Oxidative Modification to Cysteine Sulfonic Acid of Cys111 in Human Copper-Zinc Superoxide Dismutase

Noriko Fujiwara,Miyako Nakano,Shinsuke Kato,Daisaku Yoshihara,Tomomi Ookawara,Hironobu Eguchi,Naoyuki Taniguchi,Keiichiro Suzuki

doi:10.1074/jbc.m702941200

Abstract

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-SO(2)H) and to cysteine sulfonic acid (Cys-SO(3)H). The antibody raised against a synthesized peptide containing Cys(111)-SO(3)H reacted with only the Cys(111)-peroxidized SOD1 by Western blot analysis and labeled Lewy body-like 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.

Highlights

The familial form of amyotrophic lateral sclerosis (ALS) is associated with specific mutations in the SOD1 gene (SOD1) that encodes 153 amino acids [9, 10]
These results clearly indicated that Cys111 was readily oxidized to Cys-SO2H, which underwent further oxidation to Cys-SO3H without His oxidation by air, and that the peroxidation of SOD1 at Cys111 resulted in the upper band shift in reducing SDS-PAGE
Because SOD1 catalyzes the conversion of superoxide radicals into molecular oxygen and hydrogen peroxide, SOD1 is thought to be a major target of oxidative stress

Summary

EXPERIMENTAL PROCEDURES

Materials—All chemicals used in this study were obtained either from Wako Pure Chemical Industries Ltd. (Osaka, Japan), Nacalai Tesque, Inc. (Kyoto, Japan), or Sigma unless specified otherwise. The IgG was desalted with a PD-10 column and stored with 0.1 mg/ml bovine serum albumin (BSA) at deep freeze until use After washing with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG (diluted 1:5000) for anti-C111ox-SOD1 or horseradish peroxidase-conjugated anti-goat IgG (diluted 1:5000) in TBS-T containing 1% skim milk for anti-SOD1, respectively, for 2 h at room temperature. The plates were washed three times with TBS-T, and 100 ␮l of anti-C111ox-SOD1 and anti-SOD1 antibodies (diluted 1:1000 in TBS-T) was added, followed by incubation for 1 h at room temperature. The plates were washed three times with TBS-T, and 100 ␮l of horseradish peroxidase-conjugated antirabbit IgG (diluted 1:5000 in TBS-T) for anti-C111ox-SOD1 or horseradish peroxidase-conjugated anti-goat IgG (diluted 1:5000 in TBS-T) for anti-SOD1, respectively, was added and incubated for 1 h at room temperature. Protein concentrations of crude samples were determined using a BCATM protein assay kit (Pierce) with BSA as a standard

RESULTS

Findings

DISCUSSION