The folding of human active and inactive extracellular superoxide dismutases is an intracellular event

Human extracellular superoxide dismutase (EC-SOD) is a tetrameric glycoprotein responsible for the removal of superoxide generated in the extracellular space. Two different folding variants of EC-SOD exist based on the disulfide bridge connectivity, resulting in enzymatically active (aEC-SOD) and inactive (iEC-SOD) subunits. As a consequence of this, the assembly of the EC-SOD tetramers produces molecules with variable activity and may represent a way to regulate the antioxidant level in the extracellular space. To determine whether the formation of these two folding variants is an intra- or extracellular event, we analyzed the biosynthesis in human embryonic kidney 293 cells expressing wild-type EC-SOD. These analyses revealed that both folding variants were present in the intra- and extracellular spaces, suggesting that the formation is an intracellular event. To further analyze the biosynthesis, we constructed mutants with the capacity to generate only aEC-SOD (C195S) or iEC-SOD (C45S). The expression of these suggested that the cellular biosynthetic machinery supported the secretion of aEC-SOD but not iEC-SOD. The coexpression of these two mutants did not affect the expression pattern. This study shows that generation of the EC-SOD folding variants is an intracellular event that depends on a free cysteine residue not involved in disulfide bonding.     

The structure of rabbit extracellular superoxide dismutase differs from the human protein

The cDNA sequence encoding rabbit, mouse, and rat extracellular superoxide dismutase (EC-SOD) predicts that the protein contains five cysteine residues. Human EC-SOD contains an additional cysteine residue and folds into two forms with distinct disulfide bridge patterns. One form is enzymatically active (aEC-SOD), while the other is inactive (iEC-SOD). Due to the lack of the additional cysteine residue rabbit, mouse, and rat EC-SOD are unable to generate an inactive fold identical to human iEC-SOD. The amino acid sequences predict the formation of aEC-SOD only, but other folding variants cannot be ruled out based on the heterogeneity observed for human EC-SOD. To test this, we purified EC-SOD from rabbit plasma and determined the disulfide bridge pattern. The results revealed that the disulfide bridges are homogeneous and identical to human aEC-SOD. Four cysteine residues are involved in two intra-disulfide bonds while the C-terminal cysteine residue forms an intersubunit disulfide bond. No evidence for other folding variants was detected. These findings show that rabbit EC-SOD exists as an enzymatically active form only. The absence of iEC-SOD in rabbits suggests that the structure and aspects of the physiological function of EC-SOD differs significantly between rabbit and humans. This is an important notion to take when using these animals as model systems for oxidative stress.     

Extracellular superoxide dismutase

The extracellular space is protected from oxidant stress by the antioxidant enzyme extracellular superoxide dismutase (EC-SOD), which is highly expressed in selected tissues including blood vessels, heart, lungs, kidney and placenta. EC-SOD contains a unique heparin-binding domain at its carboxy-terminus that establishes localization to the extracellular matrix where the enzyme scavenges superoxide anion. The EC-SOD heparin-binding domain can be removed by proteolytic cleavage, releasing active enzyme into the extracellular fluid. In addition to protecting against extracellular oxidative damage, EC-SOD, by scavenging superoxide, preserves nitric oxide bioactivity and facilitates hypoxia-induced gene expression. Loss of EC-SOD activity contributes to the pathogenesis of a number of diseases involving tissues with high levels of constitutive extracellular superoxide dismutase expression. A thorough understanding of the biological role of EC-SOD will be invaluable for developing novel therapies to prevent stress by extracellular oxidants.     

