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J. Biol. Chem., Vol. 282, Issue 49, 35933-35944, December 7, 2007
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12
1
From the
Department of Biochemistry, Hyogo College of Medicine, Nishinomiya, Hyogo, 663-8501, Japan, the Department of Biochemistry, Osaka University Medical School and Graduate School of Medicine, Suita, Osaka, 565-0871, Japan, the ¶Department of Neuropathology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, Nishi-cho 36-1, Yonago 683-8504, Japan, and the ||Department of Disease Glycomics, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
Received for publication, April 6, 2007 , and in revised form, August 31, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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) into O2 and H2O2, thereby protecting cells against oxidative stress. On the other hand, SOD1 exhibits peroxidase activity and oxidizes various substrates in the presence of hydrogen peroxide, H2O2 (1). Although H2O2 is a substrate as well as a product of SOD1, incubation of bovine SOD1 with H2O2 caused oxidation of His118 (corresponding to His120 in human SOD1) to 2-oxohistidines, inactivating the enzyme (2). Moreover, incubation with excess H2O2 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 H2O2 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 wild-type 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. However, the role of oxidized wild-type and FALS-linked mutant SOD1s on these diseases remains unclear.
Human SOD1 has four cysteine residues, Cys6, Cys57, Cys111, and Cys146. An internal disulfide bond exists between Cys57 and Cys146 (14, 15), which contributes to the high stability of the SOD1 protein. This disulfide bond is highly conserved in SOD1s from various organisms, including yeast, plants, flies, fishes, and mammals. In contrast, two free cysteines, Cys6 and Cys111, are not conserved. Actually, yeast, fungi, and spinach (plants) have no free cysteines, and residue 6 is Ala and residue 111 is Ser in these organisms (16). More evolved organisms, such as flies, fishes, and mammals, including the Japanese monkey, have only one free cysteine, Cys6. Only humans and great apes (chimpanzee and orangutan) have two free cysteines, Cys6 and Cys111 (17). Notably, the amino acid sequence of chimpanzee SOD1 is identical to that of human SOD1. Although the evolutionary process may differ from humans and great apes, chicken SOD1 has three free cysteines, including Cys6 and Cys111. The third free Cys residue is located at the C terminus, Cys154 (18). Because free cysteines are generally reactive, and wild-type SOD1 is less thermo-stable than Ser111-SOD1 or Ala6-SOD1 (19), the A6C and S111C mutations during evolution are puzzling. In particular, Cys111 is located at the surface of the SOD1 molecule and is thought to be highly reactive. De Beus et al. (20) reported that Cys111 was modified with persulfide (S-SH) in a human SOD1 isolated from erythrocytes that is commercially available (Sigma). The persulfide SOD1 was more resistant to copper-induced aggregation than wild-type SOD1 (20). The sulfur atom of cysteine is able to assume several different oxidation states. Reversible oxidation of cysteine to disulfide (-S-S-) or sufenic acid (-SOH) is readily accomplished by thiols, such as DTT, 2-ME, glutathione, or thioredoxin. In contrast, oxidation to sulfinic acid (-SO2H) or sulfonic acid (-SO3H) is not reduced by these thiols under physiological conditions (21). For example, one cysteine in the active site of peroxiredoxin (Prx) is oxidized to sulfinic acid (-SO2H) by incubation with an excess of substrate of this enzyme, H2O2, and rereduced by a specific enzyme, sulfiredoxin, but not by general thiols (22, 23).
