Advertisement

Investigating the Effects of O-GlcNAc Modifications in Parkinson’s Disease Using Semisynthetic α-Synuclein

  • Ana Galesic
  • Matthew R. PrattEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2133)

Abstract

α-Synuclein is a small aggregation-prone protein associated with Parkinson’s disease (PD). The protein’s biochemical and biophysical properties can be heavily influenced by various types of posttranslational modification (PTMs) such as phosphorylation, ubiquitination, and glycosylation. To understand the site-specific effects of various PTMs have on the protein and its aggregation, obtaining a homogeneous sample of the protein of interest with the specific modification of interest is key. Expressed protein ligation (EPL) has emerged as robust tool for building synthetic proteins bearing site-specific modifications. Here, we outline our approach for building α-synuclein with site specific O-GlcNAc modifications, an intracellular subtype of glycosylation that has been linked to the inhibition of protein aggregation. More specifically, we provide specific protocols for the synthesis of α-synuclein bearing an O-GlcNAc modification at threonine 72, termed α-synuclein(gT72). However, this general approach utilizing two recombinant fragments and one synthetic peptide is applicable to other sites and types of modifications and should be transferable to various other protein targets, including aggregation prone proteins like tau and TDP-43.

Key words

α-synuclein Posttranslational modification Synthetic protein chemistry Expressed protein ligation O-GlcNAc 

Notes

Acknowledgments

This research was supported by the National Institutes of Health (Grant R01GM114537 to M.R.P.).

