The heparin binding site of human extracellular-superoxide dismutase.

Extracellular-superoxide dismutase (EC-SOD) is a secretory glycoprotein that is major SOD isozyme in extracellular fluids. We revealed the possible structure of the carbohydrate chain of serum EC-SOD with the serial lectin affinity technique. The structure is a biantennary complex type with an internal fucose residue attached to asparagine-linked N-acetyl-D-glucosamine and with terminal sialic acid linked to N-acetyllactosamine. EC-SOD in plasma is heterogeneous with regard to heparin affinity and can be divided into three fractions: A, without affinity; B, with intermediate affinity; and C, with high affinity. It appeared that this heterogeneity is not dependent on the carbohydrate structure upon comparison of EC-SOD A, B, and C. No effect of the glycopeptidase F treatment of EC-SOD C on its heparin affinity supported the results. A previous report showed that both lysine and arginine residues probably at the C-terminal end, contribute to heparin binding. Recombinant EC-SOD C treated with trypsin or endoproteinase Lys C, which lost three lysine residues (Lys-211, Lys-212, and Lys-220) or one lysine residue (Lys-220) at the C-terminal end, had no or weak affinity for the heparin HPLC column, respectively. The proteinase-treated r-EC-SOD C also lost triple arginine residues which are adjacent to double lysine residues. These results suggest that the heparin-binding site may occur on a cluster of basic amino acids at the C-terminal end of EC-SOD C. EC-SOD is speculated to be primarily synthesized as type C, and types A and B are probably the result of secondary modifications. It appeared that the proteolytic cleavage of the exteriorized lysine- and arginine-rich C-terminal end in vivo is a more important contributory factor to the formation of EC-SOD B and/or EC-SOD A.     

Regulation of extracellular-superoxide dismutase in rat retina pericytes

Abstract

Diabetic retinopathy (DR) is regarded as a disease of the retinal microvascular system and metabolic abnormalities that are characteristic of oxidative stress and endoplasmic reticulum (ER) stress have been identified in the retina. Pericytes are known to be susceptible to oxidative stress and selective dropout of pericytes is one of the earliest pathological changes in DR. Extracellular-superoxide dismutase (EC-SOD) is a major antioxidative enzyme and protects vascular cells from the damaging effects of superoxide. Treatment with own conditioned medium significantly decreased EC-SOD expression in pericytes, while the expression of vascular endothelial growth factor and tumor necrosis factor-α were elevated. The addition of chemical chaperone 4-phenyl butyric acid significantly suppressed the effects of conditioned medium on EC-SOD and GRP78, a prominent ER-resident chaperone. Moreover, the cell viability of pericytes changed in a manner similar to that of EC-SOD expression. These results suggest that the expressions of EC-SOD should be regulated, at least partially, through ER stress. Continuous flow of culture media neutralized the ER-stress triggered decrease of EC-SOD expression. The stagnation of factors related to ER-stress around pericytes might reduce EC-SOD expression under pathophysiological conditions such as retinal edema, and this could induce and/or promote the intraretinal microvascular impairment and development of pathogenesis in DR.     

Regulation of extracellular-superoxide dismutase in rat retina pericytes

Diabetic retinopathy (DR) is regarded as a disease of the retinal microvascular system and metabolic abnormalities that are characteristic of oxidative stress and endoplasmic reticulum (ER) stress have been identified in the retina. Pericytes are known to be susceptible to oxidative stress and selective dropout of pericytes is one of the earliest pathological changes in DR. Extracellular-superoxide dismutase (EC-SOD) is a major antioxidative enzyme and protects vascular cells from the damaging effects of superoxide. Treatment with own conditioned medium significantly decreased EC-SOD expression in pericytes, while the expression of vascular endothelial growth factor and tumor necrosis factor-α were elevated. The addition of chemical chaperone 4-phenyl butyric acid significantly suppressed the effects of conditioned medium on EC-SOD and GRP78, a prominent ER-resident chaperone. Moreover, the cell viability of pericytes changed in a manner similar to that of EC-SOD expression. These results suggest that the expressions of EC-SOD should be regulated, at least partially, through ER stress. Continuous flow of culture media neutralized the ER-stress triggered decrease of EC-SOD expression. The stagnation of factors related to ER-stress around pericytes might reduce EC-SOD expression under pathophysiological conditions such as retinal edema, and this could induce and/or promote the intraretinal microvascular impairment and development of pathogenesis in DR.     

