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Conformational change in FALS-linked mutant Cu/Zn-SOD analyzed by monoclonal antibodies

通过单克隆抗体分析 FALS 连接突变体 Cu/Zn-SOD 的构象变化

基本信息

批准号：
17500242
负责人：
FUJIWARA Noriko
金额：
$ 2.3万
依托单位：
Hyogo College of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Amyotrophic lateral sclerosis (ALS) is a neurological disease characterized by selective motor neurons in the brain and spinal cord. Although more than 100 mutations have been identified in the Copper/Zinc-superoxide dismutase (Cu/Zn-SOD) in Familial amyotrophic lateral sclerosis (FALS), the mechanism responsible for FALS remains unclear. The finding of the present study shows that FALS causing-mutant Cu/Zn-SOD proteins (FALS mutant SODs), but not wild-type SOD, are barely detected by three monoclonal antibodies (mAbs) in Western blot analyses. ELISA for denatured FALS mutant SODs by DTT, SDS or heat treatment also showed a lowered immunoreactivity against the mAbs compared with wild-type SOD. Because all the epitopes of these mAbs are mapped within the Greek key loop (residues 102-115 in human Cu/Zn-SOD), these data uggest that different conformational changes occur in the loop between wild-type and FALS mutant SODs during the unfolding process. Circular dichroism measurements revealed that FALS mutant SODs are sensitive to denaturation by DTT, SDS or heat treatment, but these results partly but not completely explain the injurious properties of FALS mutant SODs. The findings reported herein suggest that, in addition to the instability of FALS mutant SODs, subtle conformational differences in the Greek key loop between wild-type and FALS mutant SODs during/after denaturation are involved in the etiology of FALS. In addition, recent studies suggest that oxidative damage of Cu/Zn-SOD itself has own pathogenicity in ALS. Through mass spectrometry and limited proteolysis, it was determined that the mass size of the molecule is 32 and 48, and that the modification site in Cu/Zn-SOD is Cys 111 in the Greek key loop. We demonstrated that the Cys111 is selectively oxidized to cysteine sulfinic acid (Cys-S0_2H) and to cysteine sulfonic acid (Cys-SO_3H) under mild oxidizing conditions.

肌萎缩侧索硬化症（ALS）是一种神经系统疾病，其特征在于大脑和脊髓中的选择性运动神经元。虽然在家族性肌萎缩侧索硬化症（Familial Amyotrophic Lateral Sclerosis，FALS）中已发现100多个铜/锌超氧化物歧化酶（Cu/Zn-SOD）突变，但其发病机制尚不清楚。本研究的发现表明，导致突变型Cu/Zn-SOD蛋白（FALS突变SOD），而不是野生型SOD，几乎没有检测到的三个单克隆抗体（mAb）在Western印迹分析。变性的FALS突变SOD的DTT，SDS或热处理的ELISA也显示出降低的免疫反应性对单克隆抗体相比，野生型SOD。由于这些单抗的所有表位都位于Greek关键环（人Cu/Zn-SOD中的残基102-115）内，因此这些数据表明，在解折叠过程中，野生型和FALS突变SOD之间的环中发生了不同的构象变化。圆二色性测定结果表明，FALS突变SOD对DTT、SDS或热处理的变性敏感，但这些结果部分但不完全解释了FALS突变SOD的有害特性。本文报道的研究结果表明，除了不稳定性的FALS突变体的SOD，微妙的构象差异，希腊之间的关键环野生型和FALS突变体的SOD变性过程中/后参与的FALS的病因。另外，近年来的研究表明，Cu/Zn-SOD本身的氧化损伤在ALS中具有一定的致病性。通过质谱分析和有限的蛋白酶解，确定了Cu/Zn-SOD的分子质量大小为32和48，修饰位点为希腊键环中的Cys 111。在温和氧化条件下，Cys 111被选择性氧化为半胱氨酸亚磺酸（Cys-SO_2 H）和半胱氨酸磺酸（Cys-SO_3 H）。