Mapping <Emphasis Type="Italic">O</Emphasis>-GlcNAc modification sites on tau and generation of a site-specific <Emphasis Type="Italic">O</Emphasis>-GlcNAc tau antibody |
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Authors: | Scott A Yuzwa Anuj K Yadav Yuliya Skorobogatko Thomas Clark Keith Vosseller David J Vocadlo |
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Institution: | (1) Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Dr, Burnaby, BC, V5A 1S6, Canada;(2) Department of Chemistry, Simon Fraser University, 8888 University Dr, Burnaby, BC, V5A 1S6, Canada;(3) Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19129, USA; |
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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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