Different immunoreactivity against monoclonal antibodies between wild-type and mutant copper/zinc superoxide dismutase linked to amyotrophic lateral sclerosis
Different immunoreactivity against monoclonal antibodies between wild-type and mutant copper/zinc superoxide dismutase linked to amyotrophic lateral sclerosis
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DOI：
10.1074/jbc.m406106200
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发表时间：
2005-02-11
影响因子：
4.8
通讯作者：
Taniguchi, N
中科院分区：
文献类型：
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作者：
Fujiwara, N;Miyamoto, Y;Taniguchi, N
尽管在家族性肌萎缩侧索硬化症 (FAILS) 中的铜/锌超氧化物歧化酶 (Cu/Zn-SOD) 中已发现 100 多个突变，但导致 FAILS 的机制仍不清楚。本研究的结果表明，在蛋白质印迹分析中，三种单克隆抗体 (mAb) 几乎检测不到导致 FALS 的突变型 Cu/Zn-SOD 蛋白（FALS 突变型 SOD），但野生型 SOD 无法检测到。通过二硫苏糖醇、SDS 或热处理对变性 FALS 突变型 SOD 进行酶联免疫吸附测定，也显示与野生型 SOD 相比，针对 mAb 的免疫反应性降低。由于这些 mAb 的所有表位均位于希腊键环内（人 Cu/Zn-SOD 中的残基 102-115），因此这些数据表明，在展开过程中，野生型和 FAILS 突变型 SOD 之间的环中发生了不同的构象变化。圆二色性测量表明，FALS 突变型 SOD 对二硫苏糖醇、SDS 或热处理的变性敏感，但这些结果并不能完全解释变性条件下野生型和 FAILS 突变型 SOD 之间的 mAb 的不同识别能力。对单克隆抗体监测局部区域构象变化的研究可能为FALS的病因学提供新的见解。
Although more than 100 mutations have been identified in the copper/zinc superoxide dismutase (Cu/Zn-SOD) in familial amyotrophic lateral sclerosis (FAILS), the mechanism responsible for FAILS remains unclear. The finding of the present study shows that FALS-causiing 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. The enzyme-linked immunosorbent assay for denatured FALS mutant SODs by dithiothreitol, 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 suggest that different conformational changes occur in the loop between wild-type and FAILS mutant SODs during the unfolding process. Circular dichroism measurements revealed that the FALS mutant SODs are sensitive to denaturation by dithiothreitol, SDS, or heat treatment, but these results do not completely explain the different recognition by the mAbs between wild-type and FAILS mutant SODs under the denatured conditions. The study on the conformational changes in local areas monitoring with mAbs may provide a new insight into the etiology of FALS.