Epitope mapping of monoclonal antibodies to the paired helical filaments of Alzheimer's disease: identification of phosphorylation sites in tau protein

Abstract

Tau is a neuronal phosphoprotein the expression of which is developmentally regulated. A single tau isoform is expressed in fetal human brain but six isoforms are expressed in adult human brain, with the fetal isoform corresponding to the shortest adult isoform. Phosphorylation is also developmentally regulated, as fetal tau is phosphorylated at more sites than adult tau. In Alzheimer's disease, the six adult tau isoforms become hyperphosphorylated and form the paired helical filament (PHF), the major fibrous component of the neurofibrillary lesions. One way to identify phosphorylated sites in tau is to use antibodies that recognize phosphorylated residues within a specific amino acid sequence. We here characterize the two novel phosphorylation-dependent anti-tau antibodies AT270 and AT180 and identify their epitopes as containing phosphorylated Thr-181 and Thr-231 respectively. With these antibodies we show that these two threonine residues are partially phosphorylated in fetal and adult tau and almost fully phosphorylated in PHF tau. This result contrasts with previous studies of Ser-202 and Ser-396 which are partially phosphorylated in fetal tau, unphosphorylated in adult tau but almost fully phosphorylated in PHF tau.

Oxidative Stress Induces Dephosphorylation of τ in Rat Brain Primary Neuronal Cultures

“…2 for Tau-l and AT8; data not shown for RT97, 8D8, SMI3!, and SMI31O), as well as a reduction in AT18O immunoreactivity and an increased mobility of T recognised by mAb AT270 (Fig. 2) that label phosphorylated residues Thr 231 and Thr'87 on i, respectively (Goedert et al, 1994). It thus appears that production of free radicals by H 202 leads to a sitespecific dephosphorylation of~rat Ser 202/Thr205 [Tau-I/AT8 epitope (Binder et al, 1985;Goedert et al, 1995) (Goedel-f et al, 1994)], and that the increased electrophoretic mobility is due to dephosphorylation at sites other than that recognised by mAb AT270.…”

“…2) that label phosphorylated residues Thr 231 and Thr'87 on i, respectively (Goedert et al, 1994). It thus appears that production of free radicals by H 202 leads to a sitespecific dephosphorylation of~rat Ser 202/Thr205 [Tau-I/AT8 epitope (Binder et al, 1985;Goedert et al, 1995)], Ser3°tãnd Ser404 [8D8, SMl3l, and SMI3IO epitopes (Hanger et al, 1992;Lichtenberg-Kraag et al, 1992)], Thr23' I AT18O epitope (Goedert et al, 1994)], and an N-terminal site [RT97 epitope (Brion et al, 1993a)], but not Thr't' [AT270 epitope (Goedel-f et al, 1994)], and that the increased electrophoretic mobility is due to dephosphorylation at sites other than that recognised by mAb AT270. In addition to the observed 'i-dephosphorylation, we also noted a change in the immunoreactivity of the anti-MAP2 mAb, API8 (Fig.…”

“…U.S.A.). The mAbs AT8, ATI8O, and AT270 (gifts from Innogenetics, Belgium, and purchased from Autogen Bioclear) recognise phosphorylated residues Ser202/Thr205, Thr231, and Thr°'of human 7~,respectively (Goedert et al, 1994(Goedert et al, , 1995. …”

Biochemistry and Cell Biology of Tau Protein in Neurofibrillary Degeneration

“…These discoveries led to concerted efforts by cell biologists and neuroscientists to elucidate the physiological and pathological properties of Tau. Human Tau and its splicing isoforms were identified (Goedert et al 1988), Tau-specific antibodies against normal and diseased states were developed (Kosik et al 1988;Wolozin et al 1986), abnormal posttranslational modifications of Tau were identified (e.g., phosphorylation sites, kinases, phosphatases; Biernat et al 1992;Hanger et al 1992;Goedert et al 1994), and the principles of abnormal aggregation emerged Wille et al 1992).…”

“…A subset of sites have gained prominence because they are recognized by antibodies raised against AD Tau in a phosphorylation-dependent manner and are thus useful as diagnostic reagents. They include PHF1 ( pS396 þ pS404; Greenberg and Davies 1990), several of the AT series of antibodies Goedert et al 1994), for example, AT8 ( pS202 þ pT205), AT180 ( pT231 þ pS235), AT270 ( pT181) and AT100 ( pT212 þ pS214), and Sternberger monoclonal antibodies of the SMI series (e.g., SMI31, 33, 34; Lichtenberg-Kraag et al 1992; for a complete list see www.alzforum.org). Most of these sites include SP or TP motifs and are therefore targets of proline-directed kinases (e.g., GSK3b, cdc2, cdk5, MAPK, JNK).…”