An upper shifted band of human wild-type or mutant SOD1s, with the exception of mutant C111S, on SDS-PAGE has been observed under a variety of conditions: during purification (24) or when hydrogen peroxide or copper ion is added (see Fig. 1). The SOD1 in the upper band is speculated to be irreversibly linked to another molecule via a covalent bond at Cys111; however, neither the molecule nor the modification site have been identified. Ube Industries Ltd. developed recombinant human SOD1 chemically modified with 2-mercaptoethanol at Cys111 (2-ME-SOD1; Cys111-S-S-CH2CH2OH). This 2-ME-SOD1 is stable for many years in aqueous solution, showing neither degradation nor a loss of activity. Thus, in this study, the role of Cys111 in oxidative damage of human SOD1 was investigated by comparing 2-ME-SOD1 and wild-type SOD1, and the identity of the molecule that is bound to human SOD1 in the upper band on SDS-PAGE was explored. Through mass spectrometry and limited proteolysis, it was determined that the mass size of the molecule is 32 and 48 and that the modification site in SOD1 is Cys111. We demonstrated that Cys111 in human SOD1 is selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and to cysteine sulfonic acid (Cys-SO3H) even by air oxidation. Moreover, a polyclonal antibody was raised against a synthesized peptide containing Cys111-SO3H. This antibody, denoted as anti-C111ox-SOD1, reacted with the upper band (oxidized SOD1) but not the original band by Western blot analysis. Using this anti-C111ox-SOD1, the role of Cys111 on the generation of SOD1 charge isomers and the presence of oxidized SOD1 in the spinal cord of ALS mice were investigated.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-cyano-4-hydroxycinnamic acid for matrix of MALDI-TOF MS were obtained from Bruker Daltonik GmbH (Bremen, Germany). Conversion of 2-ME-SOD1 to Wild-type SOD1—2-ME-SOD1 was incubated with 20 mM 2-ME for 1 h on ice and desalted with a PD-10 column. The resultant wild-type SOD1 and 2-ME-SOD1 were used in the experiments with the exception of the experiments in Fig. 1, B and C.
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).
Oxidation of SOD1 and the Analyses with MonoQ Column—For strong oxidation, SOD1s diluted with miliQ water (1 mg/ml) were incubated with 5 mM H2O2 for 1 h at room temperature. For mild air oxidation, SOD1s diluted with miliQ water or appropriate buffer were filtered with a 0.22-µm filter (Millipore) and were slowly stirred at 30 rpm with a rotator (rotator RT-50; Taitec). The buffer or water containing oxidized SOD1s was changed with buffer A (2 mM potassium phosphate, pH 7.4) on a PD-10 column (Amersham Biosciences). The SOD1s were applied to a high performance liquid chromatograph (AKTA Explorer 10S) at a flow rate of 1 ml/min on a MonoQ column (MonoQTM 4.6/100 PE; Amersham Biosciences). After washing with buffer A, the bound proteins were eluted with a linear gradient of KCl (0-100 mM) in buffer A. Adhesive proteins were washed with 0.5 M KCl in buffer A.
Lysylendopeptidase Treatment and Peptide Analyses—SOD1 proteins were reduced with DTT, and the free sulfhydryls were carbamidemethylated by adding iodoacetamide (IA) in the dark at room temperature for 30 min. After desalting on a PD-10 column with 50 mM Tris-HCl (pH 8.8), the proteins were digested with 0.25% (w/w) lysylendopeptidase (Wako Pure Chemicals) at 37 °C for 16 h. The resultant peptides were applied to a reverse-phase high performance liquid chromatograph (AKTA Explorer 10S) at a flow rate of 1 ml/min on TSK-GEL ODS-80TM (4.6 x 250 mm; Tosoh). The peptides were separated by two linear gradients of 0-30% acetonitrile for 4 column volumes and 30-40% acetonitrile for 8 column volumes containing 0.05% trifluoroacetic acid. Peptides were detected by their absorbance at 215 nm. The peaks were subjected to the following MS analyses.
MALDI-TOF MS Analysis—MALDI-TOF MS spectra of peptides and proteins were measured on an Ultraflex TOF/TOF mass spectrometer and analyzed by the Flexcontrol 1.2 software package (Bruker Daltonics GmbH, Bremen, Germany). For analyses of peptides, ions generated by a pulsed UV laser beam (nitrogen laser; 
= 337 nm, 5 Hz) were accelerated to a kinetic energy of 20 kV in reflector mode using positive polarity. Metastable ions generated by laser-induced decomposition of the selected precursor ions were analyzed without any additional collision gas. -Cyano-4-hydroxycinnamic acid (5 mg/ml in 50% acetonitrile containing 0.1% trifluoroacetic acid) was used as a matrix for peptide analyses. For analyses of proteins, the determinations were performed in linear mode using positive polarity. Sinapinic acid (10 mg/ml in 50% acetonitrile containing 0.1% trifluoroacetic acid) was used as matrix for protein analyses. Peptide or protein samples (1 µl each) were mixed with matrix solution (4 µl each), and an aliquot (1 µl each) was applied to a polished stainless steel target (Bruker Daltonics). The mixture was dried in air at room temperature for several minutes.
