Molecular Neurobiology

, Volume 55, Issue 4, pp 3172–3184 | Cite as

Myelin Basic Protein Citrullination, a Hallmark of Central Nervous System Demyelination, Assessed by Novel Monoclonal Antibodies in Prion Diseases

  • Byungki Jang
  • Yong-Chul Jeon
  • Hae-Young Shin
  • Yun-Jung Lee
  • Hyunji Kim
  • Yoshitaka Kondo
  • Akihito Ishigami
  • Yong-Sun Kim
  • Eun-Kyoung Choi


Myelin basic protein (MBP) citrullination by peptidylarginine deiminase (PAD) enzymes leads to incomplete protein-lipid bilayer interactions and vulnerability to proteolytic enzymes, resulting in disorganization of the myelin sheath in the central nervous system. Therefore, citrullinated MBP (citMBP) has been suggested as a hallmark of demyelination, but how citMBP is implicated in prion diseases remains unknown. For the first time, we developed mouse monoclonal anti-citMBP IgG1 (clones 1B8, 1H1, and 3C6) and IgM (clone 3G5) antibodies that recognize human citMBP at its R25, R122, and R130 residues and at its C-terminal region (or the corresponding sites in mouse MBP), respectively. Using a biochemical, immunohistochemical, and immunogold-silver staining for electron microscopy techniques, we found that MBP residue R23 (corresponding to human R25) was specifically citrullinated, was stained as intense punctae in the corpus callosum, the striatum, and the cerebellar white matter, and was predominantly localized in disorganized myelin in the brains of scrapie-infected mice. In the brains of Creutzfeldt-Jakob disease (CJD) patients, MBP residues R25, R122, and R130 were markedly citrullinated and were stained as fibrils and punctae. In particular, white matter regions, such as the midbrain and the medulla, exhibited high levels of citMBP compared to other regions. However, the high levels of citMBP were not correlated with PAD2 expression. The clone 3G5 recognized significantly increased expression of the 18.5 kDa and/or 21.5 kDa variants of MBP in prion disease. Our findings suggest that significantly increased levels of citMBP may reflect demyelinating neuropathology, and that these newly developed antibodies may be useful for identifying demyelination.


Myelin basic protein Citrullination Peptidylarginine deiminase Demyelination Neurodegeneration 



