人铜锌超氧化物歧化酶cDNA的克隆,测序及表达

用逆转录聚合酶链反应（ＲＴＰＣＲ）,以人胎肝组织总ＲＮＡ为模板,扩增了人铜锌超氧化物歧化酶（ｈＣｕ,ＺｎＳＯＤ）的ｃＤＮＡ,并进行序列分析,将该ｈＣｕ,ＺｎＳＯＤｃＤＮＡ重组到Ｔ７启动子控制下的分泌型表达载体ｐＥＴ２２ｂ（＋）中,构建表达质粒ｐＥＴＳＯＤ,并转化大肠杆菌ＢＬ２１（ＤＥ３）。ＳＤＳＰＡＧＥ及蛋白质印迹分析表明,经１ｍｍｏｌ／Ｌ异丙基硫代βＤ半乳糖苷（ＩＰＴＧ）诱导后,可高效表达一分子量为１９ｋＤ的蛋白质,与抗人ＳＯＤ多抗有特异的免疫反应,表达量约为菌体总蛋白质的３０％,具有特异性ＳＯＤ酶活性,酶活力可达１７９７ｕ／ｍｌ培基。     

Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has altered redox activities and is toxic to motor neurons in vitro. Using bovine SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding activity six times faster than superoxide dismutase activity, whereas inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine. Although urate, xanthine, and ascorbate can protect superoxide dismutase activity of Cu,Zn SOD from inactivation, they were not effective at protecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydrogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible.     

Synechocystis Fe superoxide dismutase gene confers oxidative stress tolerance to Escherichia coli

The superoxide dismutase (SOD) gene (slr 1516) from the cyanobacterium Synechocystis sp. PCC 6803 was cloned and overexpressed in Escherichia coli BL 21 (DE3) using the pET-20b(+) expression vector. E. coli cells transformed with pET-SOD overexpressed the protein in cytosol, upon induction by isopropyl beta-D-thiogalactopyranoside (IPTG). The recombinant protein was purified to near homogeneity by gel filtration and ion-exchange chromatography. The SOD activity of the recombinant protein was sensitive to hydrogen peroxide and sodium azide, confirming it to be FeSOD. The pET-FeSOD transformed E. coli showed significantly higher SOD activity and tolerance to paraquat-mediated growth inhibition compared to the empty vector transformed cells. Based on these results it is suggested that overexpression of FeSOD gene from a heterologous source like Synechocystis sp. PCC 6803 may provide protection to E. coli against superoxide radical-mediated oxidative stress mediated by paraquat.     

Vaccination with live Escherichia coli expressing Brucella abortus Cu/Zn superoxide-dismutase: II. Induction of specific CD8+ cytotoxic lymphocytes and sensitized CD4+ IFN-gamma-producing cell

Previously we reported that immunization with Escherichia coli DH5alpha-expressing Brucella abortus Cu/Zn superoxide dismutase [E. coli (pBSSOD)] induces a protective immune response in BALB/c mice. Here we studied the type of immune defense that the recombinant E. coli induces in mice using as our experimental model Brucella superoxide dismutase Cu/Zn presented by J744.A1 to sensitized lymphocytes as the target of specific lysis or as cytokine inductors. The results indicate that E. coli carrying the Cu/Zn gene was able to induce specific cytotoxic T cells, mainly from CD8(+) subpopulation and IFN-gamma-producing cells belonging in their vast majority to the CD4(+) subpopulation.     

Cloning,production and characterisation of a recombinant Cu/Zn superoxide dismutase from Taenia solium

A full-length complementary DNA clone encoding a cytosolic Cu/Zn superoxide dismutase with a M(r) of 15,588 Da was isolated from a Taenia solium larvae complementary DNA library. Comparison analysis of its deduced amino acid sequence revealed a 71% identity with Schistosoma mansoni, 57.2-59.8% with mammalian and less than 54% with other helminth cytosolic Cu/Zn superoxide dismutase. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc enzymatic function are conserved. The T. solium Cu/Zn superoxide dismutase was expressed in the pRSET vector. Enzymatic and filtration chromatographic analysis showed a recombinant enzyme with an activity of 2,941 U/mg protein and a native M(r) of 37 kDa. Inhibition assays using KCN, H(2)O(2), NaN(3) and SDS indicated that Cu/Zn is the metallic cofactor in the enzyme. Thiabendazole (500 microM) and albendazole (300 microM) completely inhibited the activity of T. solium Cu/Zn superoxide dismutase. Thiabendazole had no effect on bovine Cu/Zn superoxide dismutase; in contrast, albendazole had a moderate effect on it at same concentrations. Antibodies against T. solium Cu/Zn superoxide dismutase did not affect the enzymatic function; nevertheless, it cross reacts with several Taenia species, but not with trematodes, nematodes, pig, human and bovine Cu/Zn superoxide dismutase enzymes. Western blot analysis indicated the enzyme was expressed in all stages. These results indicate that T. solium possesses a Cu/Zn superoxide dismutase enzyme that can protect him from oxidant-damage caused by the superoxide anion.     