Infusion ESI MS Analysis for Peptide Sequence—ESI mass spectra were measured on a Bruker Esquire HCT equipped with a quadrupole ion trap (Bruker Daltonics GmbH, Bremen, Germany). The solutions containing peptides digested with lysylendopeptidase were continuously introduced through the electrospray interface with a syringe infusion pump (Cole-Parmer, Vernon Hills, IL) at a flow rate of 5 µl/min. The MS conditions were as follows: nebulizer gas (N2), 10 p.s.i.; dry gas (N2), 4 liters/min; dry temperature, 250 °C; capillary voltage, 3500 V; high voltage end plate offset, -500 V; capillary exit, 190.6 V; skimmer, 40 V; trap drive, 166.7. MS/MS spectra were sequenced using BioTool 2.2 software and Sequence editor 2.2 (algorithm provided by Bruker).
Trypsin Digestion for MALDI-TOF MS—SOD1 proteins in 50 mM NH4HCO3 were digested with trypsin at 37 °C for 16 h. An aliquot of the digests (10 µl) was boiled, applied to ZipTip C18 P10 (Millipore, Bedford, MA; according to the manufacturer's protocol) for desalting, and then subjected to MALDI-TOF MS analysis. When alkylation is needed, SOD1 proteins in 50 mM NH4HCO3 were treated with excess IA in the dark at room temperature for 30 min before trypsin digestion.
In-gel Digestion of Coomassie Brilliant Blue-stained Polyacrylamide Gel for MALDI-TOF MS—The gel bands containing SOD1 protein after SDS-PAGE were clipped out and cut into small pieces in a 1.5-ml microtube. To remove Coomassie Brilliant Blue dye, the chopped gels were washed three times with 50 mM NH4HCO3 in 30% acetonitrile by shaking at room temperature for 20 min. The gels were further incubated with 500 µl of acetonitrile at room temperature for 10 min. After removing acetonitrile, the gels were incubated with an alkylating solution (500 µl) consisting of 40 mM IA, 10 mM EDTA, and 50 mM NH4HCO3 in the dark at room temperature for 30 min. After washing twice with 50 mM NH4HCO3 (500 µl) for 10 min, the gels were incubated with 0.4 µg of trypsin in 50 mM NH4HCO3 at 37 °C overnight. After removing the pieces of gels, the remained solution was concentrated with a SpeedVac concentrator and subjected to MALDI-TOF MS analyses.
Preparation of Antibody for Cys111-sulfonylated SOD1—Key-hole limpet hemocyanin-coupled peptide (residues 103-114) containing sulfonylated Cys111 (Cys111-SO3H) was obtained from Sigma. After the initial injection with the peptide-hemocyanin conjugate (200 µg of peptide) mixed with complete Freund's adjuvant, rabbits were subjected to five booster injections, each of 200 µg of peptide with incomplete Freund's adjuvant, administered (at multiple subcutaneous sites) at 1-2-week intervals. Antisera were collected 1 week after the sixth booster injection, and the IgG fraction was precipitated with 50% (w/v) ammonium sulfate. The IgG fraction passed from the wild-type SOD1 coupled to N-hydroxysucinimide-activated Sepharose was bound to the oxidized SOD1 coupled to N-hydroxysucinimide-activated Sepharose. The bound IgG was eluted with 3 M MgCl2 and collected. 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. This antibody was denoted as anti-C111ox-SOD1.
SDS-PAGE and Western Blot Analysis—Proteins were subjected to SDS-PAGE (14% gel) and then transferred to a PVDF membrane under semidry conditions by means of a Trans-blot (Bio-Rad). 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. After washing, the chemiluminescence method using an ECL or an ECL plus kit (GE Healthcare) was employed to detect peroxidase activity.