Effects of recombinant human extracellular-superoxide dismutase type C on myocardial infarct size in pigs.

The efficacy of human extracellular-superoxide dismutase type C (EC-SOD C) to limit infarct size after ischemia and reperfusion was explored and compared to that of EC-SOD C combined with catalase (CAT) and to that of CAT alone. EC-SOD C binds to heparan sulphate proteoglycan on the cell surfaces. Thirty-two pigs were subjected to 45 min of myocardial ischemia followed by 4 h of reperfusion. Control pigs (group A; n = 8) received 300 mL of saline into the great cardiac vein during a 30-min period started 5 min prior to reperfusion; pigs in group B (EC-SOD C; n = 8) got 16.6 mg of EC-SOD C; pigs in group C (EC-SOD C + CAT; n = 8) got 16.6 mg of EC-SOD C together with 150 mg of CAT. Pigs in group D (CAT; n = 8) received 150 mg of CAT. In groups B, C, and D, the drug was dissolved in saline and infused into the great cardiac. Infarct size expressed as percent of area at risk was smaller in groups B (14.5 +/- 16.7%) and C (40.8 +/- 13.3%) than in groups A (78.8 +/- 8.6%) and D (67.2 +/- 18.6%; p less than .05). Creatine kinase (CK) activity in ischemic myocardium was higher in groups B (1740 +/- 548 U/g) and C (1729 +/- 358 U/g) than in groups A (1184 +/- 237 U/g) and D (1251 +/- 434 U/g; p less than .05). There was an inverse relation (r = -.83) between infarct size and CK content. The EC-SOD C infusions resulted in only minimal increases in plasma SOD activities. In conclusion, the presence of SOD on the cell surfaces is of importance in the prevention of reperfusion injury rather than circulating SOD.     

Effects of recombinant human extracellular-superoxide dismutase type C on myocardial reperfusion injury in isolated cold-arrested rat hearts.

The efficacy of recombinant human extracellular-superoxide dismutase type C (EC-SOD C) on myocardial reperfusion injury was explored in hypothermically arrested rat hearts, as was its site of action. Forty isolated working rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion. The hearts were arrested by the administration of 10 mL of cold perfusate at the onset of ischemia. At the same time, they were randomly assigned to one of five groups; A: cold perfusate only; B: cold perfusate + EC-SOD C 10.4 mg/L (30,000 U/L); C: cold perfusate+bovine CuZn-SOD 7.5 mg/L (30,000 U/L); D: cold perfusate + EC-SOD C 10.4 mg/L + heparin 50,000U/L; E: cold perfusate + heparin 50,000 U/L. Heparin was given to prevent binding of EC-SOD C to endothelial cell surfaces. Left ventricular function was studied before ischemia and at the end of reperfusion. Percent recovery of maximal left ventricular dP/dt after reperfusion was more pronounced in group B (109 +/- 24%; p less than .05) than in groups A (42 +/- 40%), C (47 +/- 36%), D (44 +/- 33%) and E (58 +/- 25%). Likewise, percent recovery of the double product (heart rate x systolic left ventricular pressure) was better in group B (104 +/- 18%; p less than .05) than in the other groups (A: 47 +/- 37%, C: 49 +/- 36%, D: 50 +/- 35%, E: 69 +/- 31%). Compared to the preischemic level, creatine kinase increased significantly in the coronary effluent after reperfusion in groups A, C, D, and E, but not in group B. The results suggest that EC-SOD C, which attaches to the endothelial cell surfaces, might be particularly effective as protection against myocardial reperfusion injury when given together with cardioplegic solution.     

