Pathological analysis of abnormal activation for peptidylarginine deiminase in brain

瓜氨酸化蛋白是翻译后过程的产物，其中当被肽基精氨酸脱亚胺酶（PAD）以钙离子依赖性方式催化时，精氨酸残基经历修饰成瓜氨酸残基。在我们以前的报告中，PAD 2主要在大鼠大脑中表达，在神经退行性变过程的早期被激活。为了阐明瓜氨酸蛋白参与人类神经元变性，我们研究了瓜氨酸化蛋白是否在阿尔茨海默病（AD）期间产生。通过使用抗修饰瓜氨酸抗体的蛋白质印迹分析，在AD患者的海马组织中检测到不同分子量的瓜氨酸化蛋白，但在正常人中未检测到。通过双向凝胶电泳和MALDI-TOF质谱鉴定了两种瓜氨酸化蛋白，分别为波形蛋白和胶质细胞酸性蛋白（GFAP）。有趣的是，在AD和正常大脑的海马提取物中检测到了PAD 2， ...更多信息 AD组织中PAD 2的量显著增加。组织化学分析表明，瓜氨酸蛋白在整个海马，特别是在齿状回和放射层的CA1和CA2区。然而，在正常海马中未检测到瓜氨酸化蛋白。PAD2免疫反应性也普遍存在于整个AD和正常海马区。PAD2富集与瓜氨酸化蛋白阳性一致。双重免疫荧光染色显示瓜氨酸蛋白和PAD 2阳性细胞也与GFAP阳性细胞一致，但并非所有GFAP阳性细胞均为PAD 2阳性。与星形胶质细胞特异性标志蛋白GFAP一样，PAD 2主要分布在星形胶质细胞中。这些结果，瓜氨酸化蛋白的异常积累和AD患者大脑中PAD 2的异常激活，强烈表明PAD在AD的发生和发展中具有重要作用，瓜氨酸化蛋白可能成为人类神经退行性疾病的有用标志物。少

Citrullinated proteins are the products of a post-translational process in which arginine residues undergo modification into citrulline residues when catalyzed by peptidylarginine deiminases (PADs) in a calcium ion-dependent manner. In our previous report, PAD2 expressed mainly in the rat cerebrum became activated early in the neurodegenerative process. To elucidate the involvement of protein citrullination in human neuronal degeneration, we examined whether citrullinated proteins are produced during Alzheimer's disease (AD). By Western blot analysis using anti-modified citrulline antibody, citrullinated proteins of varied molecular weights were detected in hippocampal tissues from patients with AD but not normal humans. Two of the citrullinated proteins were identified as vimentin and glial fibrillary acidic protein (GFAP) by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Interestingly, PAD2 was detected in hippocampal extracts from AD and normal brains, bu … More t the amount of PAD2 in the AD tissue was markedly greater. Histochemical analysis revealed citrullinated proteins throughout the hippocampus, especially in the dentate gyrus and stratum radiatum of CA1 and CA2 areas. However, no citrullinated proteins were detected in the normal hippocampus. PAD2 immunoreactivity was also ubiquitous throughout both the AD and normal hippocampal areas. PAD2-enrichment coincided well with citrullinated protein-positivity. Double immunofluorescence staining revealed that citrullinated protein- and PAD2-positive cells also coincided with GFAP-positive cells, but not all GFAP-positive cells were positive for PAD2. Like GFAP, which is an astrocyte-specific marker protein, PAD2 is distributed mainly in astrocytes. These collective results, the abnormal accumulation of citrullinated proteins and abnormal activation of PAD2 in hippocampi of patients with AD, strongly suggest that PAD has an important role in the onset and progression of AD and that citrullinated proteins may became a useful marker for human neurodegenerative diseases. Less