Two-dimensional Gel Electrophoresis—Sample proteins were dissolved in 8 M urea, 2% (w/v) CHAPS, 0.5% (v/v) immobilized pH gradient (IPG) buffer (GE Healthcare), and 12 µl/ml DeStreakTM reagent (GE Healthcare), which forms stable disulfide bonds and prevents nonspecific Cys residue oxidation during isoelectric focusing (26). The samples were applied to 11-cm IPG strips (pH 4-7), and the strips were then isoelectrically focused on an IPGphor isoelectric focusing system (GE Healthcare) according to the following schedule: 500 V-h at 500 V for step and hold, 800 V-h at 1000 V for gradient, 8800 V-h at 6000 V for gradient, and 4000 V-h at 6000 V for step and hold. The strips were equilibrated for 20 min in 50 mM Tris-HCl (pH 8.8) containing 6 M urea, 2% (w/v) SDS, 30% (v/v) glycerol, and 1% (w/v) DTT. Second dimension separation was run on 14% SDS-polyacrylamide gels and followed by Western blot analysis. pI values of spots were calculated according to the graph showing pH as function of distance at 20 °C and 8 M urea of IPG strips (pH 4-7) provided by the GE Healthcare on-line system.
ELISA—Wild-type SOD1 was air-oxidized, treated with and without 100 mM IA, and then diluted to 500 ng/ml with phosphate-buffered saline (PBS). 100 µl of the samples were added to each well of 96-well microplates (Maxisorp; Nunc), incubated overnight at 4 °C, washed three times with TBS-T, and then blocked for 2 h at room temperature with 1% BSA in PBS. The plates were then washed three times with TBS-T, and 100 µl 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 µl of horseradish peroxidase-conjugated anti-rabbit 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. After washing five times with TBS-T, the plates were developed using 100 µl of o-phenylenediamine dihydrochloride solution, and the reaction was stopped with 25 µl of 2 M HCl. The absorbance of each well was determined at 490 nm with a SPECTRAmax PLUS384 (Molecular Devices).
Animals and Animal Tissue Preparation—Four transgenic mice carrying a high copy number of the human G93A SOD1 gene (B6SJL-TgN[SOD1-G93A]1Gur, G1H-G93A) mice, were purchased from the Jackson Laboratory (Bar Harbor, ME). Two age-matched littermates were used as controls. All animals were handled in accordance with the guidelines for care and use (Tottori University). All four G1H-G93A mice at 110 days of age neurologically exhibited hind limb paralysis, and two littermate mice at 110 days of age did not show any clinical symptoms. The G1H-G93A and littermate mice were euthanized at 110 days of age. Animals were deeply anesthetized with sodium pentobarbital (0.1 ml/100 g of body weight). After perfusion of three G1H-G93A and two littermate mice via the aorta with PBS at 37 °C, they were fixed by perfusion with 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3). The spinal cords were removed and then postfixed in the same solution. The spinal cord of one mouse for Western blot analysis was removed after perfusion with PBS, quickly frozen in liquid nitrogen, and stored at -80 °C until use.
Protein Extraction from G1H-G93A Mouse Spinal Cord—The G1H-G93A spinal cord was homogenized in ice-cold homogenization buffer, 20 mM Tris-HCl (pH 6.8) containing CompleteTM miniprotease inhibitor mixture (Roche Applied Science), and 100 mM IA for preventing the secondary oxidation of SH groups. The sample was centrifuged at 17, 000 x g at 4 °C for 30 min, and the pellet was homogenized in the ice-cold homogenization buffer containing 1% Triton X-100. The sample was centrifuged at 17,000 x g at 4 °C for 30 min, the pellet was further homogenized in the ice-cold homogenization buffer containing 1% Triton X-100 and 2% SDS, and the sample was centrifuged at 17,000 x g at 4 °C for 30 min. The supernatants of each homogenization, buffer-soluble, Triton X-100-soluble, and SDS-soluble fractions, were subjected to SDS-PAGE and Western blot analyses.
