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

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, 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, 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 Cys111 was selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and to cysteine sulfonic acid (Cys-SO3H). The antibody raised against a synthesized peptide containing Cys111-SO3H reacted with only the Cys111-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 Cys111 is a primary target for oxidative modification and plays an important role in oxidative damage to human SOD1, including familial amyotrophic lateral sclerosis mutants.This work was supported by Grants-in-aid for Scientific Research 17500242 and 19500313; a Hitech Research Center grant and the 21st Century Centers of Excellence program from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and in part by a Grant for the Research Group on Development of Novel Therapeutics for ALS from the Ministry of Health, Labor and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact

Retarded and aberrant splicings caused by single exon mutation in a phosphoglycerate kinase variant

The molecular abnormality of a phosphoglycerate kinase variant which was associated with severe tissue enzyme deficiency and episodes of muscle contractions and myoglobinuria was examined. Analysis of the patient's DNA showed the existence of a nucleotide transversion AIT --> C/G in exon 7. No other nucleotide change was detected, in the coding region of the variant gene. The mutation should produce a single amino acid substitution Glu --> Ala at protein position 251 counting from the NH2-terminal acetyl serine residue. The protein abnormality caused by the amino acid substitution cannot explain the enzyme deficiency. Northern blot hybridization indicated that the PGK mRNA content of the patient's lymphoblastoid cells was only about 10% of that of normal. Nucleotide sequence analysis revealed the existence of two PGK mRNA components in the patient's cells. The major component corresponds to the normal PGK mRNA except for A --> C change at nucleotide position 755 counting from adenine of the chain initiation codon. The minor component contains 5' region (52 bases) of intron 7 between exon 7 and exon 8, An inframe chain termination codon exists in the minor mRNA component, and the COOH-terminal half is expected to be deleted in the translation product. These results indicate that the low PGK activity in the patient's tissues is mainly due to retarded and aberrant pre-mRNA splicings caused by the change of the consensus 5' splice sequence AGgt to a nonconsensus sequence CGgt at the junction between exon 7 and intron 7 of the variant gene. (C) 1996 Academic Press, Inc