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Analysis of Covalent Modifications of Amyloidogenic Proteins Using Two-Dimensional Electrophoresis: Prion Protein and Its Sialylation

  • Elizaveta Katorcha
  • Ilia V. Baskakov
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1779)

Abstract

A number of proteins associated with neurodegenerative disease undergo several types of posttranslational modifications. They include N-linked glycosylation of the prion protein and amyloid precursor protein, phosphorylation of tau and α-synuclein. Posttranslational modifications alter physical properties of proteins including their net and surface charges, affecting their processing, life-time and propensity to acquire misfolded, disease-associated states. As such, analysis of posttranslational modifications is important for understanding the mechanisms of pathogenesis. Recent studies documented that sialylation of the disease-associated form of the prion protein or PrPSc controls the fate of prions in an organism and outcomes of prion infection. For assessing sialylation status of PrPSc, we developed a reliable protocol that involves two-dimensional electrophoresis followed by Western blot (2D). The current chapter describes the procedure for the analysis of sialylation status of PrPSc from various sources including central nervous system, secondary lymphoid organs, cultured cells, or PrPSc produced in Protein Misfolding Cyclic Amplification.

Key words

Prion proteins Prion diseases Amyloidogenic proteins Posttranslational modifications Two-dimensional electrophoresis Sialylation Sialic acid Glycosylation 

Notes

Acknowledgments

This work was supported by the National Institute of Health grant R01 NS045585.

References

  1. 1.
    Bolton DC, Meyer RK, Prusiner SB (1985) Scrapie PrP 27-30 is a sialoglycoprotein. J Virol 53:596–606PubMedPubMedCentralGoogle Scholar
  2. 2.
    Schedin-Weiss S, Winblad B, Tjernberg LO (2014) The role of protein glycosylation in Alzheimer disease. FEBS J 281:46–62CrossRefPubMedGoogle Scholar
  3. 3.
    Selden SC, Pollard TD (1983) Phosphorylation of microtubule-associated proteins regulates their interaction with actin filaments. J Biol Chem 258:7064–7071PubMedGoogle Scholar
  4. 4.
    Nakajo S, Tsukada K, Omata K et al (1993) A new brain-specific 14-kDa protein is a phosphoprotein. Its complete amino acid sequence and evidence for phosphorylation. Eur J Biochem 217:1057–1063CrossRefPubMedGoogle Scholar
  5. 5.
    Katorcha E, Makarava N, Savtchenko R et al (2014) Sialylation of prion protein controls the rate of prion amplification, the cross-species barrier, the ratio of PrPSc glycoform and prion infectivity. PLoS Pathog 10:e1004366CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Srivastava S, Makarava N, Katorcha E et al (2015) Post-conversion sialylation of prions in lymphoid tissues. Proc Natl Acad Sci U S A 112:6654–6662CrossRefGoogle Scholar
  7. 7.
    Katorcha E, Daus ML, Gonzalez-Montalban N et al (2016) Reversible off and on switching of prion infectivity via removing and reinstalling prion sialylation. Sci Rep 6:33119CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Srivastava S, Katorcha E, Daus ML et al (2017) Sialylation controls prion fate in vivo. J Biol Chem 292:2359–2368CrossRefPubMedGoogle Scholar
  9. 9.
    Baskakov IV, Katorcha E (2016) Multifaceted role of sialylation in prion diseases. Front Neurosci 10:358CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Katorcha E, Makarava N, Savtchenko R et al (2015) Sialylation of the prion protein glycans controls prion replication rate and glycoform ratio. Sci Rep 5:16912CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Makarava N, Savtchenko R, Baskakov IV (2015) Two alternative pathways for generating transmissible prion disease de novo. Acta Neuropathol Commun 3:69CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Makarava N, Savtchenko R, Baskakov IV (2013) Selective amplification of classical and atypical prions using modified protein misfolding cyclic amplification. J Biol Chem 288:33–41CrossRefPubMedGoogle Scholar
  13. 13.
    Turk E, Teplow DB, Hood LE et al (1988) Purification and properties of the cellular and scrapie hamster prion proteins. Eur J Biochem 176:21–30CrossRefPubMedGoogle Scholar
  14. 14.
    Endo T, Groth D, Prusiner SB et al (1989) Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein. Biochemistry 28:8380–8388CrossRefPubMedGoogle Scholar
  15. 15.
    Stimson E, Hope J, Chong A et al (1999) Site-specific characterization of the N-linked glycans of murine prion protein by high-performance liquid chromatography/electrospray mass spectrometry and exoglycosidase digestions. Biochemistry 38:4885–4895CrossRefPubMedGoogle Scholar
  16. 16.
    Stahl N, Baldwin MA, Teplow DB et al (1993) Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry 32:1991–2002CrossRefPubMedGoogle Scholar
  17. 17.
    Rudd PM, Endo T, Colominas C et al (1999) Glycosylation differences between the normal and pathogenic prion protein isoforms. Proc Natl Acad Sci U S A 96:13044–13049CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Katorcha E, Klimova N, Makarava N et al (2015) Knocking out of cellular neuraminidases Neu1, Neu3 or Neu4 does not affect sialylation status of the prion protein. PLoS One 10:e0143218CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Stahl N, Baldwin MA, Hecker R et al (1992) Glycosylinositol phospholipid anchors of the scrapie and cellular prion proteins contain sialic acid. Biochemistry 31:5043–5053CrossRefPubMedGoogle Scholar
  20. 20.
    Katorcha E, Srivastava S, Klimova N et al (2016) Sialylation of GPI anchors of mammalian prions is regulated in a host-, tissue- and cell-specific manner. J Biol Chem 291:17009–17019CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Makarava N, Kovacs GG, Savtchenko R et al (2012) Stabilization of a prion strain of synthetic origin requires multiple serial passages. J Biol Chem 287:30205–30214CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Gonzalez-Montalban N, Makarava N, Ostapchenko VG et al (2011) Highly efficient protein misfolding cyclic amplification. PLoS Pathog 7:e1001277CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Anatomy and Neurobiology, Center for Biomedical Engineering and TechnologyUniversity of Maryland School of MedicineBaltimoreUSA

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