Oxidative modification to cysteine sulfonic acid of Cys111 in human copper-zinc superoxide dismutase
Oxidative modification to cysteine sulfonic acid of Cys111 in human copper-zinc superoxide dismutase
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DOI：
10.1074/jbc.m702941200
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发表时间：
2007-12-07
影响因子：
4.8
通讯作者：
Suzuki, Keiichiro
中科院分区：
文献类型：
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作者：
Fujiwara, Noriko;Nakano, Miyako;Suzuki, Keiichiro
铜锌超氧化物歧化酶（SOD 1）对氧化应激起保护作用。另一方面，最近的研究表明，SOD 1本身是氧化损伤的主要靶点，在各种神经退行性疾病中具有自身的致病性，包括家族性肌萎缩侧索硬化症。在哺乳动物中，只有人类和类人猿的SOD 1分子表面有高度活性的游离半胱氨酸残基Cys（111）。本研究的目的是通过比较在Cys（111）处用2-巯基乙醇修饰的重组人SOD 1（2-ME-SOD 1）与野生型SOD 1的氧化敏感性来研究Cys（111）在SOD 1蛋白的氧化损伤中的作用。野生型SOD 1是更敏感的氧化过氧化氢生成的片段，低聚物，和电荷异构体相比，2-ME-SOD 1。此外，野生型SOD 1，而不是2-ME-SOD 1，产生一个上移动的带在还原SDS-PAGE，即使通过空气氧化。使用质谱和有限的蛋白水解，该上部条带被鉴定为SOD 1的氧化亚基; Cys（111）的巯基（Cys-SH）被选择性氧化为半胱氨酸亚磺酸（Cys-SO2 H）和半胱氨酸磺酸（Cys-SO 3 H）。通过Western印迹分析，针对含有Cys（111）-SO 3 H的合成肽产生的抗体仅与Cys（111）-过氧化的SOD 1反应，并通过免疫组织化学分析标记人SOD 1突变的肌萎缩侧索硬化小鼠脊髓中的Lewy小体样透明包涵体和空泡边缘。这些结果表明，Cys（111）是氧化修饰的主要靶点，在人类SOD 1的氧化损伤中起重要作用，包括家族性肌萎缩侧索硬化突变体。
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. Only human and great ape SOD1s among mammals have the highly reactive free cysteine residue, Cys(111), at the surface of the SOD1 molecule. The purpose of this study was to investigate the role of Cys(111) in the oxidative damage of the SOD1 protein, by comparing the oxidative susceptibility of recombinant human SOD1 modified with 2-mercaptoethanol at Cys(111) (2-ME-SOD1) to wild-type SOD1. Wild-type SOD1 was more sensitive to oxidation by hydrogen peroxide-generating fragments, oligomers, and charge isomers 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 Cys(111) was selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and to cysteine sulfonic acid (Cys-SO3H). The antibody raised against a synthesized peptide containing Cys(111)-SO3H reacted with only the Cys(111)-peroxidized SOD1 by Western blot analysis and labeled Lewy bodylike hyaline inclusions and vacuole rims in the spinal cord of human SOD1-mutated amyotrophic lateral sclerosis mice by immunohistochemical analysis. These results suggest that Cys(111) is a primary target for oxidative modification and plays an important role in oxidative damage to human SOD1, including familial amyotrophic lateral sclerosis mutants.