title: Characterization of the AT180 epitope of phosphorylated Tau protein by a combined nuclear magnetic resonance and fluorescence spectroscopy approach.
abstract: 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 Förster 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.
authors.affiliation: CNRS-UMR 8576 UGSF-IFR 147, Université des Sciences et Technologies de Lille 1, Villeneuve d'Ascq, France.
authors.forename: Laziza
authors.initials: L
authors.lastname: Amniai
authors.forename: Guy
authors.initials: G
authors.lastname: Lippens
authors.forename: Isabelle
authors.initials: I
authors.lastname: Landrieu
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016875:tau Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D016875:tau Proteins