Locations and immunoreactivities of phosphorylation sites on bovine and porcine tau proteins and a PHF-tau fragment.
Tau protein is a phosphorylated neuronal microtubule-associated protein. Tau protein is also present in the major pathological lesions of Alzheimer's disease in an insoluble hyperphosphorylated state as paired helical filaments (PHFs). We have investigated the phosphorylation state of control taus and a fragment of PHF-tau. Tau samples were digested with protease, separated by reversed-phase high-performance liquid chromatography, and analyzed by mass spectrometry and Edman microsequencing. The serine homologous with S404 of human tau 441 was phosphorylated on bovine and porcine tau and up to two phosphates were present on a peptide of amino acids 182-240 of bovine tau (193-251 of human tau 441). The serine within the KSPV motif was not phosphorylated on bovine or porcine tau. PHF-tau fragments, isolated from pronase-treated PHFs encompassed a 93-amino acid region within the microtubule binding domain. Enzymatic digestion and mass spectrometric analysis showed no phosphate was present and a second carboxyl terminus was identified at E380. Antibodies T3P and SMI34, which recognize PHF-tau and peptides phosphorylated at the sequence KSPV, both reacted with bovine and porcine tau even though the KSPV sequence was not phosphorylated. These data indicate that the 93-amino acid sequence of F5.5 tau from PHFs is not phosphorylated, and the serine equivalent to S404 of human tau is phosphorylated in bovine and porcine tau. Antibodies T3P and SMI34 react with phosphorylated epitopes that are not unique to PHF-tau and that are not necessarily at the KSPV site.
Poulter
L
L
Biotechnology Department, ICI Pharmaceuticals, Alderley Park, Macclesfield, Cheshire.
Barratt
D
D
Scott
C W
CW
Caputo
C B
CB
D000595:Amino Acid Sequence; D000818:Animals; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D001921:Brain / Q000648:ultrastructure; D001923:Brain Chemistry; D002417:Cattle; D002458:Cell Fractionation; D006801:Humans; D007118:Immunoassay; D013058:Mass Spectrometry; D008870:Microtubules / Q000737:chemistry* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D013552:Swine; D014357:Trypsin; D016875:tau Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D010446:Peptide Fragments; D010748:Phosphopeptides; D016875:tau Proteins; D014357:Trypsin