Endoplasmic reticulum stress induces retinal endothelial permeability of extracellular-superoxide dismutase

The aim of this study was to determine the reasons why the intravitreal level of extracellular-superoxide dismutase (EC-SOD) increases in proliferative diabetic retinopathy patients by the investigation of two possibilities: first, change of EC-SOD expression in the retina; and secondly, leakage of EC-SOD through the endothelial monolayer by the treatment with endoplasmic reticulum (ER) stress inducers because ER stress is known to be involved in the vascular impairment in diabetic retinopathy. Intravitreous injection of tunicamycin in mice increased the permeability of tracer dye across retinal blood vessels while the retinal EC-SOD mRNA level was not changed. The leakage of EC-SOD through the retinal endothelial cell layer was elevated by the treatment with thapsigargin or tunicamycin. The expression of claudin-5 was significantly decreased by the treatment with the ER stress inducers. These phenomena were significantly suppressed by the pre-treatment of endothelial cells with a chemical chaperone 4-phenylbutyric acid. Our observations suggest that ER stress leads to the down-regulation of claudin-5 among tight junction proteins and may induce the elevation of endothelial permeability and leakage of EC-SOD into the vitreous body.     

Nitric oxide and blood pressure in mice lacking extracellular-superoxide dismutase

Nitric oxide is a major vasorelaxant and regulator of the blood pressure. The blood vessels contain several active sources of the superoxide radical, which reacts avidly with nitric oxide to form noxious peroxynitrite. There are large amounts of extracellular-superoxide dismutase (EC-SOD) in the vascular wall. To evaluate the importance of EC-SOD for the physiology of nitric oxide, here we studied the blood pressure in mice lacking the enzyme. In chronically instrumented non-anaesthetized mice there was no difference in mean arterial blood pressure between wild-type controls and EC-SOD mutants. Extensive inhibition of nitric oxide synthases with N -monomethyl- l -arginine however resulted in a larger increase in blood pressure, and infusion of the nitric oxide donor nitrosoglutathione caused less reduction in blood pressure in the EC-SOD null mice. We interpret the alterations to be caused by a moderately increased consumption of nitric oxide by the superoxide radical in the EC-SOD null mice. One role of EC-SOD may be to preserve nitric oxide, a function that should be particularly important in vascular pathologies, in which large increases in superoxide formation have been documented.     

Endoplasmic reticulum stress induces retinal endothelial permeability of extracellular-superoxide dismutase

Abstract

The aim of this study was to determine the reasons why the intravitreal level of extracellular-superoxide dismutase (EC-SOD) increases in proliferative diabetic retinopathy patients by the investigation of two possibilities: first, change of EC-SOD expression in the retina; and secondly, leakage of EC-SOD through the endothelial monolayer by the treatment with endoplasmic reticulum (ER) stress inducers because ER stress is known to be involved in the vascular impairment in diabetic retinopathy. Intravitreous injection of tunicamycin in mice increased the permeability of tracer dye across retinal blood vessels while the retinal EC-SOD mRNA level was not changed. The leakage of EC-SOD through the retinal endothelial cell layer was elevated by the treatment with thapsigargin or tunicamycin. The expression of claudin-5 was significantly decreased by the treatment with the ER stress inducers. These phenomena were significantly suppressed by the pre-treatment of endothelial cells with a chemical chaperone 4-phenylbutyric acid. Our observations suggest that ER stress leads to the down-regulation of claudin-5 among tight junction proteins and may induce the elevation of endothelial permeability and leakage of EC-SOD into the vitreous body.     

Enhanced alloxan-induced beta-cell damage and delayed recovery from hyperglycemia in mice lacking extracellular-superoxide dismutase.