Immunohistochemical Analysis—After fixation, the specimens were embedded in paraffin, cut into 5-µm-thick sections, and examined for immunohistochemical analysis. Sections were deparaffinized and then washed in PBS. Normal serum homologous with the secondary antibody diluted in 1% BSA-containing PBS (BSA-PBS) was used as a blocking reagent. Tissue sections were incubated with anti-C111ox-SOD1 (diluted 1:1000 in BSA-PBS) at 4 °C for 18 h. Bound antibody was visualized by the avidin-biotin-immunoperoxidase complex (ABC) method using the appropriate Vectastain Elite ABC rabbit IgG kit (Vector Laboratories) and 3,3'-diaminobenzidine tetrahydrochloride (Wako, Osaka, Japan) as a chromogen. The endogenous peroxidase activity was quenched by incubation for 30 min with 3% H2O2 after the secondary antibody treatment to prevent nonspecific oxidation before anti-C111ox-SOD1 treatment.
SOD1 Activity—SOD1 activity was assayed using the xanthine-xanthine oxidase/cytochrome c system as described previously (27).
Protein Assay—SOD1 protein concentrations were estimated using a dimeric molar extinction at 280 nm of 10,800 M-1 cm-1 (28). Protein concentrations of crude samples were determined using a BCATM protein assay kit (Pierce) with BSA as a standard.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Additional 2-ME Treatment Recovered the 2-ME-SOD1 to Wild-type SOD1—Next, the ability to remove 2-ME from Cys111 was examined. 2-ME-SOD1 was incubated with 0 (H2O), 2, 20, and 200 mM 2-ME for 1 h on ice; the excess 2-ME was removed on a PD-10 column with miliQ water, and then the mass of the proteins was analyzed. As shown in supplemental Fig. S2A, the incubation with more than 20 mM 2-ME decreased the mass of the protein (from 15,871 to 15,795 m/z). The difference in the mass was 76 m/z, indicating that incubation with 20 mM 2-ME removes 2-ME from Cys111. Thus, to confirm this finding, the 2-ME-treated SOD1s were digested with trypsin after incubation with IA, and the mass of the resulting peptides was determined. Removal of 2-ME from Cys111, would allow the SH group of Cys111 to be carbamidemethylated by IA, yielding a mass of 2514 (2456 plus 58) m/z.As shown in supplemental Fig. S2B, the mass of the tryptic peptide containing Cys111 (residues Asp92-Arg115) treated with more than 20 mM 2-ME was 2514 m/z. In contrast, the mass of the 2-ME-SOD1 treated with H2O was 2533 m/z, indicating that the SH group of Cys111 remained bound to 2-ME. These results show that incubation of 2-ME-SOD1 with 2-ME in excess of 20 mM removed 2-ME from Cys111, converting 2-ME-SOD1 to wild-type SOD1. The SOD activities of 2-ME-SOD1 and the wild-type SOD1 were 4181 and 4056 units/mg, respectively, indicating that both SOD1s have similar activities.
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Role of Cys111 in the Generation of Negatively Charged Molecules after Oxidation—To examine the role of Cys111 in the generation of negatively charged molecules after oxidation, 2-ME-SOD1 and wild-type SOD1 were incubated with 5 mM H2O2 for 1 h and were applied to a MonoQ column. Some of the fractions were then subjected to reducing SDS-PAGE and Western blotting. Since incubation with 5 mM H2O2 caused oxidation of almost all histidine and cysteine residues in bovine SOD1 (3), it is thought that negatively charged molecules were generated in both SOD1s. As shown in the upper panels of Fig. 2, A and B, several peaks containing oxidized SOD1 proteins were eluted with similar patterns in both SOD1s. However, the results of Western blotting were quite different (lower panels in Fig. 2, A and B). In oxidized 2-ME-SOD1, only one fragment (labeled with an asterisk) from the first peak and slight polymer bands from the last fractions, which were obtained by washing the column with 0.5 M KCl, were observed (Fig. 2A). The single fragment resulting from oxidation of 2-ME-SOD1 has been identified by Ookawara et al. (5) as a large fragment cleaved between Pro62 and His63. Because Ookawara et al. (5) also used recombinant human SOD1 (2-ME-SOD1) obtained from Ube Industries Ltd., it can be concluded that the identity of the single fragment in the present study and that of Ookawara et al. (5) are the same. In contrast, oxidation of the wild-type SOD1 resulted in not only the upper band but also in several additional fragments and polymer bands (Fig. 2B). Oxidation of Cys111 may become a trigger of fragmentation and polymerization. Zhang et al. (8) reported that a covalently cross-linked dimer (polymer) of human SOD1 was induced by bicarbonate and H2O2. Therefore, the effects of bicarbonate on the oxidation of 2-ME-SOD1 and wild-type SOD1 were investigated. However, no difference in dimer formation between the two SOD1 variants was observed (data not shown), suggesting that the cross-linkage between monomers was not mediated by Cys111.