This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2009822 and NRF-2015R1D1A1A01059584) and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI16C0965).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Boggs JM (2006) Myelin basic protein: A multifunctional protein. Cell Mol Life Sci 63:1945–1961CrossRefPubMedGoogle Scholar
  2. 2.
    Jahn O, Tenzer S, Werner HB (2009) Myelin proteomics: Molecular anatomy of an insulating sheath. Mol Neurobiol 40:55–72CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Morell P, Quarles RH (1999) Characteristic composition of myelin. In: Siegel GJ, Agranoff BW, Albers RW et al (eds) Basic neurochemistry: Molecular, cellular and medical aspects, 6th edn. Lippincott-Raven, Philadelphia, pp. 183–185Google Scholar
  4. 4.
    Vassall KA, Bamm VV, Harauz G (2015) MyelStones: The executive roles of myelin basic protein in myelin assembly and destabilization in multiple sclerosis. Biochem J 472:17–32CrossRefPubMedGoogle Scholar
  5. 5.
    Harauz G, Boggs JM (2013) Myelin management by the 18.5-kDa and 21.5-kDa classic myelin basic protein isoforms. J Neurochem 125:334–361CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Harauz G, Musse AA (2007) A tale of two citrullines--structural and functional aspects of myelin basic protein deimination in health and disease. Neurochem Res 32:137–158CrossRefPubMedGoogle Scholar
  7. 7.
    Harauz G, Ladizhansky V, Boggs JM (2009) Structural polymorphism and multifunctionality of myelin basic protein. Biochemistry 48:8094–8104CrossRefPubMedGoogle Scholar
  8. 8.
    Kim JK, Mastronardi FG, Wood DD, Lubman DM, Zand R, Moscarello MA (2003) Multiple sclerosis: An important role for post-translational modifications of myelin basic protein in pathogenesis. Mol Cell Proteomics 2:453–462CrossRefPubMedGoogle Scholar
  9. 9.
    Vossenaar ER, Zendman AJ, van Venrooij WJ, Pruijn GJ (2003) PAD, a growing family of citrullinating enzymes: Genes, features and involvement in disease. BioEssays 25:1106–1118CrossRefPubMedGoogle Scholar
  10. 10.
    Moscarello MA, Wood DD, Ackerley C, Boulias C (1994) Myelin in multiple sclerosis is developmentally immature. J Clin Invest 94:146–154CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Wood DD, Moscarello MA (1989) The isolation, characterization, and lipid-aggregating properties of a citrulline containing myelin basic protein. J Biol Chem 264:5121–5127PubMedGoogle Scholar
  12. 12.
    Moscarello MA, Mastronardi FG, Wood DD (2007) The role of citrullinated proteins suggests a novel mechanism in the pathogenesis of multiple sclerosis. Neurochem Res 32:251–256CrossRefPubMedGoogle Scholar
  13. 13.
    Mastronardi FG, Noor A, Wood DD, Paton T, Moscarello MA (2007) Peptidyl argininedeiminase 2 CpG island in multiple sclerosis white matter is hypomethylated. J Neurosci Res 85:2006–2016CrossRefPubMedGoogle Scholar
  14. 14.
    Wood DD, Bilbao JM, O'Connors P, Moscarello MA (1996) Acute multiple sclerosis (Marburg type) is associated with developmentally immature myelin basic protein. Ann Neurol 40:18–24CrossRefPubMedGoogle Scholar
  15. 15.
    Jang B, Jin JK, Jeon YC, Cho HJ, Ishigami A, Choi KC, Carp RI, Maruyama N et al (2010) Involvement of peptidylarginine deiminase-mediated post-translational citrullination in pathogenesis of sporadic Creutzfeldt-Jakob disease. Acta Neuropathol 119:199–210CrossRefPubMedGoogle Scholar
  16. 16.
    Jang B, Kim E, Choi JK, Jin JK, Kim JI, Ishigami A, Maruyama N, Carp RI et al (2008) Accumulation of citrullinated proteins by up-regulated peptidylarginine deiminase 2 in brains of scrapie-infected mice: A possible role in pathogenesis. Am J Pathol 173:1129–1142CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Jang B, Shin HY, Choi JK, Nguyen du PT, Jeong BH, Ishigami A, Maruyama N, Carp RI et al (2011) Subcellular localization of peptidylarginine deiminase 2 and citrullinated proteins in brains of scrapie-infected mice: Nuclear localization of PAD2 and membrane fraction-enriched citrullinated proteins. J Neuropathol Exp Neurol 70:116–124CrossRefPubMedGoogle Scholar
  18. 18.
    Ishigami A, Ohsawa T, Hiratsuka M, Taguchi H, Kobayashi S, Saito Y, Murayama S, Asaga H et al (2005) Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer's disease. J Neurosci Res 80:120–128CrossRefPubMedGoogle Scholar
  19. 19.
    Colby DW, Prusiner SB (2011) Prions. Cold Spring Harb Perspect Biol 3:a006833CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Caverzasi E, Mandelli ML, DeArmond SJ, Hess CP, Vitali P, Papinutto N, Oehler A, Miller BL et al (2014) White matter involvement in sporadic Creutzfeldt-Jakob disease. Brain 137:3339–3354CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Jang B, Jeon YC, Choi JK, Park M, Kim JI, Ishigami A, Maruyama N, Carp RI et al (2012) Peptidylarginine deiminase modulates the physiological roles of enolase via citrullination: Links between altered multifunction of enolase and neurodegenerative diseases. Biochem J 445:183–192Google Scholar