Enhanced bleomycin-induced pulmonary damage in mice lacking extracellular superoxide dismutase

Extracellular superoxide dismutase (EC-SOD) is highly expressed in the extracellular matrix of lung and vascular tissue. Localization of EC-SOD to the matrix of the lung may protect against oxidative tissue damage that leads to pulmonary fibrosis. This study directly examines the protective role of EC-SOD in a bleomycin model of pulmonary fibrosis and the effect of this enzyme on oxidative protein fragmentation. Mice null for ec-sod display a marked increase in lung inflammation at 14 d post-bleomycin treatment as compared to their wild-type counterparts. Hydroxyproline analysis determined that both wild-type and ec-sod null mice display a marked increase in interstitial fibrosis at 14 d post-treatment, and the severity of fibrosis is significantly increased in ec-sod null mice compared to wild-type mice. To determine if the lack of EC-SOD promotes bleomycin-induced oxidative protein modification, 2-pyrrolidone content (as a measure of oxidative protein fragmentation at proline residues) was assessed in lung tissue from treated mice. 2-Pyrrolidone levels in the lung hydrolysates from ec-sod null mice were increased at both 7 and 14 d post-bleomycin treatment as compared to wild-type mice, indicating EC-SOD can inhibit oxidative fragmentation of proteins in this specific model of oxidative stress.     

Expression profiles of extracellular superoxide dismutase during mouse organogenesis

Although extracellular superoxide dismutase (EC-SOD), which scavenges the superoxide anion in extracellular spaces, has previously been implicated in the prenatal pulmonary response to oxidative stress in the developing lungs, little is currently known regarding the schematic expression pattern and the roles played by EC-SOD during embryogenesis. In an effort to characterize the pattern of EC-SOD expression during mouse organogenesis, quantitative RT-PCR, Western blotting, and in situ hybridization analyses were conducted in mouse embryos and extraembryonic tissues including placenta on embryonic days (Eds) 7.5-18.5. EC-SOD mRNA and protein were expressed in all the embryos and extraembryonic tissues examined. The mRNA level was higher in the embryos than the extraembryonic tissues on Eds 7.5-10.5, but after Ed 13.5, it evidenced an increasing pattern in the extraembryonic tissues. EC-SOD immunoreactivity also increased in the extraembryonic tissues after Ed 13.5. During organogenesis, EC-SOD mRNA was expressed principally in the ectoplacental cone, amnion, and neural ectoderm on Ed 7.5 and in the neural folds and primitive streak on Ed 8.5. On Eds 9.5-12.5, EC-SOD mRNA was expressed abundantly in the nervous tissues and forelimb and hindlimb buds. On Eds 13.5-18.5, EC-SOD mRNA was observed at high levels in the airway epithelium of lung, liver, the intestinal epithelium, skin, vibrissae, the metanephric corpuscle of kidney, the nasal cavity, and the labyrinth trophoblast, spongiotrophoblast, and blood cells in placenta. Our overall results indicate that EC-SOD is expressed spatiotemporally in developing embryos and surrounding extraembryonic tissues during mouse organogenesis, thus suggesting that EC-SOD may be relevant to organogenesis, playing the role of an antioxidant enzyme against endogenous and exogenous oxygen stresses.     

Oxidative stress,NO* and smooth muscle cell extracellular superoxide dismutase expression

Oxygen free radicals apparently play important roles in diseases of the blood vessel wall and increased secretion of superoxide radicals occurs in many situations. The vascular wall contains large amounts of extracellular superoxide dismutase (EC-SOD). The synthesis of the enzyme by the smooth muscle cells (SMC) is modulated by cytokines, growth factors, and vasoactive factors.Here we studied the effects of oxidants (pyrogallol, xanthine oxidase, Cu and Fe), antioxidants (SOD, catalase, and ascorbate), glutathione modulation (n-acetylcysteine and buthionine sulfoximine) and nitric oxide on EC-SOD expression by human vascular SMCs. Generally, the responses in EC-SOD synthesis were small, and no changes were noted in mRNA levels. High concentrations of some of the agents caused reductions in EC-SOD synthesis, mostly concomitantly with toxic effects on the cells. Cell cultures are normally ascorbate deficient, and addition of ascorbate to approach physiological levels doubled the EC-SOD content. Iron ions up-regulated EC-SOD synthesis but also blocked the secretion of the enzyme. Only down-regulation was found by NO*-releasing compounds.In conclusion, there is limited response to oxidant stress of EC-SOD synthesis by SMCs on a cell-autonomous level. The synthesis appears mainly regulated by factors coordinating concerted tissue responses.     