Alloxan is a diabetogenic agent which apparently acts through formation of superoxide radicals formed by redox cycling. Superoxide radicals are also formed by a variety of mechanisms in hyperglycemia. We exposed extracellular-superoxide dismutase (EC-SOD) null mutant and wild-type mice to alloxan, and followed up both the initial diabetes induction and the long-term course of the hyperglycemia. The null mutant mice responded with a modestly enhanced hyperglycemia compared to the wild type controls. In the long-term follow-up all mice eventually regained glycemic control, although it took longer for individuals with higher initial hyperglycemia. This delaying effect of the hyperglycemia was much more pronounced in the null mutant mice. These data suggest that the difference in initial diabetes induction between the groups is due to interception by EC-SOD of extracellular superoxide radicals produced by alloxan. The delayed recovery in the null mutant mice suggests that superoxide radicals released as a result of hyperglycemia impair beta-cell regeneration and that EC-SOD provides some protection. Mouse islets were found to contain little EC-SOD, whereas the content of the cytosolic Cu- and Zn-containing SOD was very high. This low EC-SOD activity may contribute to the high alloxan susceptibility of beta-cells, and may also cause a high susceptibility to superoxide radicals produced by activated inflammatory leukocytes and in hyperglycemia.     

Heparin-, dextran sulfate- and protamine-induced release of extracellular-superoxide dismutase to plasma in pigs

Intravenous heparin has previously been shown to release the high-heparin-affinity fraction C of extracellular-superoxide dismutase (EC-SOD, EC 1.15.1.1) to plasma in man and other mammals. This paper reports on further studies of the phenomena in the pig. A dose-response curve of the effect of heparin revealed that 1000 IU/kg body weight is needed for maximal release of EC-SOD C. This dose is an order of magnitude larger than that needed for the maximal release to plasma of factors such as lipoprotein lipase, hepatic lipase, and diamine oxidase, which are distributed between plasma and endothelium similarly to EC-SOD C. Thus EC-SOD C appears to have an unusually high affinity for endothelial cell-surface sulfated glycosaminoglycans relative to the affinity for heparin. There was no significant difference in releasing potency between unfractionated heparin and heparin subfractions with high or low affinity for antithrombin III. The heparin structure conferring high-affinity binding to antithrombin III is thus not specifically involved in binding to EC-SOD C. The non-biosynthetic compound dextran sulfate 5000 was an order of magnitude more efficient than heparin. Protamine displayed dual effects. Given alone in high dose it released EC-SOD to plasma, probably due to binding to endothelial cell-surface sulfated glycosaminoglycans displacing fraction C of the enzyme. When given after heparin, in a dose just below that expected to neutralize the heparin, protamine reversed the heparin-induced EC-SOD release.     

Enzymic and nonenzymic properties of human beta-thrombin

Autolysis or tryptic hydrolysis converts human alpha-thrombin to its beta-derivative and subsequently to gamma-thrombin. Human beta-thrombin was obtained by tryptic digestion of alpha-thrombin and isolated by BioRex chromatography. The kinetic parameters for human alpha- and beta-thrombins with H-D-phenylalanyl-L-pipecolyl-L-arginine-para-nitroanilide were similar, as well as the rate of inactivation by tosyl-lysine chloromethyl ketone. By contrast, the rate of inactivation by diisopropyl fluorophosphate was reduced by half, and the inhibition constant for benzamidine was increased 2.5-fold. Moreover, the beta cleavages induced a drastic reduction in reactivity toward protein C, affinity for thrombomodulin, and fibrinogen clotting activity. Unlike alpha-thrombin, beta-thrombin was not protected from inhibition by diisopropyl fluorophosphate in the presence of fibrinogen and failed to bind to fibrin-Sepharose. Our results indicate that the beta cleavages induce multiple defects in the functions of human thrombin. Although the three catalytic residues remain in an active configuration, subtle changes are induced in the microenvironment of the active serine. However, the drastic reduction of fibrinogen clotting activity should rather be ascribed to major alterations observed in both the fibrinopeptide groove and the fibrin recognition site. These observations provide further evidence for a double-site mechanism in the interaction of fibrinogen with thrombin.     

