60. Yadav S, Shire SJ, Kalonia DS. Factors affecting the viscosity in high concentration solutions of different monoclonal antibodies. J Pharm Sci. 2010;99(12):4812–29.

    Article  CAS  PubMed  Google Scholar 

  61. Yadav S, Laue TM, Kalonia DS, Singh SN, Shire SJ. The influence of charge distribution on self-association and viscosity behavior of monoclonal antibody solutions. Mol Pharm. 2012;9(4):791–802.

    Article  CAS  PubMed  Google Scholar 

  62. Yadav S, Liu J, Shire SJ, Kalonia DS. Specific interactions in high concentration antibody solutions resulting in high viscosity. J Pharm Sci. 2010;99(3):1152–68.

    Article  CAS  PubMed  Google Scholar 

  63. Harding S. Dilute solution viscometry of food biopolymers. Functional properties of food macromolecules. 1998;1–49.

  64. Bull HB. The electroviscous effect in egg albumin solutions. Trans Faraday Soc. 1940;35:80–4.

    Article  Google Scholar 

  65. Komatsubara M, Suzuki K, Nakajima H, Wada Y. Electroviscous effect of lysozyme in aqueous solutions. Biopolymers. 1973;12(8):1741–6.

    Article  CAS  PubMed  Google Scholar 

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ACKNOWLEDGMENTS AND DISCLOSURES

We thank Paul Carmillo and Yan Gao for performing the KIRA assays, Susan Foley for performing analytical measurements, Tony Rossomando for helpful discussions, and Tia Estey for her continuous support. KDR acknowledges support from The Marie Curie Actions Programme of the E.U (Grant No. PCIG09-GA-2011-294214) and the Danish Council for Independent Research Natural Sciences (Steno Grant No. 11- 104058).

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Authors and Affiliations

  1. Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark

    Qiu Danwen, Christian Code & Kasper D. Rand

  2. Biogen, Cambridge, Massachusetts, USA

    Chao Quan, Bang-Jin Gong, Joseph Arndt, Blake Pepinsky & Damian Houde

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  1. Qiu Danwen
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  2. Christian Code
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  3. Chao Quan
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Correspondence to Kasper D. Rand or Damian Houde.

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Danwen, Q., Code, C., Quan, C. et al. Investigating the Role of Artemin Glycosylation. Pharm Res 33, 1383–1398 (2016). https://doi.org/10.1007/s11095-016-1880-x

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