Conformation of paired helical filaments blocks dephosphorylation of epitopes shared with fetal tau except Ser199/202 and Ser202/Thr205

To determine if the high phosphate content of paired helical filaments (PHF s) in Alzheimer's disease (AD) is a result of limited access to filament ph osphorylation sites, we studied in vitro dephosphorylation of intact PHFs, PHFs with filamentous structure abolished by formic acid treatment (PHFFA) and fetal human tau protein. Samples were treated with alkaline phosphatase for up to 24 h at 37 degrees C and then immunoblotted with eight well char acterized tau antibodies, that recognize two phosphorylation-insensitive si tes and six phosphorylation-sensitive epitopes at Thr181, Ser199/202, Ser20 2/Thr205, Thr231, Ser262/356 and Ser396/404. Intact PHFs were effectively d ephosphorylated only at the two N-terminal epitopes Ser199/202 and Ser202/T hr205, with little change in electrophoretic mobility. In contrast, PHFFA w ere dephosphorylated at all epitopes, with particular effectiveness at thos e in the C-terminus and with significant increase in electrophoretic mobili ty. The fetal tau epitopes were effectively dephosphorylated except at Thr1 81 and Thr231 with marked increase in mobility. The extent of dephosphoryla tion of PHFFA was equal or more effective than in fetal tau, except for Thr 181 that was minimally dephosphorylated in both proteins. The results indic ate that intact PHFs, but not PHFFA or fetal tau display differential depho sphorylation of the N- and C-terminal epitopes. The results confirm that th e filamentous conformation may significantly contribute to hyperphosphoryla tion of PHFs in the C-terminus. The filamentous conformation, however, does not limit access to two N-terminal epitopes Ser199/202 and Ser202/Thr205. The access to these sites in AD may be limited by other factors, e.g., inhi bition of phosphatase binding. (C) 2000 Elsevier Science B.V. All rights re served.