Effects of homocysteine on the binding of extracellular-superoxide dismutase to the endothelial cell surface

Homocysteine is known to be a risk factor for several vascular diseases. Previously, we found a significant association between plasma homocysteine and plasma extracellular-superoxide dismutase (EC-SOD) levels. The binding of EC-SOD to human and bovine aortic endothelial cell cultures showed significant decreases after incubation with 10 microM homocysteine, whereas the expression of EC-SOD in fibroblast cell cultures was inhibited with a high concentration (1 mM) of homocysteine. Furthermore, binding of EC-SOD to heparin immobilized on plates was decreased with homocysteine. These observations suggested that homocysteine decreases the binding of EC-SOD to vascular endothelial cell surfaces by degradation of endothelial heparan sulfate proteoglycan, which results in a loss of the ability to protect endothelial cell surfaces from oxidative stress.     

Induction of manganese superoxide dismutase in cultured human trophoblast during in vitro differentiation.

The antioxidant responses of human cell differentiation and membrane fusion are not known and may be important in understanding cellular response to injury in the human placenta. We studied the regulation of antioxidant enzymes in human trophoblasts which differentiate from mononucleated cellular trophoblasts to synctium in vivo and in culture. We characterized morphological and biochemical differentiation of cultured trophoblasts from term placenta in the presence or absence of serum, on different growth surfaces, and with a range of plating densities. Culture of cellular trophoblasts consistently and transiently induced the mRNAs of the mitochondrial antioxidant manganese superoxide dismutase (Mn SOD) but not the mRNAs for the antioxidant enzymes copper zinc SOD or catalase. Fibrin and type I collagen substrates modulated only the expression of the placental specific proteins, human chorionic gonadotropin, and human placental lactogen. Both Mn SOD induction and terminal differentiation, as reflected by human chorionic gonadotropin expression, were dependent on trophoblastic plating density. Increased levels of a smaller Mn SOD mRNA species correlated temporally with an increase in Mn SOD enzyme activity in cultured trophoblasts. These results demonstrate that Mn SOD gene expression and enzyme activity precede or are coordinately regulated with morphological and biochemical trophoblastic differentiation.     

The in vitro glycation of human serum albumin in the presence of Zn(II)

Amino groups of human serum albumin (HSA) can react non-enzymatically with carbonyl groups of reducing sugars to form advanced glycation end products (AGEs). These AGEs contribute to many of the chronic complications of diabetes including atherosclerosis, cataract formation and renal failure. The current study focused on in vitro non-enzymatic reactivity of glyceraldehyde (GA) and methylglyoxal (MG) with HSA and evaluated the rate and extent of AGE formation in the presence of varied concentrations of Zn(II). At normal physiological conditions, GA and MG readily react with HSA. The presence of Zn(II) in HSA-GA or HSA-MG incubation mixtures reduced AGE formation. This finding was confirmed by UV and fluorescence spectrometry, HPLC techniques, and matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF). HPLC studies revealed decreased adduct formation of the glycated protein in the presence of Zn(II). The inhibition of AGE formation was intense at elevated Zn(II) concentrations. The results of this study suggest that Zn(II) may prove to be a potent agent in reducing AGE formation.     

Effect of nonenzymatic glycation of albumin and superoxide dismutase by glucuronic acid and suprofen acyl glucuronide on their functions in vitro.