Extracellular superoxide dismutase exists as an octamer

Human extracellular superoxide dismutase (EC-SOD) is involved in the defence against oxidative stress induced by the superoxide radical. The protein is a homotetramer stabilised by hydrophobic interactions within the N-terminal region. During the purification of EC-SOD from human aorta, we noticed that material with high affinity for heparin-Sepharose formed not only a tetramer but also an octamer. Analysis of the thermodynamic stability of the octamer suggested that the C-terminal region is involved in formation of the quaternary structure. In addition, we show that the octamer is composed of both aEC-SOD and iEC-SOD folding variants. The presence of the EC-SOD octamer with high affinity may represent a way to influence the local concentration of EC-SOD to protect tissues specifically sensitive to oxidative damage.     

Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis

Bleomycin administration results in well-described intracellular oxidative stress that can lead to pulmonary fibrosis. The role of alveolar interstitial antioxidants in this model is unknown. Extracellular superoxide dismutase (EC-SOD) is the primary endogenous extracellular antioxidant enzyme and is abundant in the lung. We hypothesized that EC-SOD plays an important role in attenuating bleomycin-induced lung injury. Two weeks after intratracheal bleomycin administration, we found that wild-type mice induced a 106 +/- 25% increase in lung EC-SOD. Immunohistochemical staining revealed that a large increase in EC-SOD occurred in injured lung. Using mice that overexpress EC-SOD specifically in the lung, we found a 53 +/- 14% reduction in bleomycin-induced lung injury assessed histologically and a 17 +/- 6% reduction in lung collagen content 2 wk after bleomycin administration. We conclude that EC-SOD plays an important role in reducing the magnitude of lung injury from extracellular free radicals after bleomycin administration.     

Secretion of extracellular superoxide dismutase in neonatal lungs

Extracellular superoxide dismutase (EC-SOD), the only known enzymatic scavenger of extracellular superoxide, may modulate reactions of nitric oxide (NO) in the lungs by preventing reactions between superoxide and NO. The regulation of EC-SOD has not been examined in developing lungs. We hypothesize that EC-SOD plays a pivotal role in the response to increased oxygen tension and NO in the neonatal lung. This study characterizes rabbit EC-SOD and investigates the developmental regulation of EC-SOD activity, protein expression, and localization. Purified rabbit EC-SOD was found to have several unique biochemical attributes distinct from EC-SOD in other species. Rabbit lung EC-SOD contains predominantly uncleaved subunits that do not form disulfide-linked dimers. The lack of intersubunit disulfide bonds may contribute to the decreased heparin affinity and lower EC-SOD content in rabbit lung. EC-SOD activity in rabbit lungs is low before birth and increases soon after gestation. In addition, the enzyme is localized intracellularly in preterm and term rabbit lungs. Secretion of active EC-SOD into the extracellular compartment increases with age. The changes in EC-SOD localization and activity have implications for the neonatal pulmonary response to oxidative stress and the biological activity of NO at birth.     

Decreased pulmonary extracellular superoxide dismutase during systemic inflammation

Oxidative damage is a major cause of lung injury during systemic inflammatory response syndrome. In this study, the expression of an antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), and its protective role against pulmonary oxidative damage were investigated using mouse models of systemic inflammation. Intraperitoneal injection with bacterial endotoxin lipopolysaccharides (LPS; 20 mg/kg) caused oxidative damage in lungs as assessed by increased tyrosine nitration in proteins. LPS administration also resulted in a rapid and significant loss of more than 80% of pulmonary EC-SOD in a time- and dose-dependent manner, but other types of SODs, cytoplasmic CuZn-SOD and mitochondrial Mn-SOD, were not affected. EC-SOD protein is most abundant in lungs but also present at high levels in other tissues such as heart and white fat; however, the LPS-mediated decrease in this enzyme was most apparent in the lungs. Intravenous injection of mice with tumor necrosis factor alpha (10 microg per mouse) also caused a 60% decrease in EC-SOD in the lungs, suggesting that the EC-SOD down-regulation is mediated by this LPS-inducible inflammatory cytokine. A protective role for EC-SOD against LPS-mediated systemic inflammation was shown by an increased survival rate (75% vs 29% in 5 days) and decreased pulmonary oxidative damage in EC-SOD transgenic mice that overexpress the human EC-SOD gene. These results demonstrate that the inflammation-mediated EC-SOD down-regulation has a major pathophysiological impact during the systemic inflammatory response syndrome.     