  22. 22.
    Shimada N, Handa S, Uchida Y, Fukuda M, Maruyama N, Asaga H, Choi EK, Lee J et al (2010) Developmental and age-related changes of peptidylarginine deiminase 2 in the mouse brain. J Neurosci Res 88:798–806PubMedGoogle Scholar
  23. 23.
    Carp RI, Merz PA, Kascsak RJ, Merz GS, Wisniewski HM (1985) Nature of the scrapie agent: Current status of facts and hypotheses. J Gen Virol 66:1357–1368CrossRefPubMedGoogle Scholar
  24. 24.
    Bruce ME, McConnell I, Fraser H, Dickinson AG (1991) The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: Implications for the nature of the agent and host control of pathogenesis. J Gen Virol 72:595–603CrossRefPubMedGoogle Scholar
  25. 25.
    Matsuo A, Lee GC, Terai K, Takami K, Hickey WF, McGeer EG, McGeer PL (1997) Unmasking of an unusual myelin basic protein epitope during the process of myelin degeneration in humans: A potential mechanism for the generation of autoantigens. Am J Pathol 150:1253–1266PubMedPubMedCentralGoogle Scholar
  26. 26.
    Bremer J, Baumann F, Tiberi C, Wessig C, Fischer H, Schwarz P, Steele AD, Toyka KV et al (2010) Axonal prion protein is required for peripheral myelin maintenance. Nat Neurosci 13:310–318CrossRefPubMedGoogle Scholar
  27. 27.
    Nishida N, Tremblay P, Sugimoto T, Shigematsu K, Shirabe S, Petromilli C, Erpel SP, Nakaoke R et al (1999) A mouse prion protein transgene rescues mice deficient for the prion protein gene from Purkinje cell degeneration and demyelination. Lab Investig 79:689–697PubMedGoogle Scholar
  28. 28.
    Erickson AK, Payne DM, Martino PA, Rossomando AJ, Shabanowitz J, Weber MJ, Hunt DF, Sturgill TW (1990) Identification by mass spectrometry of threonine 97 in bovine myelin basic protein as a specific phosphorylation site for mitogen-activated protein kinase. J Biol Chem 265:19728–19735PubMedGoogle Scholar
  29. 29.
    Hirschberg D, Rådmark O, Jörnvall H, Bergman T (2003) Thr94 in bovine myelin basic protein is a second phosphorylation site for 42-kDa mitogen-activated protein kinase (ERK2). J Protein Chem 22:177–181CrossRefPubMedGoogle Scholar
  30. 30.
    Lee HP, Jun YC, Choi JK, Kim JI, Carp RI, Kim YS (2005) Activation of mitogen-activated protein kinases in hamster brains infected with 263K scrapie agent. J Neurochem 95:584–593CrossRefPubMedGoogle Scholar
  31. 31.
    Frid K, Einstein O, Friedman-Levi Y, Binyamin O, Ben-Hur T, Gabizon R (2015) Aggregation of MBP in chronic demyelination. Ann Clin Transl Neurol 2:711–721CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Dendrou CA, Fugger L, Friese MA (2015) Immunopathology of multiple sclerosis. Nat Rev Immunol 15:545–558CrossRefPubMedGoogle Scholar
  33. 33.
    Gao J, Xu D (2012) Correlation between posttranslational modification and intrinsic disorder in protein. Pac Symp Biocomput 94–103Google Scholar
  34. 34.
    Micu I, Jiang Q, Coderre E, Ridsdale A, Zhang L, Woulfe J, Yin X, Trapp BD et al (2006) NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia. Nature 439:988–992PubMedGoogle Scholar
  35. 35.
    Moscarello MA, Lei H, Mastronardi FG, Winer S, Tsui H, Li Z, Ackerley C, Zhang L et al (2013) Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis. Dis Models Mech 6:467–478CrossRefGoogle Scholar
  36. 36.
    Witalison EE, Cui X, Hofseth AB, Subramanian V, Causey CP, Thompson PR, Hofseth LJ (2015) Inhibiting protein arginine deiminases has antioxidant consequences. J Pharmacol Exp Ther 353:64–70CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Jang B, Kim HW, Kim JS, Kim WS, Lee BR, Kim S, Kim H, Han SJ et al (2015) Peptidylarginine deiminase inhibition impairs toll-like receptor agonist-induced functional maturation of dendritic cells, resulting in the loss of T cell-proliferative capacity: A partial mechanism with therapeutic potential in inflammatory settings. J Leukoc Biol 97:351–362CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Byungki Jang
    • 1
  • Yong-Chul Jeon
    • 1
  • Hae-Young Shin
    • 1
  • Yun-Jung Lee
    • 1
  • Hyunji Kim
    • 1
  • Yoshitaka Kondo
    • 2
  • Akihito Ishigami
    • 2
  • Yong-Sun Kim
    • 1
    • 3
  • Eun-Kyoung Choi
    • 1
    • 4
  1. 1.Ilsong Institute of Life ScienceHallym UniversityAnyang, Gyeonggi-doRepublic of Korea
  2. 2.Molecular Regulation of AgingTokyo Metropolitan Institute of GerontologyTokyoJapan
  3. 3.Department of Microbiology, College of MedicineHallym UniversityChuncheon, Gangwon-doRepublic of Korea
  4. 4.Department of Biomedical GerontologyGraduate School of Hallym UniversityChuncheon, Gangwon-doRepublic of Korea

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