Acyl glucuronides bind irreversibly to plasma proteins, and one mechanism proposed for this covalent binding is similar to that for glycation of protein by reducing sugars. Because glycation of protein by glucose and other reducing sugars can alter protein function, this lead to the hypothesis that the glycation of proteins by acyl glucuronides may cause similar effects. When human serum albumin (HSA) was incubated with 0.5 M glucose for 5 days, the unbound fractions of diazepam and warfarin were increased by 41 and 35%, respectively, less than that caused by glucuronic acid which increased the unbound fractions by 90% for diazepam and 420% for warfarin. When HSA was incubated with suprofen glucuronide (SG) at a much lower concentration of 0.005 M for only 24 h, the effects on the unbound fractions of diazepam and warfarin to HSA were altered dramatically with increases of 340 and 230%, respectively. After incubation of superoxide dismutase (SOD) with 0.5 or 1 M reducing sugars for 14 days, the enzyme activity decreased to 82 and 61% of initial levels at day 14, respectively, whereas glucuronic acid almost completely inactivated the enzyme activity over the same period. Even at a very low concentration (0.005 M) of SG, SOD activity was reduced significantly to 11% of initial levels by day 14, which was comparable to the effect by 0.5 and 1.0 M concentrations of glucuronic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix associated laser desorption/ionization time of flight mass spectrometry indicated that several equivalents of reducing sugars or SG became attached to albumin after incubation. These results suggest that acyl glucuronides may affect the function of proteins by the formation of glycated protein in vivo and may be associated with the toxicity of xenobiotics metabolized to labile acyl glucuronides.     

Interrupting the hydrogen bond network at the active site of human manganese superoxide dismutase.

Histidine 30 in human manganese superoxide dismutase (MnSOD) is located at a site partially exposed to solvent with its side chain participating in a hydrogen-bonded network that includes the active-site residues Tyr(166) and Tyr(34) and extends to the manganese-bound solvent molecule. We have replaced His(30) with a series of amino acids and Tyr(166) with Phe in human MnSOD. The crystal structure of the mutant of MnSOD containing Asn(30) superimposed closely with the wild type, but the side chain of Asn(30) did not participate in the hydrogen-bonded network in the active site. The catalytic activity of a number of mutants with replacements at position 30 and for the mutant containing Phe(166) showed a 10-40-fold decrease in k(cat). This is the same magnitude of decrease in k(cat) obtained with the replacement of Tyr(34) by Phe, suggesting that interrupting the hydrogen-bonded active-site network at any of the sites of these three participants (His(30), Tyr(34), and Tyr(166)) leads to an equivalent decrease in k(cat) and probably less efficient proton transfer to product peroxide. The specific geometry of His(30) on the hydrogen bond network is essential for stability since the disparate mutations H30S, H30A, and H30Q reduce T(m) by similar amounts (10-16 degrees C) compared with wild type.     

Role of hydrogen bonding in the active site of human manganese superoxide dismutase

The side chain of Gln143, a conserved residue in manganese superoxide dismutase (MnSOD), forms a hydrogen bond with the manganese-bound solvent and is critical in maintaining catalytic activity. The side chains of Tyr34 and Trp123 form hydrogen bonds with the carboxamide of Gln143. We have replaced Tyr34 and Trp123 with Phe in single and double mutants of human MnSOD and measured their catalytic activity by stopped-flow spectrophotometry and pulse radiolysis. The replacements of these side chains inhibited steps in the catalysis as much as 50-fold; in addition, they altered the gating between catalysis and formation of a peroxide complex to yield a more product-inhibited enzyme. The replacement of both Tyr34 and Trp123 in a double mutant showed that these two residues interact cooperatively in maintaining catalytic activity. The crystal structure of Y34F/W123F human MnSOD at 1.95 A resolution suggests that this effect is not related to a conformational change in the side chain of Gln143, which does not change orientation in Y34F/W123F, but rather to more subtle electronic effects due to the loss of hydrogen bonding to the carboxamide side chain of Gln143. Wild-type MnSOD containing Trp123 and Tyr34 has approximately the same thermal stability compared with mutants containing Phe at these positions, suggesting the hydrogen bonds formed by these residues have functional rather than structural roles.