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

使用抗氧化 SOD1 的新型抗体（抗 C111ox-SOD1）研究氧化 SOD1 对 ALS 的作用

• 批准号: 19500313
• 负责人: FUJIWARA Noriko
• 金额: $ 2.83万
• 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: 2007
• 资助国家: 日本
• 起止时间: 2007 至 2009
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-19500313/
• 关键词: スーパーオキシドジスムターゼ; Cu; Zn-SOD; 抗体; 筋萎縮性側索硬化症(ALS); 酸化的翻訳後修飾; MALDI-TOF-MS; 筋萎縮性側索硬化症; ALS; 酸化; 構造変化; チャージアイソマー

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.

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，并且在各种神经退行性疾病中具有其自身的致病性，包括家族性肌萎缩侧索硬化症（FALS）。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。本研究的目的是研究Cys 111在SOD 1蛋白的氧化损伤中的作用，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的氧化易感性。与2-ME-SOD 1相比，野生型SOD 1对过氧化氢氧化更敏感，产生片段和寡聚体。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。经质谱分析和有限蛋白酶解鉴定，该条带为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-SO_2 H）和半胱氨酸磺酸（Cys-SO_3 H）。Western blot分析表明，抗Cys 111-SO_3H合成肽的抗体仅与Cys 111过氧化的SOD 1反应，免疫组化分析表明，该抗体可标记人SOD 1突变型ALS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是氧化修饰的主要目标，并在氧化损伤中起重要作用，人类SOD 1包括FALS突变体。

项目成果

Protective role of glutathione S-transferase A4 induced in copper/zinc-superoxide dismutase knockout mice

谷胱甘肽 S-转移酶 A4 对铜/锌超氧化物歧化酶敲除小鼠的保护作用

• 发表时间: 2009
• 期刊: Free Radic. Biol. Med. 47
• 作者: Yoshihara D.;Fujiwara N.;Ookawara T.;Kato S.;Sakiyama H.;Yokoe S.;Eguchi H.;Suzuki K.
• 通讯作者: Suzuki K.

ALSにおける酸化ストレスおよび酸化型SODIの関与

氧化应激和氧化 SODI 在 ALS 中的作用

• 发表时间: 2009
• 期刊: 実験医学(増刊) 27
• 作者: Yoshihara D.;Fujiwara N.;Ookawara T.;Kato S.;Sakiyama H.;Yokoe S.;Eguchi H.;Suzuki K;藤原範子
• 通讯作者: 藤原範子

Cys111 in human copper-zinc-superoxide dismutase is a primary target for peroxidation to cysteine sulfonic acid

人铜锌超氧化物歧化酶中的 Cys111 是过氧化为半胱氨酸磺酸的主要靶标

• 发表时间: 2008
• 作者: Fujiwara N.;Nakano M.;Yoshihara D.;Ookawara T.;Eguchi H.;Taniguchi N.;Suzuki K.
• 通讯作者: Suzuki K.

Oxidative modification of Cys111 residue in human Copper/zinc superoxide dismutase and its immuno-probe

人铜/锌超氧化物歧化酶Cys111残基的氧化修饰及其免疫探针

• 发表时间: 2009
• 作者: Kawaguchi,K.;et al;Fujiwara N.
• 通讯作者: Fujiwara N.

Oxidative modification to cysteine sulfonic acid of Cys111 in human copper-zinc superoxide dismutase

• DOI: 10.1074/jbc.m702941200
• 发表时间: 2007-12-07
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Fujiwara, Noriko;Nakano, Miyako;Suzuki, Keiichiro
• 通讯作者: Suzuki, Keiichiro