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To obtain greater quantities of oxidized peptides, SOD1 proteins in peaks a and b separated with the MonoQ column (Fig. 3B) were reduced by DTT, alkylated with IA, and digested with lysylendopeptidase, but not with trypsin. The resultant peptides were applied to a reverse-phase high performance liquid chromatograph (ODS column). As shown in Fig. 4A, the HPLC elution profiles were nearly identical, but two additional peaks (d and e) were observed after the last peak (c) only in digests from peak b of the MonoQ column (i.e. the lower panel). Peak c has already been identified as residues 92-122 containing carbamidemethylated Cys111 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. 4B). Each fraction was subjected to MALDI-TOF MS analyses. As a result, peptide c in fractions 1 and 2 corresponded to residues 92-122 containing carbamidemethylated Cys111 (3320.5 m/z), as expected. Peptide d, in fractions 2 and 3, and peptide e, in fractions 3 and 4, gave masses corresponding to residues 92-122 plus 32 (3295.5 m/z) and residues 92-122 plus 48 (3311.6 m/z), respectively (Fig. 4C).
To directly demonstrate the formation of Cys111-SO2H and Cys111-SO3H, these peptides (c-e), were further analyzed by infusion ESI MS/MS. This method was used to ascertain the site of oxidative modification of SOD1 by determination of the amino acid sequence of the peptides. The amino acid sequence of the peptide 92-122 was determined based on the assumption that Cys111 was modified with carbamidemethyl (Fig. 5B), sulfinic acid (SO2H) (Fig. 5C), and sulfonic acid (SO3H) (Fig. 5D), respectively. The mass of peptide c (3320.5 m/z), Cys-carbamidemethyl ([M + 2H]2+ = 1661.2 m/z) gave the major fragment ions, y5 (611.3 m/z), y10 (1152.0 m/z), y12 (1424.6 m/z), y13 (1561.7 m/z), and y21* ([M + 2H]2+ = 1161.0 m/z), indicating that Cys111 was carbamidemethylated, as expected (Fig. 5E). The mass of peptide d (3295.5 m/z) ([M + 2H]2+ = 1648.3 m/z) gave the major fragment ions, y11 (1264.6, m/z), y12 (1399.7 m/z), y13 (1536.7 m/z), and b20 (2031.6 m/z), which was identified to be residues 92-122 containing Cys111-SO2H (Fig. 5F). Furthermore, the mass of peptide e (3311.6 m/z) ([M + 2H]2+ = 1657.2 m/z) gave the major fragment ions, y9 (1038.6 m/z), y12 (1415.6 m/z), y13 (1552.7 m/z), y18 (2012.0 m/z), and y21 (2310.8 m/z), indicating that Cys111 was oxidized to Cys-SO3H (Fig. 5G). Analyses based on the assumption that His110 and/or His120 were oxidized to 2-oxo-histidine showed that the corresponding y ions and b ions were absent (data not shown). 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.