Crystallization and preliminary X-ray analysis of the monomeric Cu,Zn superoxide dismutase from Escherichia coli.

The Cu,Zn superoxide dismutase (Cu,Zn SOD) originally isolated from the periplasmic space of Escherichia coli has been cloned and overexpressed in the E. coli strain BMH 71/18. The protein has been purified as a single component of 17,000 Da, corresponding to one subunit of the common dimeric eukaryotic Cu,Zn SODs. Large crystals of the purified protein have been grown in the presence of polyethylene glycol 4,000 at pH 8.5; the crystals belong to the monoclinic space group P2(1), with unit cell constants a = 33.1 A, b = 52.6 A, c = 43.3 A, beta = 111.4 degrees. One SOD subunit is contained in the asymmetric unit, yielding a Vm value of 2.1 A3/Da; the crystals diffract X-rays beyond 2.0 A resolution.     

Superoxide dismutases enhance the rate of autoxidation of 3-hydroxyanthranilic acid

The autoxidation of 3-hydroxyanthranilate to cinnabarinate at 37 degrees C and at pH 7.4 is hastened by superoxide dismutase (SOD). The Cu,Zn-containing enzyme from bovine erythrocytes and the Mn-containing enzyme from Escherichia coli were equally effective in this regard; whereas the H2O2-inactivated Cu,Zn enzyme was ineffective. Catalase appears to augment the effect of superoxide dismutase, because it prevents the bleaching of cinnabarinate by H2O2. It follows that O2-, which is a product of the autoxidation, slows the net autoxidation by engaging in back reactions and that SOD increases the rate of autoxidation by removal of O2- and thus by prevention of these back reactions.     

Vibrio cholerae periplasmic superoxide dismutase: isolation of the gene and overexpression of the protein

Superoxide dismutases are ubiquitous enzymes which play an important role in protecting cells against oxidative damage and which have also been shown to contribute to the pathogenicity of many bacterial species. Here we demonstrate that Vibrio cholerae, the causative agent of cholerae, expresses an active periplasmic Cu,Zn superoxide dismutase. Moreover, we have set up an expression system yielding large amounts of V. cholerae recombinant Cu,Zn superoxide dismutase in the periplasm of Escherichia coli and a procedure to obtain the enzyme in a highly purified form. Unlike the bovine enzyme, V. cholerae Cu,Zn superoxide dismutase has been proved to be highly resistant to inactivation by hydrogen peroxide. This property, which appears to be common to other bacterial enzymes of this class, might improve the ability of Cu,Zn superoxide dismutase to protect bacteria against the reactive oxygen species produced by phagocytes.     

Involvement of redox events in caspase activation in zinc-depleted airway epithelial cells

Airway epithelial cells (AEC) contain both pro- and anti-apoptotic factors but little is known about mechanisms regulating apoptosis of these cells. In this study we have examined the localization of pro-caspase-3 and Zn(2+), a cellular regulator of pro-caspase-3, in primary sheep and human AEC. Zn(2+) was concentrated in both cytoplasmic vesicles and ciliary basal bodies, in the vicinity of both pro-caspase-3 and the antioxidant Cu/Zn superoxide dismutase (Cu/Zn SOD). Depletion of intracellular Zn(2+) in sheep AEC, using the membrane permeant Zn(2+) chelator TPEN, increased lipid peroxidation in the apical cell membranes (as assessed by immunofluorescence with anti-hydroxynonenal) as well as increasing activated pro-caspase-3 and apoptosis. There were smaller increases in caspase-2 and -6 but not other caspases. Activation of caspase-3 in TPEN-treated AEC was inhibited strongly by N-acetylcysteine and partially by vitamin C and vitamin E. These findings suggest that cytoplasmic pro-caspase-3 is positioned near the lumenal surface of AEC where it is under the influence of Zn(2+) and other anti-oxidants.     

Thermal stability and redox properties of M. tuberculosis CuSOD

The superoxide dismutase from Mycobacterium tuberculosis is the only Cu-containing superoxide dismutase that lacks zinc in the active site. To explore the structural properties of this unusual enzyme, we have investigated its stability by differential scanning calorimetry. We have found that the holo-enzyme is significantly more stable than the apo-protein or the partially metallated enzyme, but that its melting temperature is markedly lower than that of all the other characterized eukaryotic and prokaryotic Cu,Zn superoxide dismutases. We have also observed that, unlike the zinc-free eukaryotic or bacterial enzymes, the active site copper of the mycobacterial enzyme is not reduced by ascorbate, confirming that its redox properties are comparable to those typical of the enzymes containing zinc in the active site. Our findings highlight the role of zinc in conferring stability to Cu,Zn superoxide dismutases and indicate that the structural rearrangements observed in M. tuberculosis Cu,SOD compensate for the absence of zinc in achieving a fully active enzyme.     

