Different Immunoreactivity against Monoclonal Antibodies between Wild-type and Mutant Copper/Zinc Superoxide Dismutase Linked to Amyotrophic Lateral Sclerosis

Fujiwara, Noriko; Miyamoto, Yasuhide; Ogasahara, Kyoko; Takahashi, M.; Takamiya, Rina; Suzuki, Keiichiro; Taniguchi, Naoyuki

doi:10.1074/jbc.m406106200

Cited by 11 publications

(20 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…the lower panel). Peak c has already been identified as residues 92-122 containing carbamidemethylated Cys 111 in previous work (25). When fractions containing the additional peaks d and e were reapplied to the ODS column, four fractions containing three distinct peaks were obtained (Fig.…”

Section: Role Of Cysmentioning

confidence: 56%

“…Production and Purification of Wild-type and Mutant SOD1 Proteins-Overproduction of SOD1s by the baculovirus/insect cells system and purification of SOD1 proteins were carried out as described previously (25).…”

Section: Methodsmentioning

confidence: 99%

“…It is thought that the two bands are unable to separate on the gradient gels. Next, various purified wild-type and mutant human SOD1 proteins expressed in the baculovirus/insect cell system (25) were oxidized with 1 mM H 2 O 2 , followed by reducing SDS-PAGE and Western blotting. As shown in Fig.…”

Section: Upper Band Of Oxidized Human Sod1 On Sds-page Involves Cysmentioning

confidence: 99%

“…After blocking by incubation with 5% skim milk in Tris-buffered saline (TBS; 20 mM Tris-HCl, pH 8.0, 0.15 M NaCl) for 2 h at room temperature, the membrane was incubated with anti-C111ox-SOD1 (diluted 1:1000), or a goat polyclonal antibody against full-length human SOD1 (25), anti-SOD1 (diluted 1:1000), in TBS containing 0.05% Tween 20 (TBS-T) and 1% skim milk for 2 h at room temperature or for 18 h at 4°C. 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.…”

mentioning

confidence: 99%

See 3 more Smart Citations

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

Fujiwara

Nakano

Kato

et al. 2007

Journal of Biological Chemistry

Self Cite

126

142

View full text Add to dashboard Cite

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). (2). Moreover, incubation with excess H 2 O 2 caused oxidation of almost all histidine and cysteine residues (3), fragmentation (4, 5) and aggregation (6, 7) of SOD1 itself. Co-incubation with bicarbonate and H 2 O 2 also induced bicarbonate radical anion formation, resulting in oligomerization of human SOD1 (8).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). To date, more than 110 familial ALS (FALS)-causing mutations in SOD1 have been identified (available on the World Wide Web); however, the mechanism by which SOD1 mutants induce ALS remains unknown. The presence of intracellular aggregates that contain SOD1 in spinal cord motor neurons is thought to be a pathological hallmark of ALS. In particular, FALS-linked mutant SOD1s are prone to misfolding and aggregation (11,12). Recently, Ezzi et al. (7) reported that even wild-type SOD1 results in aggregation after oxidation, and the oxidized wildtype SOD1 gains properties like FALS mutant SOD1s. In addition to ALS, oxidative damaged SOD1 proteins were detected in the brains of patients with Alzheimer and Parkinson diseases (13). These findings suggest that oxidized SOD1 plays a role in the pathophysiology of various neurodegenerative diseases.

show abstract

Section: Role Of Cysmentioning

confidence: 56%

Section: Methodsmentioning

confidence: 99%

Section: Upper Band Of Oxidized Human Sod1 On Sds-page Involves Cysmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Fujiwara

Nakano

Kato

et al. 2007

Journal of Biological Chemistry

Self Cite

126

142

View full text Add to dashboard Cite

show abstract

“…Each of these latter mutSOD1s in the metallated state has properties very similar to wtSOD1; however, the stabilities of their apoproteins differ widely. wtSOD1 and mutSOD1s show different immunoreactivities against commercial antibodies both in denaturing and reducing conditions (13) and nonreducing conditions (A.F., M.C., and M.T.C., unpublished work). Therefore, for this study, we made C-terminus myc-tagged ( Fig.…”

