The role of oxidized SOD1 on ALS using the new antibody against oxidized SOD1 (anti-C111ox-SOD1)

铜锌超氧化物歧化酶（SOD1）对氧化应激具有保护作用。另一方面，最近的研究表明SOD1本身是氧化损伤的主要靶点，在包括家族性肌萎缩侧索硬化症（FALS）在内的各种神经退行性疾病中具有自身的致病性。哺乳动物中只有人类和类人猿的SOD1分子表面有高活性的游离半胱氨酸残基Cys111。本研究的目的是通过比较2-巯基乙醇修饰的重组人SOD1 （2-ME-SOD1）与野生型SOD1的氧化敏感性，探讨Cys111在SOD1蛋白氧化损伤中的作用。与2- me型SOD1相比，野生型SOD1对过氧化氢产生的片段和低聚物更敏感。此外，野生型SOD1在空气氧化作用下对SDS-PAGE的还原也会产生上移位带，而2-ME-SOD1则不会。通过质谱分析和有限的蛋白水解，鉴定出该上带为SOD1的氧化亚基；Cys111的巯基（Cys-SH）被选择性氧化为半胱氨酸亚磺酸（Cys-SO_2H）和半胱氨酸磺酸（Cys-SO_3H）。经Western blot分析，该抗体仅与cys111 -过氧化SOD1反应，免疫组化分析，该抗体在人SOD1突变的ALS小鼠脊髓中标记出lewy -body样透明包涵体和液泡边缘。这些结果表明，Cys111是氧化修饰的主要靶点，在包括FALS突变体在内的人类SOD1的氧化损伤中起重要作用。

Copper-zinc superoxide dismutase (SOD1) plays a protective role against oxidative stress. On the other hand, recent studies suggest that SOD1 itself is a major target of oxidative damage and has its own pathogenicity in various neurodegenerative diseases, including familial amyotrophic lateral sclerosis (FALS). Only human and great ape SOD1s among mammals have the highly reactive free cysteine residue, Cys111, at the surface of the SOD1 molecule. The purpose of this study was to investigate the role of Cys111 in the oxidative damage of the SOD1 protein, by comparing the oxidative susceptibility of recombinant human SOD1 modified with 2-mercaptoethanol at Cys111 (2-ME-SOD1) to wild-type SOD1. Wild-type SOD1 was more sensitive to oxidation by hydrogen peroxide generating fragments and oligomers compared with 2-ME-SOD1. Moreover, wild-type SOD1, but not 2-ME-SOD1, generated an upper shifted band in reducing SDS-PAGE even by air oxidation. Using mass spectrometry and limited proteolysis, this upper band was identified as an oxidized subunit of SOD1 ; the sulfhydryl group (Cys-SH) of Cys111 was selectively oxidized to cysteine sulfinic acid (Cys-SO_2H) and to cysteine sulfonic acid (Cys-SO_3H). The antibody raised against a synthesized peptide containing Cys111-SO_3H reacted with only the Cys111-peroxidized SOD1 by Western blot analysis, and labeled Lewy-body-like hyaline inclusions and vacuole rims in the spinal cord of human SOD1-mutated ALS mice by immunohistochemical analysis. These results suggest that Cys111 is a primary target for oxidative modification and plays an important role in oxidative damage to human SOD1 including FALS mutants.