Mapping O-GlcNAc modification sites on tau and generation of a site-specific O-GlcNAc tau antibody.
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.
Yuzwa
Scott A
SA
Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada.
Yadav
Anuj K
AK
Skorobogatko
Yuliya
Y
Clark
Thomas
T
Vosseller
Keith
K
Vocadlo
David J
DJ
D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis*; D001921:Brain / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods*; D011817:Rabbits; D051381:Rats; D016875:tau Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D016875:tau Proteins; D000117:Acetylglucosamine