Recombinant human extracellular superoxide dismutase produced in milk of transgenic rabbits

Expression of human extracellular superoxide dismutase (EC-SOD), a glycosylated, tetrameric metalloprotein, was targeted to the lactating mammary gland of transgenic rabbits. Efficient expression of the recombinant whey acidic protein/ec-sod gene was achieved and up to 3 mg ml–1 of the enzyme was secreted into the milk. Rabbit milk-produced recombinant EC-SOD was primarily found in the whey and purified by a two-step chromatographic method. To evaluate the rabbit milk-produced human EC-SOD, comparisons with native and Chinese hamster ovary cell (CHO)-produced EC-SOD were performed. All proteins were tetrameric and N-glycosylated. The behaviour on SDS-PAGE and size-exclusion chromatography indicated that the masses, and thereby the extent of post-translational modification of the proteins was similar. The monosaccharide composition of both recombinant EC-SOD variants was analysed and indicated similarities in the attached N-glycans on the two proteins. Furthermore, the peptide maps of the three EC-SOD variants revealed that all proteins had similar polypeptide backbones     

The heparin-binding domain of extracellular superoxide dismutase C and formation of variants with reduced heparin affinity.

A fundamental property of the secretory tetrameric extracellular superoxide dismutase (EC-SOD) is its affinity for heparin and analogues, in vivo, mediating attachment to heparan sulfate proteoglycans located on cell surfaces and in the connective tissue matrix. EC-SOD is in vivo heterogeneous with regard to heparin affinity and can be divided into subclasses; A which lacks heparin affinity, B with intermediate affinity, and C with strong heparin affinity. The EC-SOD C subunits contain 222 amino acids and among the last 20 carboxyl-terminal amino acids, 10 are positively charged and six of these are located in a cluster in positions 210-215. To analyze if this local accumulation of basic amino acids is responsible for heparin binding we produced three series of recombinant EC-SOD (rEC-SOD) variants, six containing amino acid exchanges in the carboxyl-terminal end, four with truncations, and two with both truncations and substitutions. Exchange of positively or negatively charged amino acids on the carboxyl-terminal side of the cluster results in only minor modifications in heparin affinity, whereas substitution of three of the amino acids in the cluster abrogates the heparin binding. Insertions of stop codons at different positions resulted in either C or A but not B class EC-SOD. In an attempt to produce EC-SODs with intermediate heparin affinities, plasmids defining C and A class EC-SOD were cotransfected into Chinese hamster ovary cells. In addition to the parental A and C class EC-SOD forms, two variants with intermediate heparin affinities were formed. Coincubation of EC-SOD C and A resulted in the appearance of one heterotetramer with intermediate affinity for heparin. We conclude that the cluster of six basic amino acids forms the essential part of the heparin-binding domain and that the composition of the four subunits in the EC-SOD tetramer determines the affinity for heparin. This domain is different from heparin-binding domains of other proteins, and its localization allows the distribution of EC-SOD in vivo to be regulated by proteolytic processing.     