References

  1. 1.
    Lashuel HA, Overk CR, Oueslati A, Masliah E (2013) The many faces of α-synuclein: from structure and toxicity to therapeutic target. Nat Rev Neurosci 14:38–48.  https://doi.org/10.1038/nrn3406CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Wilhelm BG, Mandad S, Truckenbrodt S, Kröhnert K, Schäfer C, Rammner B et al (2014) Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science 344:1023–1028.  https://doi.org/10.1126/science.1252884CrossRefPubMedGoogle Scholar
  3. 3.
    Wang C, Zhao C, Li D, Tian Z, Lai Y, Diao J et al (2016) Versatile structures of α-synuclein. Front Mol Neurosci 9:48.  https://doi.org/10.3389/fnmol.2016.00048CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Jao CC, Hegde BG, Chen J, Haworth IS, Langen R (2008) Structure of membrane-bound alpha-synuclein from site-directed spin labeling and computational refinement. Proc Natl Acad Sci USA 105:19666–19671.  https://doi.org/10.1073/pnas.0807826105CrossRefPubMedGoogle Scholar
  5. 5.
    Varkey J, Isas JM, Mizuno N, Jensen MB, Bhatia VK, Jao CC et al (2010) Membrane curvature induction and tubulation are common features of synucleins and apolipoproteins. J Biol Chem 285:32486–32493.  https://doi.org/10.1074/jbc.M110.139576CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Mizuno N, Varkey J, Kegulian NC, Hegde BG, Cheng N, Langen R et al (2012) Remodeling of lipid vesicles into cylindrical micelles by α-synuclein in an extended α-helical conformation. J Biol Chem 287:29301–29311.  https://doi.org/10.1074/jbc.M112.365817CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Emanuele M, Chieregatti E (2015) Mechanisms of alpha-synuclein action on neurotransmission: cell-autonomous and non-cell autonomous role. Biomolecules. 5:865–892.  https://doi.org/10.3390/biom5020865CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Fink AL (2006) The aggregation and fibrillation of α-synuclein. Acc Chem Res 39:628–634.  https://doi.org/10.1021/ar050073tCrossRefPubMedGoogle Scholar
  9. 9.
    Wood SJ, Wypych J, Steavenson S, Louis JC, Citron M, Biere AL (1999) alpha-synuclein fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of Parkinson’s disease. J Biol Chem 274:19509–19512.  https://doi.org/10.1074/jbc.274.28.19509CrossRefPubMedGoogle Scholar
  10. 10.
    Oueslati A, Fournier M, Lashuel HA (2010) Role of post-translational modifications in modulating the structure, function and toxicity of alpha-synuclein: implications for Parkinson's disease pathogenesis and therapies. Prog Brain Res. 183:115–145.  https://doi.org/10.1016/S0079-6123(10)83007-9CrossRefPubMedGoogle Scholar
  11. 11.
    Schmid AW, Fauvet B, Moniatte M, Lashuel HA (2013) Alpha-synuclein post-translational modifications as potential biomarkers for Parkinson disease and other synucleinopathies. Mol Cell Proteomics 12:3543–3558.  https://doi.org/10.1074/mcp.R113.032730CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Wang Z, Park K, Comer F, Hsieh-Wilson LC, Saudek CD, Hart GW (2009) Site-specific GlcNAcylation of human erythrocyte proteins potential biomarker(s) for diabetes. Diabetes 58:309–317.  https://doi.org/10.2337/db08-0994CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Wang Z, Udeshi ND, O'Malley M, Shabanowitz J, Hunt DF, Hart GW (2010) Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry. Mol Cell Proteomics 9:153–160.  https://doi.org/10.1074/mcp.M900268-MCP200CrossRefPubMedGoogle Scholar
  14. 14.
    Alfaro JF, Gong C-X, Monroe ME, Aldrich JT, Clauss TRW, Purvine SO et al (2012) Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. Proc Natl Acad Sci USA 109:7280–7285.  https://doi.org/10.1073/pnas.1200425109CrossRefPubMedGoogle Scholar
  15. 15.
    Morris M, Knudsen GM, Maeda S, Trinidad JC, Ioanoviciu A, Burlingame AL et al (2015) Tau post-translational modifications in wild-type and human amyloid precursor protein transgenic mice. Nat Neurosci. 18:1183–1189.  https://doi.org/10.1038/nn.4067CrossRefPubMedGoogle Scholar
  16. 16.
    Wang S, Yang F, Petyuk VA, Shukla AK, Monroe ME, Gritsenko MA et al (2017) Quantitative proteomics identifies altered O-GlcNAcylation of structural, synaptic and memory-associated proteins in Alzheimer’s disease. J Pathol 243:78–88.  https://doi.org/10.1002/path.4929CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Bond MR, Hanover JA (2015) A little sugar goes a long way: the cell biology of O-GlcNAc. J Cell Biol 208:869–880.  https://doi.org/10.1083/jcb.201501101CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Yang X, Qian K (2017) Protein O-GlcNAcylation: emerging mechanisms and functions. Nat Rev Mol Cell Biol 18:452–465.  https://doi.org/10.1038/nrm.2017.22CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    O’Donnell N, Zachara NE, Hart GW, Marth JD (2004) Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability. Mol Cell Biol 24:1680–1690CrossRefGoogle Scholar
  20. 20.
    Wang AC, Jensen EH, Rexach JE, Vinters HV, Hsieh-Wilson LC (2016) Loss of O-GlcNAc glycosylation in forebrain excitatory neurons induces neurodegeneration. Proc Natl Acad Sci USA 113:15120–15125.  https://doi.org/10.1073/pnas.1606899113CrossRefPubMedGoogle Scholar
  21. 21.
    Liu F, Iqbal K, Grundke-Iqbal I, Hart G, Gong C (2004) O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer’s disease. Proc Natl Acad Sci USA 101:10804–10809CrossRefGoogle Scholar
  22. 22.
    Aguilar AL, Hou X, Wen L, Wang PG, Wu P (2017) A chemoenzymatic histology method for O-GlcNAc detection. ChemBioChem 18:2416–2421.  https://doi.org/10.1002/cbic.201700515CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Yuzwa SA, Shan X, Macauley MS, Clark T, Skorobogatko Y, Vosseller K et al (2012) Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation. Nat Chem Biol 8:393–399.  https://doi.org/10.1038/nchembio.797CrossRefPubMedGoogle Scholar
  24. 24.
    Yuzwa SA, Cheung AH, Okon M, McIntosh LP, Vocadlo DJ (2014) O-GlcNAc modification of tau directly inhibits its aggregation without perturbing the conformational properties of tau monomers. J Mol Biol. 426:1736–1752.  https://doi.org/10.1016/j.jmb.2014.01.004CrossRefPubMedGoogle Scholar
  25. 25.
    Marotta NP, Lin YH, Lewis YE, Ambroso MR, Zaro BW, Roth MT et al (2015) O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease. Nat Chem. 7:913–920.  https://doi.org/10.1038/nchem.2361CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Lewis YE, Galesic A, Levine PM, De Leon CA, Lamiri N, Brennan CK et al (2017) O-GlcNAcylation of α-synuclein at serine 87 reduces aggregation without affecting membrane binding. ACS Chem Biol 12:1020–1027.  https://doi.org/10.1021/acschembio.7b00113CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Levine PM, Galesic A, Balana AT, Mahul-Mellier A-L, Navarro MX, De Leon CA et al (2019) α-Synuclein O-GlcNAcylation alters aggregation and toxicity, revealing certain residues as potential inhibitors of Parkinson’s disease. Proc Natl Acad Sci USA 116:1511–1519.  https://doi.org/10.1073/pnas.1808845116CrossRefPubMedGoogle Scholar
  28. 28.
    Zhang J, Lei H, Chen Y, Ma Y-T, Jiang F, Tan J et al (2017) Enzymatic O-GlcNAcylation of α-synuclein reduces aggregation and increases SDS-resistant soluble oligomers. Neurosci Lett 655:90–94.  https://doi.org/10.1016/j.neulet.2017.06.034CrossRefPubMedGoogle Scholar
  29. 29.
    Dawson P, Muir T, Clark-Lewis I, Kent S (1994) Synthesis of proteins by native chemical ligation. Science 266:776–779CrossRefGoogle Scholar
  30. 30.
    Muir TW, Sondhi D, Cole PA (1998) Expressed protein ligation: a general method for protein engineering. Proc Natl Acad Sci USA 95:6705–6710CrossRefGoogle Scholar
  31. 31.
    De Leon CA, Lang G, Saavedra MI, Pratt MR (2018) Simple and efficient preparation of O- and S-GlcNAcylated amino acids through InBr3-catalyzed synthesis of β-N-acetylglycosides from commercially available reagents. Org Lett 20:5032–5035.  https://doi.org/10.1021/acs.orglett.8b02182CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of Southern CaliforniaLos AngelesUSA
  2. 2.Department of Biological SciencesUniversity of Southern CaliforniaLos AngelesUSA

Personalised recommendations