Kato K, Inaguma Y, Ito H, Iida K, Iwamoto I, Kamei K, Ochi N, Ohta H, Kishikawa M
Institute for Developmental Research, Aichi Human Service Center, Aichi, Japan.
J Neurochem. 2001 Feb;76(3):730-6. doi: 10.1046/j.1471-4159.2001.00038.x.
The phosphorylation state of alphaB-crystallin accumulated in the brains of two patients with Alexander's disease (one infantile and one juvenile type) was determined by means of SDS-PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against alphaB-crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25-28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of alphaB-crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser-59 in both cases. A significant level of phosphorylation at Ser-45 but not Ser-19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. alphaB-crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. AlphaB-crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimer's disease and aged controls. The phosphorylation site was mostly at Ser-59 in all cases. Immunohistochemically, alphaB-crystallin was stained in Rosenthal fibers in brains of patients with Alexander's disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser-59. These results indicate that the major phosphorylation site in alphaB-crystallin in brains of patients with Alexander's disease or Alzheimer's disease as well as in aged controls is Ser-59.
通过对两名亚历山大病患者（一名婴儿型和一名少年型）脑提取物的可溶和不溶部分进行SDS-PAGE或等电聚焦，随后用抗αB-晶状体蛋白及三个磷酸化丝氨酸残基各自的特异性抗体进行蛋白质免疫印迹分析，确定了在其大脑中积累的αB-晶状体蛋白的磷酸化状态。同样通过蛋白质免疫印迹分析估计了同一部分中25 - 28 kDa的哺乳动物小热休克蛋白（Hsp27）的水平。在脑匀浆的不溶部分检测到了大部分αB-晶状体蛋白，并且在两种情况下均优先在Ser-59处观察到磷酸化。还检测到Ser-45处有显著水平的磷酸化，但Ser-19处未检测到。在不溶部分发现了相当水平的Hsp27。在少年型患者的脑脊液中检测到了αB-晶状体蛋白及其磷酸化形式。在阿尔茨海默病患者和老年对照的脑匀浆不溶部分中也可检测到相当水平的αB-晶状体蛋白及其磷酸化形式。在所有情况下，磷酸化位点主要在Ser-59。免疫组织化学显示，αB-晶状体蛋白在亚历山大病患者大脑的罗森塔尔纤维中被染色，其周边部分被识别磷酸化Ser-59的抗体免疫染色。这些结果表明，亚历山大病患者、阿尔茨海默病患者以及老年对照大脑中αB-晶状体蛋白的主要磷酸化位点是Ser-59。
