New phosphorylation sites identified in hyperphosphorylated tau (paired helical filament-tau) from Alzheimer's disease brain using nanoelectrospray mass spectrometry.

Paired helical filaments (PHFs) are the structural constituents of neurofibrillary tangles in Alzheimer's disease and are composed of hyperphosphorylated forms of the microtubule-associated protein tau (PHF-tau). Pathological hyperphosphorylation of tau is believed to be an important contributor to the destabilisation of microtubules and their subsequent disappearance from tangle-bearing neurons in Alzheimer's disease, making elucidation of the mechanisms that regulate tau phosphorylation an important research goal. Thus, it is essential to identify, preferably by direct sequencing, all of the sites in PHF-tau that are phosphorylated, a task that is incomplete because of the difficulty to date of purifying insoluble PHF-tau to homogeneity and in sufficient quantities for structural analysis. Here we describe the solubilisation of PHF-tau followed by its purification by Mono Q chromatography and reversed-phase HPLC. Phosphopeptides from proteolytically digested PHF-tau were sequenced by nanoelectrospray mass spectrometry. We identified 22 phosphorylation sites in PHF-tau, including five sites not previously identified. The combination of our new data with previous reports shows that PHF-tau can be phosphorylated on at least 25 different sites.

Phosphorylation of tau by cyclic-AMP-dependent protein kinase.

Alzheimer's disease paired helical filaments contain abnormally phosphorylated tau (PHF-tau) which has reduced electrophoretic mobility on sodium dodecyl sulphate polyacrylamide electrophoresis. We have investigated the effects of cyclic-AMP-dependent protein kinase (PKA) on recombinant human tau isoforms and two recombinant tau fragments. PKA phosphorylated tau and reduced its electrophoretic mobility, phosphorylation towards the C-terminus of tau having a major influence on this property. Substitution of serine396 (phosphorylated in PHF-tau) or serine416 (phosphorylated by calcium/calmodulin kinase II) by alanine demonstrated that these are not major sites for PKA phosphorylation. Although the phosphorylated forms of tau generated by PKA are not identical to those of PHF-tau, PKA may be involved in the generation of PHF-tau in Alzheimer's disease via phosphorylation of additional, as yet unidentified, sites on tau.

Neurofilament monoclonal antibodies RT97 and 8D8 recognize different modified epitopes in paired helical filament-tau in Alzheimer's disease.

Neurofibrillary tangles in Alzheimer's disease have been previously found to be labeled by some neurofilament antibodies that also recognize tau proteins. We have studied the reactivity of two such monoclonal antibodies, RT97 and 8D8, and of an anti-ubiquitin serum with the abnormal paired helical filaments (PHF)-tau (A68) polypeptides known to be the main component of the PHFs constituting the neurofibrillary tangles. 8D8 recognized the three major PHF-tau polypeptides, but RT97 reacted only with the two larger PHF-tau species. PHF-tau polypeptides were labeled by 8D8 and RT97 much more strongly than normal human tau and this labeling was decreased after alkaline phosphatase treatment. Anti-ubiquitin and anti-phosphotyrosine antibodies did not label PHF-tau polypeptides. The immunoreactivity of proteolytic fragments of PHF-tau polypeptides was studied with RT97, 8D8, and a panel of tau antibodies. The epitope for 8D8 on PHF-tau was localized between amino acids 222 and 427 in the carboxyl half of tau. The RT97 epitope on PHF-tau was localized in the amino domain of tau, probably in the 29-amino-acid insertion (insert 1) found towards the amino terminus of some tau isoforms. These results show that the basis for the labeling of neurofibrillary tangles by antibodies 8D8 and RT97 to neurofilament is their ability to react with PHF-tau polypeptides by recognizing sites specifically modified on PHF-tau, including a site specific to some tau isoforms.

Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries.

We present a simple, efficient and rapid method for affinity-purifying antibodies from a relatively crude antiserum in quantities large enough to screen a DNA expression library. The method presents a very convenient way to remove crossreacting or contaminating antibody specificities. The affinity matrix, antigen non-covalently bound to nitrocellulose, is prepared by the electrophoretic separation of antigen by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by the transfer of antigen to nitrocellulose. The matrix can be used repeatedly. A brief wash with 6 M guanidine hydrochloride is included between steps to remove residual antibodies which bind with high affinity to nitrocellulose-bound antigen. Various buffer solutions were assessed as antibody/antigen-dissociating agents. Glycine/HCl buffer, pH 2.5, appeared to be the most efficient in our hands, although a number of other less efficient dissociating reagents, including 4.5 M magnesium chloride, pH 7.5, 6 M urea, pH 7, and 0.05 M diethylamine, pH 11.5, also could be used; these may be the elution conditions of choice for other antibody/antigen combinations. The use of affinity-purified antibody solutions instead of the corresponding antisera gave increased signal-to-noise ratios with the detection systems that are commonly used to identify positive signals in screening expression libraries. Protein A- and goat anti-rabbit-alkaline phosphatase conjugates gave the most sensitive signals.