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Amino Acids

, Volume 40, Issue 3, pp 857–868 | Cite as

Mapping O-GlcNAc modification sites on tau and generation of a site-specific O-GlcNAc tau antibody

  • Scott A. Yuzwa
  • Anuj K. Yadav
  • Yuliya Skorobogatko
  • Thomas Clark
  • Keith Vosseller
  • David J. Vocadlo
Original Article

Abstract

The microtubule-associated protein tau is known to be post-translationally modified by the addition of N-acetyl-d-glucosamine monosaccharides to certain serine and threonine residues. These O-GlcNAc modification sites on tau have been challenging to identify due to the inherent complexity of tau from mammalian brains and the fact that the O-GlcNAc modification typically has substoichiometric occupancy. Here, we describe a method for the production of recombinant O-GlcNAc modified tau and, using this tau, we have mapped sites of O-GlcNAc on tau at Thr-123 and Ser-400 using mass spectrometry. We have also detected the presence of a third O-GlcNAc site on either Ser-409, Ser-412, or Ser-413. Using this information we have raised a rabbit polyclonal IgG antibody (3925) that detects tau O-GlcNAc modified at Ser-400. Further, using this antibody we have detected the Ser-400 tau O-GlcNAc modification in rat brain, which confirms the validity of this in vitro mapping approach. The identification of these O-GlcNAc sites on tau and this antibody will enable both in vivo and in vitro experiments designed to understand the possible functional roles of O-GlcNAc on tau.

Keywords

O-GlcNAc Tau Antibody Mass spectrometry Alzheimer disease 

Notes

Acknowledgments

This work was supported by grants from the Canadian Institutes of Health (CIHR) and the Scottish Rite Charitable Foundation. DJV is a Canada Research Chair in Chemical Glycobiology and a Scholar of the Michael Smith Health Research Foundation. SAY is a recipient of a doctoral award from the Alzheimer Society of Canada. Ramesh Kaul and Garrett Whitworth are thanked for assistance with the peptide deprotection.

Supplementary material

726_2010_705_MOESM1_ESM.doc (142 kb)
Supplementary material 1 (DOC 142 kb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Scott A. Yuzwa
    • 1
  • Anuj K. Yadav
    • 2
  • Yuliya Skorobogatko
    • 3
  • Thomas Clark
    • 2
  • Keith Vosseller
    • 3
  • David J. Vocadlo
    • 1
    • 2
  1. 1.Department of Molecular Biology and BiochemistrySimon Fraser UniversityBurnabyCanada
  2. 2.Department of ChemistrySimon Fraser UniversityBurnabyCanada
  3. 3.Department of Biochemistry and Molecular BiologyDrexel University College of MedicinePhiladelphiaUSA

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