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

The action of N-acetylglucosaminyltransferase-V is prevented by the bisecting GlcNAc residue at the catalytic step

AbstractUsing a purified protein and bisected acceptor oligosaccharides, we demonstrate that N-acetylglucosaminyltransferase (GnT)-V transfers a N-acetylglucosamine residue via a β1,6-linkage to the bisected oligosaccharides. We also kinetically characterized the substrate specificity of GnT-V with respect to the bisected oligosaccharide. Although the Km values for the bisected acceptors were comparable to that for a non-bisected acceptor, the Vmax values for the bisected acceptors were much lower than that for the non-bisected acceptor. These findings suggest that the acceptor specificity of GnT-V is determined by the catalytic process rather than by its binding to the substrate. It was also found that the presence of the 2-N-acetyl group in the bisecting monosaccharide moiety plays a critical role in determining the catalytic efficiency of the enzyme

Cu,Zn-SOD deficiency induces the accumulation of hepatic collagen

<p>Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic diseases, and results in the development of fibrosis. Oxidative stress is thought to be one of the underlying causes of NAFLD. Copper/zinc superoxide dismutase (SOD1) is a primary antioxidative enzyme that scavenges superoxide anion radicals. Although SOD1 knockout (KO) mice have been reported to develop fatty livers, it is not known whether this lack of SOD1 leads to the development of fibrosis. Since the accumulation of collagen typically precedes liver fibrosis, we assessed the balance between the synthesis and degradation of collagen in liver tissue from SOD1 KO mice. We found a higher accumulation of collagen in the livers of SOD1 KO mice compared to wild type mice. The level of expression of HSP47, a chaperone of collagen, and a tissue inhibitor (TIMP1) of matrix metalloproteinases (a collagen degradating enzyme) was also increased in SOD1 KO mice livers. These results indicate that collagen synthesis is increased but that its degradation is inhibited in SOD1 KO mice livers. Moreover, SOD1 KO mice liver sections were extensively modified by advanced glycation end products (AGEs), which suggest that collagen in SOD1 KO mice liver might be also modified with AGEs and then would be more resistant to the action of collagen degrading enzymes. These findings clearly show that oxidative stress plays an important role in the progression of liver fibrosis.</p