Transcriptional activation of the human Cu/Zn superoxide dismutase gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin through the xenobiotic-responsive element

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced during biological oxidations and environmental stress. Here we have investigated the effect of the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the promoter of the Cu/Zn superoxide dismutase (SOD1) gene in HepG2 and HeLa cells using the chloramphenicol acetyltransferase gene as a reporter. The SOD1 promoter was activated 4- to 5-fold by TCDD treatment, in a concentration-dependent manner. In addition, the level of SOD1 mRNA and the enzymatic activity of the SOD1 protein were also enhanced on exposure of the cells to TCDD. Functional analysis of the regulatory region of the SOD1 gene by deletion and point mutation, and the use of a heterologous promoter system, showed that the SOD1 gene was transactivated by TCDD via the xenobiotic-responsive element (XRE). Gel mobility shift assays also confirmed the induction and the inducible binding of a receptor-ligand complex to XRE. Yeast cells that overexpress hSOD1 appeared to be more resistant to TCDD than the wild type. These results demonstrate that SOD1 is induced by TCDD via the XRE. The induced SOD1 may accelerate the neutralization of the superoxide anion and thus reduce the oxidative damage associated with dioxin toxicity.     

cDNA cloning,high-level expression,purification, and characterization of an avian Cu,Zn superoxide dismutase from Peking duck

As a special species of avian, Peking duck is often used as a model for exploring effective factors against cardio-cerebrovascular diseases, and therefore investigations of antioxidant enzymes including superoxide dismutase are intriguing. By using 3(')-RACE with a gene-specific primer, a cDNA encoding duck Cu,Zn SOD was amplified from the total RNA extracted from Peking duck liver. Three free cysteine residues are found in the deduced amino acid sequence of duck SOD, among which Cys153 at the carbonyl-terminal is a distinctive feature. Production with a high yield of recombinant duck Cu,Zn SOD was achieved in Escherichia coli after the reconstituted expression vector pET-3a-dSOD was transformed into the bacterial strain BL21(DE3)pLysS. After two steps of anion exchange chromatography, a great quantity of the purified enzyme (100mg/L fermented culture) with an enzymatic activity comparable to that of native duck and bovine SOD was finally obtained. Duck SOD is a homodimer with 153 residues for each subunit. The molecular mass of the recombinant enzyme is 15,540.0Da measured by mass spectrum, which well coincides with the estimated size of the sequence but significantly differs from that of the native counterpart. Five charge isomers were observed on isoelectricfocusing (IEF). The most interesting observation is that the thermal stability of duck SOD is much lower than that of the bovine enzyme as revealed by irreversible heat inactivation at 70 degrees C. These properties are discussed in relation to the distinctive free Cys residues in duck Cu,Zn SOD.     

17β-Estradiol-mediated increase in Cu/Zn superoxide dismutase expression in the brain: a mechanism to protect neurons from ischemia

A number of studies have demonstrated that 17β-estradiol (E(2)) protects the brain from ischemia and yet the mechanism by which this hormone brings about its protective effect is unclear. Interestingly, like E(2), overexpression of the oxidative stress response protein Cu/Zn superoxide dismutase (SOD1), which plays a critical role in regulating reactive oxygen species, also protects the brain from ischemia. Because we previously showed that E(2) treatment of cultured mammary cells increases SOD1 expression, we hypothesized that E(2) might increase SOD1 expression in the brain and that this E(2)-mediated increase in SOD1 expression might help to protect the brain from ischemia. We now show that SOD1 is expressed in cortical neurons, that SOD1 expression is increased by exposure of brain slice cultures to E(2), and that the E(2)-mediated increase in SOD1 expression is further augmented by exposure of brain slice cultures to increased superoxide levels or oxygen and glucose deprivation. Importantly, when cortical neurons are exposed to increased superoxide levels and markers of protein and DNA damage, nitrotyrosine and 8-oxoguanine, respectively, are measured, both protein and DNA damage are reduced. In fact, E(2) reduces nitrotyrosine and 8-oxoguanine levels in brain slice cultures regardless of whether they have or have not been exposed to increased superoxide levels. Likewise, when brain slice cultures are treated with E(2) and deprived of oxygen and glucose, 8-oxoguanine levels are reduced. Taken together, these studies provide a critical link between E(2) treatment, SOD1 expression, and neuroprotection and help to define a mechanism through which E(2)-mediated neuroprotection may be conferred.     