The C-terminal proteolytic processing of extracellular superoxide dismutase is redox regulated

The antioxidant protein extracellular superoxide dismutase (EC-SOD) encompasses a C-terminal region that mediates interactions with a number of ligands in the extracellular matrix (ECM). This ECM-binding region can be removed by limited proteolysis before secretion, thus supporting the formation of EC-SOD tetramers with variable binding capacity. The ECM-binding region contains a cysteine residue (Cys219) that is known to be involved in an intersubunit disulfide bridge. We have determined the redox potential of this disulfide bridge and show that both EC-SOD dimers and EC-SOD monomers are present within the intracellular space. The proteolytic processing of the ECM-binding region in vitro was modulated by the redox status of Cys219, allowing cleavage under reducing conditions only. When wild-type EC-SOD or the monomeric variant Cys219Ser was expressed in mammalian cells proteolysis did not occur. However, when cells were exposed to oxidative stress conditions, proteolytic processing was observed for wild-type EC-SOD but not for the Cys219Ser variant. Although the cellular response to oxidative stress is complex, our data suggest that proteolytic removal of the ECM-binding region is regulated by the intracellular generation of an EC-SOD monomer and that Cys219 plays an important role as a redox switch allowing the cellular machinery to secrete cleaved EC-SOD.     

Interactions between human extracellular superoxide dismutase C and sulfated polysaccharides

The high heparin affinity subtype C of the secretory enzyme extracellular superoxide dismutase (EC-SOD) exists in the body mainly complexed with extracellular sulfated glycosaminoglycans (SGAGs). Addition of sulfated polysaccharides to EC-SOD C resulted in a prompt partial inhibition of the enzymic activity, in most cases amounting to 10-17%, but with the large dextran sulfate 500,000 amounting to 35%. Complex formation between heparin and EC-SOD C could also be observed as increases in apparent molecular weight of the enzyme. The findings suggest that the binding sites for SGAGs on EC-SOD C are localized far from the active site and that EC-SOD in vivo associated with SGAGs should retain the major part of its enzymic activity. Studies with amino acid-specific reagents suggested that both lysine and arginine residues are involved in the binding of SGAGs. In particular, modification of only a few lysine residues/subunit resulted in loss of high SGAG affinity, whereas arginine modification resulted in loss of not only SGAG affinity but also enzymic activity. We propose that this is due to modification of Arg-186, which is homologous to the highly conserved arginine in the entrance to the active site of the copperzinc-SODs.     

Extracellular superoxide dismutase in biology and medicine

Accumulated evidence has shown that reactive oxygen species (ROS) are important mediators of cell signaling events such as inflammatory reactions (superoxide) and the maintenance of vascular tone (nitric oxide). However, overproduction of ROS such as superoxide has been associated with the pathogenesis of a variety of diseases including cardiovascular diseases, neurological disorders, and pulmonary diseases. Antioxidant enzymes are, in part, responsible for maintaining low levels of these oxygen metabolites in tissues and may play key roles in controlling or preventing these conditions. One key antioxidant enzyme implicated in the regulation of ROS-mediated tissue damage is extracellular superoxide dismutase (EC-SOD). EC-SOD is found in the extracellular matrix of tissues and is ideally situated to prevent cell and tissue damage initiated by extracellularly produced ROS. In addition, EC-SOD is likely to play an important role in mediating nitric oxide-induced signaling events, since the reaction of superoxide and nitric oxide can interfere with nitric oxide signaling. This review will discuss the regulation of EC-SOD and its role in a variety of oxidant-mediated diseases.     

Leukocyte-derived extracellular superoxide dismutase does not contribute to airspace EC-SOD after interstitial pulmonary injury