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Two-dimensional Gel Electrophoresis Characterization of Oxidized SOD1—It is well known that human, bovine, and recombinant human SOD1 proteins have several charge isomers detected by HPLC, isoelectric gel focusing, or two-dimensional gel electrophoresis (26, 30, 31). The reason for the heterogeneity is still unknown, although some hypotheses, such as different metallation, different conformation, and different oxidation of Cys residues, were presented (13, 26). Thus, two-dimensional gel electrophoresis of oxidized SOD1 was performed to examine the effects of Cys111 oxidation on the generation of charge isomers. As shown in Fig. 7A, wild-type SOD1 presented the main spot 3 (pI 5.15) and three tiny spots, 1 (pI 4.92), 2 (pI 5.02), and 4 (pI 5.8). 2-ME-SOD1 also presented four similar spots (Fig. 7B). Three of them, spots 1-3, were thought to correspond to three isomers of recombinant human SOD1 with pI of 4.99, 5.06, and 5.14, which was previously determined by isoelectric gel electrophoresis (30). When 2-ME-SOD1 was oxidized by H2O2, three major spots, 5 (pI 5.32), 6 (pI 5.52) and 7 (pI 5.65), were newly generated between spots 3 and 4 (Fig. 7C). In contrast, when wild-type SOD1 was oxidized by H2O2, further new spots, 5' (pI 5.3), 6' (pI 5.46), and 7' (pI 5.6), which were probably their upper and acidic shifted spots of the spots 5 (pI 5.32), 6 (pI 5.52), and 7 (pI 5.65), respectively, were generated (Fig. 7, D and E). Furthermore, two major spots, 1' and 2', and weak spot 3', just above spots 1, 2, and 3, respectively, were also detected by anti-C111ox-SOD1 (Fig. 7E). Air oxidation of wild-type SOD1 generated only the two major spots, 1' and 2', and one tiny spot, 7' (Fig. 7, F and G). Therefore, these results indicated that spots 1', 2', and 7' were generated by the oxidation of Cys111 and that the generation of spots 5, 6, and 7 (Fig. 7C) was caused by the oxidation of other amino acid residues.
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25 kDa band in the Triton X-100-soluble fraction (Fig. 8A, left). Although some weak bands were detected, the upper band of oxidized SOD1 was not detected in all fractions. Immunostaining with anti-SOD1 detected a large amount of human SOD1 overexpressed in the G1H-G93A mouse and mouse SOD1 (Fig. 8A, right). In contrast, anti-C111ox-SOD1 detected the upper band of oxidized SOD1 (positive control) but neither the reduced form of G93A-SOD1 nor mouse SOD1 (Fig. 8A, left). Therefore, the 25 kDa band, selectively recognized by anti-C111ox-SOD1, was thought to be some molecule-bound oxidized SOD1. Basso et al. (26) detected mono- and polyubiquitinated SOD1 (24, 32, 40, 48 kDa spots in two-dimensional gel electrophoresis) in the Triton X-100-insoluble fraction of G93A transgenic mice spinal cords. Thus, the 25 kDa band was speculated to be monoubiquitinated SOD1, and then the immunostaining with anti-ubiquitin on the same membrane was performed. Although the 25 kDa band appeared to be one of the ubiquitinated proteins (data not shown), evidence of monoubiquitination has not been obtained. We are currently exploring the identity of the molecule bound to Cys111-peroxidized SOD1. Finally, an immunohistochemical study of paraffin-embedded spinal cord sections of G1H-G93A mice was performed. The G1H-G93A mice examined at 110 days of age revealed severe loss of anterior horn cells with gliosis and both Lewy body-like hyaline inclusions (LBHIs) and vacuolation pathologies (32). The anti-C111ox-SOD1 selectively labeled the LBHIs in the neuropil and in the cytoplasm of the neurons (Fig. 8B) and the rim of the vacuoles in the neuropil (Fig. 8C). When the paraffin sections were incubated with BSA-PBS alone or with anti-C111ox-SOD1 pretreated with an excess amount of air-oxidized SOD1 or peptide containing sulfonylated Cys111, no staining was detected. The spinal cords of the two littermates exhibit neither distinct histopathological changes nor staining with anti-C111ox-SOD1. These results suggested that the Cys111-peroxidized SOD1 was involved in the formation of the LBHIs and the vacuoles in ALS spinal cords.
