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

由于成对螺旋细丝的不溶性，传统的分析方法不能用于分析它们的亚基组成。在我们寻找抗PHF活性多肽（可能是PHF的前体）的过程中，我们发现tau，一种神经元特异性微管相关蛋白，被强烈标记为抗PHF。为了验证tau本身，而不是与tau具有共同抗原决定因子的其他蛋白质被整合到PHF中的假设，我们研究了PHF是否仍然保留tau的一些特性，特别是磷酸化诱导的构象变化。识别磷酸化和非磷酸化形式的tau蛋白的PHF抗血清通过顺序应用于两个亲和柱：去磷酸化tau蛋白和磷酸化tau蛋白柱进行分离。从第二柱洗脱的抗体只与磷酸化的tau蛋白反应。由于PHF抗血清中的这些抗体可以识别磷酸化tau蛋白的独特构象，因此PHF本身可能含有磷酸化tau蛋白。为了进一步确认假设的有效性，我们需要证明PHF消化中的特定序列与tau的序列相同。PHF经浓甲酸处理后，用赖氨酸内肽酶酶切，反相高效液相色谱法分离得到的蛋白水解片段。纯化的人tau多肽也进行了类似的处理。分析了两种HPLC图谱的离体部分的氨基酸组成和序列，发现PHF和tau有两个独立的片段共享。因此，可以确定的是，tau本身被整合到PHF中。然而，在PHF中tau蛋白中存在什么样的改变仍然是未知的。

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.