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

用蛋白酶鉴定源自阿尔茨海默病配对螺旋丝的蛋白水解片段

基本信息

批准号：
60480224
负责人：
IHARA Yasuo
金额：
$ 4.16万
依托单位：
Tokyo Metropolitan Institute of Gerontology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1986
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-60480224/
关键词：
Alzheimer's disease paired helical filaments tau protein リン酸化

项目摘要

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.

由于成对螺旋丝的不溶性，常规分析方法无法应用于其亚基组成的分析。在我们寻找抗PHF反应性多肽（PHF的可能前体）的过程中，我们发现tau（一种神经元特异性微管相关蛋白）被抗PHF强烈标记。为了检验 tau 本身（而不是与 tau 具有共同抗原决定簇的其他蛋白质）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 tau 形式的 PHF 抗血清通过顺序应用到两个亲和柱上进行分级：去磷酸化 tau 柱和磷酸化 tau 柱。从第二根柱洗脱的抗体仅与磷酸化 tau 反应。由于 PHF 抗血清中的这些抗体可以识别磷酸化 tau 的独特构象，因此 PHF 本身很可能含有磷酸化 tau。为了进一步证实该假设的有效性，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，用赖氨酰内肽酶消化，并通过反相HPLC分离所得蛋白水解片段。纯化的人 tau 多肽也进行类似的处理。对两种 HPLC 谱中的共洗脱级分进行氨基酸组成和序列分析，发现 PHF 和 tau 共有两个独立片段。因此，可以确定 tau 本身已整合到 PHF 中。然而，PHF 中 tau 蛋白存在哪些类型的改变仍有待了解。