期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 412, 期 4, 页码 743-746出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2011.08.046
关键词
Tau protein; Monoclonal antibody; Epitope mapping; Nuclear magnetic resonance spectroscopy; Phosphorylation; Fluorescence spectroscopy
资金
- Region Nord
- CNRS
- Pasteur Institute of Lille
- European Community
- French Research Ministry
- University of Sciences and Technologies of Lille I
- TGE RMN THC, France [FR-3050]
- Region Nord-CNRS
We present here the characterization of the epitope recognized by the AT180 monoclonal antibody currently used to define an Alzheimer's disease (AD)-related pathological form of the phosphorylated Tau protein. Some ambiguity remains as to the exact phospho-residue(s) recognized by this monoclonal: pThr231 or both pThr231 and pSer235. To answer this question, we have used a combination of nuclear magnetic resonance (NMR) and fluorescence spectroscopy to characterize in a qualitative and quantitative manner the phospho-residue(s) essential for the epitope recognition. Data from the first step of NMR experiments are used to map the residues bound by the antibodies, which were found to be limited to a few residues. A fluorophore is then chemically attached to a cystein residue introduced close-by the mapped epitope, at arginine 221, by mutagenesis of the recombinant protein. The second step of Forster resonance energy transfer (FRET) between the AT180 antibody tryptophanes and the phospho-Tau protein fluorophore allows to calculate a dissociation constant Kd of 30 nM. We show that the sole pThr231 is necessary for the AT180 recognition of phospho-Tau and that phosphorylation of Ser235 does not interfere with the binding. (C) 2011 Elsevier Inc. All rights reserved.
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