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DISCUSSION |
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Although more than 110 FALS mutations in the SOD1 have been identified, the mechanism by which the FALS-linked mutant SOD1s cause motor neuron degeneration is not completely understood. Two hypotheses have been proposed explaining the toxic gain of function that is associated with these mutations (12, 41). The "copper hypothesis" proposes that copper, either bound to or released from FALS-linked mutant SOD1s generates reactive oxygen species harmful to motor neurons (42-44). The "aggregation hypothesis" supposes that FALS-linked mutant SOD1s are structurally unstable and tend to aggregate, resulting in degeneration of neuronal cells analogous to that observed in other neurodegenerative disorders such as Alzheimer, Parkinson, and Huntington diseases (33, 45, 46). However, recent reports suggest that these two hypotheses are interrelated. Copper ion oxidized Cys111 in human SOD1 (Fig. 1). Oxidation may result in misfolding and aggregation even in wild-type SOD1 (6, 47). Oxidized wild-type SOD1 exhibits characteristics of FALS-linked mutant SOD1s: conjugation with polyubiquitin, interaction with Hsp70 or chromogranin B, and toxic effects on motor neurons (7). Although this study demonstrated that the SH of Cys111 underwent irreversible peroxidation to Cys-SO2H and to Cys-SO3H, Cys111 may also participate in disulfide bond linkage with other cysteine residues and oligomerization (48). Furukawa et al. (49-51) showed that incorrect intermolecule disulfide cross-linking of immature, misfolded FALS-linked mutant SOD1s leads to formation of insoluble aggregates. On the other hand, an immunohistochemical study using the anti-C111ox-SOD1 revealed that Cys111-peroxidized mutant SOD1 accumulated in the vacuole structures and LBHIs (Fig. 8, B and C). Because the most characteristic neuropathological findings in ALS model mice are LBHIs and vacuoles (32, 33), the peroxidation of Cys111 may contribute to the pathology of the degeneration/death of FALS motor neurons. However, the amount of the Cys111-peroxidized SOD1 appears to be quite limited (Fig. 8A). Large quantities of thiol compounds, such as glutathione and cysteine, probably protect the SH of Cys111 in healthy cells. The surviving neuron cells were not immunostained by anti-C111ox-SOD (Fig. 8, B and C). Ferri et al. (48) proposed that Cys111 was a key mediator of mitochondrial association of SOD1 and subsequent mitochondrial dysfunction, because the C111S mutant was less associated with mitochondria. Recently, it was also reported that A4V/C111S protein was more stable than A4V protein in cells (52). However, it is still an open question whether Cys111 is essential for the etiology of ALS, because mice that express mouse SOD1-G85R developed ALS-like symptoms, although Ser, not Cys, is at residue 111 of mouse SOD1 (53). It is thought that Cys111 may enhance human ALS development. A comparative study of symptoms between mice expressing the ALS mutation with C111S (G93A/C111S etc.) and traditional ALS model mice (G93A etc.) would answer the question.
In summary, we demonstrated that Cys111 in human SOD1 is a primary target for oxidation and is readily oxidized to Cys111-SO3H. The specific antibody against the Cys111-SO3H will be a useful tool for detecting oxidized human SOD1. Precise studies of the role of oxidized SOD1 in ALS are currently under way.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.
1 These authors contributed equally to this work.
2 To whom correspondence should be addressed. Tel.: 81-798-45-6357; Fax: 81-798-46-3164; E-mail: noriko-f{at}hyo-med.ac.jp.
3 The abbreviations used are: SOD, superoxide dismutase; ALS, amyotrophic lateral sclerosis; FALS, familial amyotrophic lateral sclerosis; MALDI, matrix-assisted laser desorption/ionization; TOF, time of flight; ESI, electrospray ionization; MS, mass spectrometry; MS/MS, tandem MS; TBS, Tris-buffered saline; PVDF, polyvinylidene fluoride; DTT, dithiothreitol; 2-ME, 2-mercaptoethanol; IA, iodoacetamide; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; Prx, peroxiredoxin; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; IPG, immobilized pH gradient; LBHI, Lewy body-like hyaline inclusion.
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