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

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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.

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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.

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Authors and Affiliations

  1. Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Dr, Burnaby, BC, V5A 1S6, Canada

    Scott A. Yuzwa & David J. Vocadlo

  2. Department of Chemistry, Simon Fraser University, 8888 University Dr, Burnaby, BC, V5A 1S6, Canada

    Anuj K. Yadav, Thomas Clark & David J. Vocadlo

  3. Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA, 19129, USA

    Yuliya Skorobogatko & Keith Vosseller

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  1. Scott A. Yuzwa
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  2. Anuj K. Yadav
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  3. Yuliya Skorobogatko
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  4. Thomas Clark
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  5. Keith Vosseller
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  6. David J. Vocadlo
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Correspondence to David J. Vocadlo.

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Yuzwa, S.A., Yadav, A.K., Skorobogatko, Y. et al. Mapping O-GlcNAc modification sites on tau and generation of a site-specific O-GlcNAc tau antibody. Amino Acids 40, 857–868 (2011). https://doi.org/10.1007/s00726-010-0705-1

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  • DOI: https://doi.org/10.1007/s00726-010-0705-1

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