Advertisement

Antibodies and Activity Measurements for the Detection of O-GlcNAc Transferase and Assay of its Substrate, UDP-GlcNAc

  • Tony Lefebvre
  • Ludivine Drougat
  • Stephanie Olivier-Van Stichelen
  • Jean-Claude Michalski
  • Anne-Sophie Vercoutter-Edouart
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1022)

Abstract

Since the discovery of O-GlcNAc modification (O-GlcNAcylation) 20 years ago, much attention has been given to OGT (O-GlcNAc transferase), the unique enzyme responsible for the nuclear and cytosolic O-GlcNAcylation processes. This review focuses on protocols that are routinely used to analyze OGT expression and activity. First are detailed techniques using rabbit polyclonal anti-OGT antibodies, namely, Western blot, (co-)immunoprecipitation, and immunofluorescence. We also describe the measurement of OGT activity by using synthetic peptides as acceptors and radiolabeled UDP-GlcNAc. Finally, a sensitive HPAEC-based technique to measure the cellular content of UDP-GlcNAc, the donor substrate of OGT, is described in detail.

Key words

O-GlcNAc transferase Polyclonal anti-OGT antibodies OGT activity assay UDP-GlcNAc content assay 

References

  1. 1.
    Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-­Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res 37:D233–D238PubMedCrossRefGoogle Scholar
  2. 2.
    Hanover JA, Yu S, Lubas WB, Shin SH, Ragano-­Caracciola M, Kochran J, Love DC (2003) Mitochondrial and nucleocytoplasmic isoforms of O-linked GlcNAc transferase encoded by a single mammalian gene. Arch Biochem Biophys 409:287–297PubMedCrossRefGoogle Scholar
  3. 3.
    Hu Y, Suarez J, Fricovsky E, Wang H, Scott BT, Trauger SA, Han W, Hu Y, Oyeleye MO, Dillmann WH (2009) Increased enzymatic O-GlcNAcylation of mitochondrial proteins impairs mitochondrial function in cardiac myocytes exposed to high glucose. J Biol Chem 284:547–555PubMedCrossRefGoogle Scholar
  4. 4.
    Torres CR, Hart GW (1984) Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc. J Biol Chem 259:3308–3317PubMedGoogle Scholar
  5. 5.
    Haltiwanger RS, Blomberg MA, Hart GW (1992) Glycosylation of nuclear and cytoplasmic proteins. Purification and characterizat­ion of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase. J Biol Chem 267:9005–9013PubMedGoogle Scholar
  6. 6.
    Kreppel LK, Blomberg MA, Hart GW (1997) Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats. J Biol Chem 272: 9308–9315PubMedCrossRefGoogle Scholar
  7. 7.
    Dehennaut V, Hanoulle X, Bodart JF, Vilain JP, Michalski JC, Landrieu I, Lippens G, Lefebvre T (2008) Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry. Biochem Biophys Res Commun 369:539–546PubMedCrossRefGoogle Scholar
  8. 8.
    Perez-Cervera Y, Harichaux G, Schmidt J, Debierre-Grockiego F, Dehennaut V, Bieker U, Meurice E, Lefebvre T, Schwarz RT (2011) Direct evidence of O-GlcNAcylation in the apicomplexan Toxoplasma gondii: a biochemical and bioinformatic study. Amino Acids 40:847–856PubMedCrossRefGoogle Scholar
  9. 9.
    Dehennaut V, Slomianny MC, Page A, Vercoutter-­Edouart AS, Jessus C, Michalski JC, Vilain JP, Bodart JF, Lefebvre T (2008) Identification of structural and functional O-linked N-acetylglucosamine-bearing proteins in Xenopus laevis oocyte. Mol Cell Proteomics 7:2229–2245PubMedCrossRefGoogle Scholar
  10. 10.
    Lubas WA, Hanover JA (2000) Functional expression of O-linked GlcNAc transferase. Domain structure and substrate specificity. Domain structure and substrate specificity. J Biol Chem 275:10983–10988PubMedCrossRefGoogle Scholar
  11. 11.
    Guinez C, Mir AM, Leroy Y, Cacan R, Michalski JC, Lefebvre T (2007) Hsp70-GlcNAc-binding activity is released by stress, proteasome inhibition, and protein misfolding. Biochem Biophys Res Commun 361: 414–420PubMedCrossRefGoogle Scholar
  12. 12.
    Dehennaut V, Lefebvre T, Leroy Y, Vilain JP, Michalski JC, Bodart JF (2009) Survey of O-GlcNAc level variations in Xenopus laevis from oogenesis to early development. Glycoconj J 26:301–311PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Tony Lefebvre
    • 1
  • Ludivine Drougat
    • 1
  • Stephanie Olivier-Van Stichelen
    • 1
  • Jean-Claude Michalski
    • 1
  • Anne-Sophie Vercoutter-Edouart
    • 1
  1. 1.Unit of Structural and Functional GlycobiologyUniversity of Lille 1Villeneuve d’AscqFrance

Personalised recommendations