Mapping O-GlcNAc modification sites on tau and generation of a site-specific O-GlcNAc tau antibody
- 1.2k Downloads
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.
KeywordsO-GlcNAc Tau Antibody Mass spectrometry Alzheimer disease
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.
- Lim KH, Ha CH, Chang HI (2002) Production of O-GlcNAc modified recombinant proteins in Escherichia coli. J Microbiol Biotech 12(2):306–311Google Scholar
- Wang Z, Udeshi ND, O’Malley M, Shabanowitz J, Hunt DF, Hart GW (2010) Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry. Mol Cell Proteomics 9(1):153–160CrossRefPubMedGoogle Scholar