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 ser(396) to thr(413) tau region contains two or more phosphorylated residues and eight possible ser/thr phosphorylation sites, Immunologic studies and mass spectroscopy have identified ser(396) 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 ser(404) but not the corresponding non-phosphorylated peptide, PHF-9 demonstrated no immunoreactivity with a synthetic peptide phosphorylated at ser(396) indicating that the PHF-9 epitope is C-terminal to ser(396), In conclusion, the present study describes a Mab, PHF-9, which recognizes phosphorylated ser(404) of tau independently of phosphorylated ser(396) and indicates that tau ser(404) is phosphorylated in PHF. (C) 1996 Wiley-Liss, Inc.