Monoclonal antibody PHF-9 recognizes phosphorylated serine 404 of tau protein and labels paired helical filaments.
Paired helical filaments (PHFs) purified from alzheimer's brain consist of hyperphosphorylated microtubule-associated protein tau. In PHF, phosphorylation occurs at ser/thr tau residues. Several of these ser/thr phosphorylation sites lie immediately C-terminal to the tau tubulin binding domain. The C-terminal ser396 to thr413 tau region contains two or more phosphorylated residues and eight possible ser/thr phosphorylation sites. Immunologic studies and mass spectroscopy have identified ser396 as one of the phosphorylation sites but identification of more C-terminal phosphorylated residues has been hampered by the lack of monoclonal antibodies (Mabs) that recognize defined epitopes in this region. We have raised Mabs against PHF purified from Alzheimer's brain. One of these Mabs, PHF-9, showed phosphorylation-dependent binding to purified PHF and recognized a phosphorylated epitope in the C-terminal portion of cyanogen bromide-digested PHF. Epitope mapping studies employing synthetic tau phosphopeptides indicated that PHF-9 labeled a 13-mer tau peptide phosphorylated at ser404 but not the corresponding non-phosphorylated peptide. PHF-9 demonstrated no immunoreactivity with a synthetic peptide phosphorylated at ser396 indicating that the PHF-9 epitope is C-terminal to ser396. In conclusion, the present study describes a Mab, PHF-9, which recognizes phosphorylated ser404 of tau independently of phosphorylated ser396 and indicates that tau ser404 is phosphorylated in PHF.
Department of Psychiatry, University of Cincinnati College of Medicine, Ohio 45267-0559, USA.
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D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006801:Humans; D008969:Molecular Sequence Data; D016501:Neurites / Q000737:chemistry*; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry*; D016875:tau Proteins / Q000737:chemistry* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D016875:tau Proteins; D012694:Serine