CALPAIN-INDUCED PROTEOLYSIS OF NORMAL HUMAN TAU AND TAU ASSOCIATED WITH PAIRED HELICAL FILAMENTS
CALPAIN-INDUCED PROTEOLYSIS OF NORMAL HUMAN TAU AND TAU ASSOCIATED WITH PAIRED HELICAL FILAMENTS
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DOI：
10.1111/j.1432-1033.1995.009_1.x
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发表时间：
1995-10-01
期刊：
影响因子：
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通讯作者：
KSIEZAKREDING, H
中科院分区：
文献类型：
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作者：
YANG, LS;KSIEZAKREDING, H
阿尔茨海默病中神经元缠结（NFT）的主要成分是成对螺旋丝（PHF）束，其主要由高度磷酸化的tau蛋白（PHF-tau）组成。为了进一步了解PHF在NFT中积累的机制，我们研究了钙蛋白酶诱导的高度纯化和主要非聚集的PHF和正常tau蛋白的蛋白水解，这些蛋白具有从胎儿（F-tau）或成人脑（N-tau）分离的各种磷酸盐含量。使用蛋白质印迹分析和一组位点特异性tau抗体（Alz 50、Tau-2、Tau 14、Tau-1、AT 8、：E-11、AH-1和PHF-1）通过tau免疫反应性的降低来确定蛋白水解的程度。我们发现，在30 ℃下孵育10分钟期间，N-tau和F-tau的全长多肽在体外被钙蛋白酶（钙蛋白酶II，3.3单位/mg蛋白）类似地快速蛋白水解，并且它们的半衰期（t（1/2））分别为1.5分钟和1.8分钟。免疫印迹分析表明，tau的全长多肽首先降解成大小与内源性产生的相似的大片段，然后降解成较小的片段。由于内源性和体外产生的tau片段都保留了N末端表位，因此结果表明大多数钙蛋白酶敏感位点可能位于tau分子的C末端。相比之下，PHF对降解具有极强的抗性，并且仅5倍高浓度的钙蛋白酶（16.7单位/mg蛋白质）诱导PHF的部分蛋白水解。一个主要的钙蛋白酶产生的片段是一个45-kDa的多肽来源于PHF-tau的C-末端区域，它形成了一个核心的细丝。这些结果表明，潜在的钙蛋白酶消化位点在细丝核心的不可及性可能有助于PHF的钙蛋白酶的阻力，并随后导致PHF在阿尔茨海默病的积累。这些结果还表明，tau蛋白的过度磷酸化可能与PHF对钙蛋白酶降解的抗性有关。超微结构检查显示，与以前的研究胰蛋白酶。钙蛋白酶不改变纤维的形态外观;与钙蛋白酶孵育后，大多数PHF保持短而分散，聚集成NFT样簇的PHF数量没有显着增加。结果表明，钙蛋白酶在促进纤维聚集和成簇中的作用是有限的。
The major components of neurofibrillary tangles (NFT) in Alzheimer's disease are bundles of paired helical filaments (PHF) which are primarily composed of highly phosphorylated tau proteins (PHF-tau). To further understand the mechanism of PHF accumulation in NFT, we examined the calpain-induced proteolysis of highly purified and primarily non-aggregated PHF and normal tau proteins with various contents of phosphate isolated from either fetal (F-tau) or adult human brain (N-tau). The extent of proteolysis was determined by decreases in tau immunoreactivity using Western-blot analysis and a panel of site-specific tau antibodies (Alz 50, Tau-2, Tau 14, Tau-1, AT8,:E-11, AH-1 and PHF-1). We found that full-size polypeptides of N-tau and F-tau were similarly and rapidly proteolyzed in vitro by calpain (calpain II, 3.3 units/mg protein) during a 10-min incubation at 30 degrees C, and that their half lives (t(1/2)) were 1.5 min and 1.8 min, respectively. Analysis of immunoblots suggests that full length polypeptides of tau are first degraded into large fragments similar in size to that generated endogenously, then into smaller fragments. Since both endogenous and in-vitro-generated tau fragments retained N terminal epitopes, the results suggest that most of the calpain-sensitive sites may be located in the C-terminal half of the tau molecule. In contrast, PHF were extremely resistant to degradation and only a fivefold higher concentration of calpain (16.7 units/mg protein) induced partial proteolysis of PHF. A major calpain-generated fragment was a 45-kDa polypeptide derived from the C-terminal region of PHF-tau, which forms a core of filaments. The results suggest that the inaccessibility of potential calpain-digestion sites in the filament core could contribute to the resistance of PHF to calpain and subsequently lead to the accumulation of PHF in Alzheimer's disease. The results also suggest that hyperphosphorylation of tau may be mariginally involved in the resistance of PHF to degradation by calpain. Ultrastructural examination revealed that, in contrast to previous studies with trypsin. calpain did not alter the morphologic appearance of filaments; after incubation with calpain, the majority of PHF remained short and disperse and the number of PHF aggregated into NFT-like clusters was not significantly increased. The results suggest that the role of calpain in promoting the aggregation and clustering of filaments is limited.