Section: Mutsod1s Show Diverse Properties With Respect To Superoxidementioning

confidence: 99%

Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials

Ferri¹,

Cozzolino

Crosio

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

234

211

View full text Add to dashboard Cite

Recent studies suggest that the toxicity of familial amyotrophic lateral sclerosis mutant Cu, Zn superoxide dismutase (SOD1) arises from its selective recruitment to mitochondria. Here we demonstrate that each of 12 different familial ALS-mutant SOD1s with widely differing biophysical properties are associated with mitochondria of motoneuronal cells to a much greater extent than wild-type SOD1, and that this effect may depend on the oxidation of Cys residues. We demonstrate further that mutant SOD1 proteins associated with the mitochondria tend to form cross-linked oligomers and that their presence causes a shift in the redox state of these organelles and results in impairment of respiratory complexes. The observation that such a diverse set of mutant SOD1 proteins behave so similarly in mitochondria of motoneuronal cells and so differently from wild-type SOD1 suggests that this behavior may explain the toxicity of ALS-mutant SOD1 proteins, which causes motor neurons to die. motor neuron ͉ neurodegeneration ͉ amyotrophic lateral sclerosis I n the familial form of ALS (fALS), which is linked to mutations in the Cu, Zn superoxide dismutase (SOD1) gene, it is generally considered that the pathological phenotype is because of the acquisition by the mutant SOD1 protein of new properties that transform it from a highly stable, dimeric antioxidant enzyme into a protein with a propensity to form toxic aggregates and cause oxidative damage to neuronal tissue (1). More than 100 different ALS-causing mutations in the SOD1 gene have been reported to date, and the properties of many of the fALS-linked mutant SOD1 proteins (mutSOD1s) have been studied (2-8) in a concerted effort to identify properties common to the mutant proteins but not shared by wild-type SOD1 (wtSOD1), which might explain their toxicity. Instead of similarities, however, biochemical and biophysical studies of the mutSOD1s have revealed a wide range of differences; in fact, some of the mutSOD1s have been found to be very similar to wtSOD1 in all of the properties that have been measured (1). For example, mutSOD1s with substitutions at or near the metalbinding region have altered metal-binding properties and a tendency to crystallize in filamentous structures, but other mutSOD1s with substitutions remote from the metal-binding region are very similar to wtSOD1 in their crystal structures (9-11). Likewise, the global stabilities of some of the metal ion-free mutSOD1 apoproteins are severely compromised, but others have apoproteins that are nearly identical to or even more stable than apo-wtSOD1 (7). Therefore, other abnormal properties of the mutant proteins must be sought to explain the toxicity of all of the mutSOD1s.We reasoned that the common properties shared by the ALS-mutSOD1 proteins and not by wtSOD1 protein might only become apparent when the mutant proteins were studied within the cellular context and also, possibly, only within motoneuronal cells. We have therefore built a collection of inducible cell lines, derived from a mouse motoneuronal line...

show abstract

Disease progression of human SOD1 (G93A) transgenic ALS model rats

et al. 2006

View full text Add to dashboard Cite

The recent development of a rat model of amyotrophic lateral sclerosis (ALS) in which the rats harbor a mutated human SOD1 (G93A) gene has greatly expanded the range of potential experiments, because the rats' large size permits biochemical analyses and therapeutic trials, such as the intrathecal injection of new drugs and stem cell transplantation. The precise nature of this disease model remains unclear. We described three disease phenotypes: the forelimb-, hindlimb-, and general-types. We also established a simple, non-invasive, and objective evaluation system using the body weight, inclined plane test, cage activity, automated motion analysis system (SCANET), and righting reflex. Moreover, we created a novel scale, the Motor score, which can be used with any phenotype and does not require special apparatuses. With these methods, we uniformly and quantitatively assessed the onset, progression, and disease duration, and clearly presented the variable clinical course of this model; disease progression after the onset was more aggressive in the forelimb-type than in the hindlimb-type. More importantly, the disease stages defined by our evaluation system correlated well with the loss of spinal motor neurons. In particular, the onset of muscle weakness coincided with the loss of approximately 50% of spinal motor neurons. This study should provide a valuable tool for future experiments to test potential ALS therapies.

show abstract

Different Immunoreactivity against Monoclonal Antibodies between Wild-type and Mutant Copper/Zinc Superoxide Dismutase Linked to Amyotrophic Lateral Sclerosis

Cited by 11 publications

References 45 publications

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

Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials

Disease progression of human SOD1 (G93A) transgenic ALS model rats

Contact Info

Product

Resources

About