Identification of proteolytic fragments derived from Alzheimer's paired helical filaments with proteases

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Because of the insolubility of paired helical filaments, the conventional analytical methods cannot be applied to the analysis of their subunit composition. In the course of our search for antiPHF reactive polypeptides, possible precursors fo PHF, we found that tau, a neuron-specific microtubule-associated protein, is strongly labeled with antiPHF. To test the hypothesis that tau itself, not other proteins sharing common antigenic determinants with tau, is integrated into PHF, we investigated whether PHF still retain some properties of tau, especially a phosphorylation-induced conformational change. PHF antisera recognizing both phosphorylated and non-phosphorylated forms of tau were fractionated by sequential application on the two affinity columns: dephosphorylated tau and phosphorylated tau columns. The antibodies eluted from the second column reacted exclusively with phosphorylated tau. Since those antibodies in PHF antisera can recognize a unique conformation of phosphorylated tau, it is likely that PHF contain phosphorylated tau itself. To further confirm the validity of the hypothesis, we need to prove particular sequences in the PHF digests identical to those of tau. PHF, after treatment of concentrated formic acid, were digested with lysyl-endopeptidase, and resultant proteolytic fragments were separated by reversed phase HPLC. Purified human tau polypeptides were similarly processed. Coeluted fractions in both HPLC profiles were analyzed for amino acid compositions and sequences and two independent fragments werefound to be shared by PHF and tau. Thus, it is definitive that tau itself is integrated into PHF. However, it remains to be known what kinds of alterations are present in tau in PHF.