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is abundant in the lung and is known to limit inflammation and fibrosis following numerous pulmonary insults. Previous studies have reported a loss of full-length EC-SOD from the pulmonary parenchyma with accumulation of proteolyzed EC-SOD in the airspace after an interstitial lung injury. However, following airspace only inflammation, EC-SOD accumulates in the airspace without a loss from the interstitium, suggesting this antioxidant may be released from an extrapulmonary source. Because leukocytes are known to express EC-SOD and are prevalent in the bronchoalveolar lavage fluid (BALF) after injury, it was hypothesized that these cells may transport and release EC-SOD into airspaces. To test this hypothesis, C57BL/6 wild-type and EC-SOD knockout mice were irradiated and transplanted with bone marrow from either wild-type mice or EC-SOD knockout mice. Bone marrow chimeric mice were then intratracheally treated with asbestos and killed 3 and 7 days later. At both 3 and 7 days following asbestos injury, mice without pulmonary EC-SOD expression but with EC-SOD in infiltrating and resident leukocytes did not have detectable levels of EC-SOD in the airspaces. In addition, leukocyte-derived EC-SOD did not significantly lessen inflammation or early stage fibrosis that resulted from asbestos injury in the lungs. Although it is not influential in the asbestos-induced interstitial lung injury model, EC-SOD is still known to be present in leukocytes and may play an influential role in attenuating pneumonias and other inflammatory diseases.     

Expression of extracellular superoxide dismutase by human cell lines.

Extracellular superoxide dismutase (EC-SOD) is the major SOD isoenzyme in extracellular fluids, but occurs also in tissues. The sites and characteristics of the synthesis of the enzyme are unknown. The occurrence of EC-SOD in cultures of a large panel of human cell lines was assayed by means of an e.l.i.s.a. Unlike the situation for the intracellular isoenzymes CuZn-SOD and Mn-SOD, expression of EC-SOD occurs in only a few cell types. None of the ten investigated suspension-growing cell lines produced EC-SOD. Among normal diploid anchorage-dependent cell lines, expression was found in all 25 investigated fibroblast cell lines, in the two glia-cell lines, but not in six endothelial-cell lines, two epithelial-cell lines or in two amnion-derived lines. Among neoplastic anchorage-dependent cell lines expression was found in 13 out of 29. EC-SOD was secreted into the culture medium by cell lines expressing the enzyme. The rate of EC-SOD synthesis varied by nearly 100-fold among the fibroblast lines and remained essentially constant in the individual lines during long-term culture. In the nine investigated cases, the secreted EC-SOD was of the high-heparin-affinity C type. It is suggested that tissue EC-SOD is secreted by a few well-dispersed cell types, such as fibroblasts and glia cells, to diffuse subsequently around and reversibly bind to heparan sulphate proteoglycan ligands in the glycocalyx of the surface of most tissue cell types and in the interstitial matrix.     

Regulation by cytokines of extracellular superoxide dismutase and other superoxide dismutase isoenzymes in fibroblasts.

The influence of cytokines on extracellular superoxide dismutase (EC-SOD) expression by human dermal fibroblasts was investigated. The expression was markedly stimulated by interferon-gamma (IFN-gamma), was varying between fibroblast lines stimulated or depressed by interleukin-1 alpha (IL-1 alpha), was intermediately depressed by tumor necrosis factor-alpha (TNF-alpha), and markedly depressed by transforming growth factor-beta (TGF-beta). TNF-alpha, however, enhanced the stimulation by a high dose of IFN-gamma, whereas TGF-beta markedly depressed the stimulations given by IFN-gamma and IL-1 alpha. The ratio between the maximal stimulation and depression observed was around 30-fold. The responses were generally slow and developed over periods of several days. There were no effects of IFN-alpha, IL-2, IL-3, IL-4, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, human growth hormone, Escherichia coli lipopolysaccharide, leukotriene B4, prostaglandin E2, formylmethionylleucylphenylalanine, platelet-activating factor, and indomethacin. The cytokines influencing the EC-SOD expression are also known to influence superoxide production by leukocytes and other cell types, and the EC-SOD response pattern is roughly compatible with the notion that its function is to protect cells against extracellular superoxide radicals. The results show that EC-SOD is a participant in the complex inflammatory response orchestrated by cytokines. The CuZn-SOD activity of the fibroblasts was not influenced by any of the cytokines, whereas the Mn-SOD activity was depressed by TGF-beta. TNF-alpha, IL-1 alpha, and IFN-gamma stimulated the Mn-SOD activity, as previously known, and these responses were reduced by TGF-beta. The different responses of the three SOD isoenzymes illustrate their different physiological roles.