Superoxide Dismutase Concentration and Activity in Familial Amyotrophic Lateral Sclerosis

Abstract: Some cases of autosomal-dominant familial amyotrophic lateral sclerosis (FALS) have been associated with mutations in SOD1 , the gene that encodes Cu/Zn superoxide dismutase (Cu/Zn SOD). We determined the concentrations (µg of Cu/Zn SOD/mg of total protein), specific activities (U/µg of total protein), and apparent turnover numbers (U/µmol of Cu/Zn SOD) of Cu/Zn SOD in erythrocyte lysates from patients with known SOD1 mutations. We also measured the concentrations and activities of Cu/Zn SOD in FALS patients with no identifiable SOD1 mutations, sporadic ALS (SALS) patients, and patients with other neurologic disorders. The concentration and specific activity of Cu/Zn SOD were decreased in all patients with SOD1 mutations, with mean reductions of 51 and 46%, respectively, relative to controls. In contrast, the apparent turnover number of the enzyme was not altered in these patients. For the six mutations studied, there was no correlation between enzyme concentration or specific activity and disease severity, expressed as either duration of disease or age of onset. No significant alterations in the concentration, specific activity, or apparent turnover number of Cu/Zn SOD were detected in the FALS patients with no identifiable SOD1 mutations, SALS patients, or patients with other neurologic disorders. That Cu/Zn SOD concentration and specific activity are equivalently reduced in erythrocytes from patients with SOD1 mutations suggests that mutant Cu/Zn SOD is unstable in these cells. That concentration and specific activity do not correlate with disease severity suggests that an altered, novel function of the enzyme, rather than reduction of its dismutase activity, may be responsible for the pathogenesis of FALS.     

铜锌超氧化物歧化酶(SOD)研究进展——从基因到功能

超氧化物歧化酶(SOD,EC 1.15.1.1),己经在多种组织中发现,它能将O2.-催化生成H2O2及O2.迄今为止,已经从哺乳动物体内分离出三种SOD:CuZnSOD(SOD1)、MnSOD(SOD2)TLEC-SOD(胞外超氧化物歧化酶,SOD3),各自具有不同的生化及分子特性.CuZnSOD(SOD1),是一类含有Cu及Zn原子的二聚体,存在于特定细胞的基质内,约占SOD总量的90%.在胞质及周质中,SOD以二聚体形式存在,而在线粒体及质外,则以四聚体形式存在.在保护脑、肺及其它组织的氧化应激中,CuZnSOD被认为起着保护作用.运动神经元肌萎缩侧索硬化症(ALS),据称也与同源二聚体CuZnSOD的错误折叠有关,己经报导,有多个CuZnSOD基因位点突变与ALS有关.本文将从基因的结构、表达、调节及蛋白的结构与功能等方面,对CuZnSOD进行简要论述.     

Dominant role of copper in the kinetic stability of Cu/Zn superoxide dismutase

Mutations in Cu/Zn superoxide dismutase (SOD) are involved in some cases of familial amyotrophic lateral sclerosis, and it appears that misfolding and aggregation, perhaps mediated by abnormal binding or loss of copper (Cu) and/or zinc (Zn), may play a pathological role. It is known that the absence of both metals kinetically destabilizes wild type and mutant SOD leading to a 60-fold increase in their rate of unfolding. Here, the individual contributions of Cu and Zn to the kinetic stability of SOD were investigated, and the results show that Cu plays a greater role. Thus, the deficiency of Cu or Zn, especially the former, will compromise the kinetic stability of SOD, thereby increasing the probability that pathogenic mutants and even the WT protein may misfold and self-assemble into toxic species.     

Electron transfer facilitated by superoxide dismutase: a model for membrane redox systems?

Membranes, which are an amalgam of proteins and lipids, effect electron transfer through largely unknown mechanisms. Using albumin with bound fatty acids as a model, we have investigated the possible role of these two membrane constituents in electron transfer. In the presence of albumin: fatty acid, there is substantial enhancement of the reduction of ferricytochrome C by ferrous iron. To assess the possible role of free superoxide in cytochrome C reduction, we added mammalian copper/zinc containing superoxide dismutase (Cu/Zn SOD), which catalyzes the transfer of electrons between superoxide anion radicals, forming oxygen and hydrogen peroxide. Surprisingly, in the presence of either albumin or fatty acid free albumin, Cu/Zn SOD actually accelerates electron transfer from ferrous iron to ferricytochrome C. By contrast, neither inactive Cu/Zn SOD nor active manganese SOD facilitates the ferrous iron-dependent reduction of cytochrome C. These results suggest that, in some circumstances, Cu/Zn SOD may transfer electrons to alternative acceptors and that such transfer depends upon the unique reduction/oxidation reaction mechanism of Cu/Zn SOD. If so, this ubiquitous enzyme could be involved in regulating cellular electron transfer reactions as well as acting as a superoxide 'detoxify-ing' agent.