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

肌萎缩侧索硬化症(ALS)是一种以大脑和脊髓中选择性运动神经元为特征的神经系统疾病。家族性肌萎缩侧索硬化症(FALS)患者的铜/锌超氧化物歧化酶(Cu/Zn-SOD)基因突变已超过100个，但其发病机制尚不清楚。本研究的结果表明，在Western印迹分析中，三种单抗几乎不能检测到引起突变的铜/锌超氧化物歧化酶蛋白(FALS突变型SODS)，而不是野生型的超氧化物歧化酶。与野生型SOD相比，经DTT、SDS或热处理变性的FALS突变型SOD对mAbs的免疫反应性也有所降低。由于这些单抗的所有表位都被映射到希腊关键环内(人类铜/锌-超氧化物歧化酶中的102-115残基)，这些数据提示在展开过程中野生型和fals突变超氧化物歧化酶之间的环中发生了不同的构象变化。圆二色谱分析表明，Fals突变超氧化物歧化产物对DTT、十二烷基硫酸钠和热处理的变性很敏感，但这些结果部分但不完全解释了Fals突变超氧化物歧化产物的伤害性。这些结果表明，除了fals突变超氧化物歧化酶的不稳定性外，野生型和fals突变超氧化物歧化酶在变性过程中和变性后希腊密钥环的细微构象差异也参与了fals的病因学。此外，最近的研究表明，在ALS中，铜/锌-超氧化物歧化酶本身的氧化损伤也具有一定的致病性。通过质谱分析和限制性蛋白水解法确定了该分子的质量大小分别为32和48，并确定其修饰位点为希腊关键环上的Cys 111。我们证明了在温和的氧化条件下，Cys111可以选择性地氧化成半胱氨酸亚磺酸(Cys-S02H)和半胱氨酸磺酸(Cys-SO3H)。

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.