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

1992 ◽  
Vol 267 (25) ◽  
pp. 18205-18209
Author(s):  
J Sandström ◽  
L Carlsson ◽  
S.L. Marklund ◽  
T Edlund
Keyword(s):  
Superoxide Dismutase ◽  
Binding Domain ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Binding ◽  
Heparin Binding Domain ◽  
Heparin Affinity

scholarly journals Heparin-affinity patterns and composition of extracellular superoxide dismutase in human plasma and tissues

1993 ◽  
Vol 294 (3) ◽  
pp. 853-857 ◽  
Author(s):  
J Sandström ◽  
K Karlsson ◽  
T Edlund ◽  
S L Marklund
Keyword(s):  
Superoxide Dismutase ◽  
Umbilical Cord ◽  
Heparan Sulphate ◽  
Complex Mixture ◽  
Distribution Volume ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Binding ◽  
Intracellular Space ◽  
Heparin Binding Domain ◽  
Heparin Affinity

The tetrameric extracellular superoxide dismutase (EC-SOD) in human tissues and plasma has previously been found to be heterogenous with regard to heparin affinity and could be divided into at least three classes: A, lacking heparin affinity; B, with weak affinity; and C, with strong affinity. Using rigorous extraction conditions and an extensive set of anti-proteolytic agents, tissue EC-SOD is now shown to be almost exclusively of native homotetrameric C-class. Plasma EC-SOD on the other hand is shown to be mainly composed of a complex mixture of heterotetramers with modifications probably residing in the C-terminal heparin-binding domain. Proteolytic truncations appear to be a major cause of this heterogeneity. The findings suggest that, since 99% of the EC-SOD in the human body exists in the extravascular space of tissue, EC-SOD is primarily synthesized in tissues and secreted as homotetrameric native EC-SOD C. This tissue EC-SOD C should exist almost completely sequestered by heparin sulphate proteoglycans. C-terminal modifications subsequently occurring in the EC-SOD C would weaken the binding to heparan sulphate proteoglycan, facilitate entrance to the vasculature through capillaries and lymph flow, and finally result in the heterogeneous plasma EC-SOD pattern. With the new extraction and analysis procedure, the tissue content of EC-SOD is found to be higher than previously reported. It is found, for example, when compared with Mn-SOD, to be higher in umbilical cord and uterus, about equal in placenta and testis and as high as that of CuZn-SOD in umbilical cord. The findings suggest that the protection level against superoxide radicals provided by EC-SOD in the tissue interstitial space, given the small distribution volume, is not much less prominent than that bestowed on the intracellular space by CuZn-SOD and Mn-SOD.

scholarly journals The Heparin-binding Domain of Extracellular Superoxide Dismutase Is Proteolytically Processed Intracellularly during Biosynthesis

1999 ◽  
Vol 274 (21) ◽  
pp. 14818-14822 ◽  
Author(s):  
Jan J. Enghild ◽  
Ida B. Thøgersen ◽  
Tim D. Oury ◽  
Zuzana Valnickova ◽  
Peter Højrup ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Binding Domain ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Binding ◽  
Heparin Binding Domain

scholarly journals Proteolytic modification of the heparin-binding affinity of extracellular superoxide dismutase

1993 ◽  
Vol 290 (2) ◽  
pp. 623-626 ◽  
Author(s):  
K Karlsson ◽  
A Edlund ◽  
J Sandström ◽  
S L Marklund
Keyword(s):  
Superoxide Dismutase ◽  
Binding Affinity ◽  
Heparan Sulphate ◽  
Limited Proteolysis ◽  
Size Exclusion ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Binding ◽  
Binding Domains ◽  
Heparin Affinity

The heparin-binding affinity of the tetrameric extracellular superoxide dismutase (EC-SOD) is a result of the cooperative effect of the heparin-binding domains of the subunits, located in the hydrophilic, strongly positively charged C-terminal ends. EC-SOD C, the high-heparin-affinity type, exposed to immobilized trypsin and plasmin was found to rapidly lose its affinity for heparin, without any loss of enzymic activity or major change in molecular mass as judged by size-exclusion chromatography. Heparin and dextran sulphate 5000 inhibited the proteolysis, suggesting that EC-SOD C sequestered by heparan sulphate proteoglycan in vivo is partially protected against proteolysis. The loss of heparin-affinity occurred with the stepwise formation of intermediates, and the pattern upon chromatography on heparin-Sepharose and subsequent immunoblotting was compatible with the notion that the changes are due to sequential truncations of heparin-binding domains from subunits composing the EC-SOD tetramers. A similar pattern with intermediates and apparent truncations has previously been found with EC-SOD of human plasma. The findings show that the unique design of the heparin-binding domain of EC-SOD allows easy modification of the heparin-affinity by means of limited proteolysis, and suggest that such proteolysis is a major contributor to the heterogeneity in heparin-affinity of EC-SOD in mammalian plasma.

scholarly journals Amelioration of the severity of heparin-binding antithrombin mutations by posttranslational mosaicism

Blood ◽  
2012 ◽  
Vol 120 (4) ◽  
pp. 900-904 ◽  
Author(s):  
Irene Martínez-Martínez ◽  
José Navarro-Fernández ◽  
Alice Østergaard ◽  
Ricardo Gutiérrez-Gallego ◽  
José Padilla ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Binding Domain ◽  
Heparin Binding ◽  
Wild Type ◽  
Antithrombin Deficiency ◽  
Thrombotic Risk ◽  
Heparin Binding Domain ◽  
New Type ◽  
Fluorescence Kinetic ◽  
Heparin Affinity

The balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural β-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and β-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (KD, 107.9 ± 3nM) was restored in the β-L99F glycoform (KD, 53.9 ± 5nM) to values close to the activity of α-wild type (KD, 43.9 ± 0.4nM). Accordingly, the β-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional β-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin mutations.

Gene transfer of extracellular superoxide dismutase protects against vascular dysfunction with aging

2006 ◽  
Vol 290 (6) ◽  
pp. H2600-H2605 ◽  
Author(s):  
Kathryn A. Brown ◽  
Yi Chu ◽  
Donald D. Lund ◽  
Donald D. Heistad ◽  
Frank M. Faraci
Keyword(s):  
Superoxide Dismutase ◽  
Gene Transfer ◽  
Vascular Function ◽  
Vascular Dysfunction ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Binding ◽  
Aged Rats ◽  
Young Rats ◽  
Heparin Binding Domain ◽  
Old Rats

Aging is an independent risk factor for cardiovascular disease, but mechanisms leading to vascular dysfunction have not been fully elucidated. Recent studies suggest that oxidative stress may increase in blood vessels during aging. Levels of superoxide are influenced by the activity of SODs. The goal of this study was to examine the effect of extracellular superoxide dismutase (ECSOD) on superoxide levels and vascular function in an animal model of aging. Aortas from young (4–8 mo old) and old (29–31 mo old) Fischer 344 rats were examined in vitro. Relaxation of aorta to ACh was impaired in old rats compared with young rats; e.g., 3 μM ACh produced 57 ± 4% (mean ± SE) and 84 ± 2% relaxation in old and young rats, respectively ( P < 0.0001). Three days after gene transfer of adenovirus expressing human ECSOD (AdECSOD), the response to ACh was not affected in young rats but was improved in old rats. There was no difference in relaxation to the endothelium-independent dilator sodium nitroprusside between young, aged, and AdECSOD-treated old rats. Superoxide levels (lucigenin-enhanced chemiluminescence) were significantly increased in aged rats compared with young rats. After gene transfer of ECSOD to aged rats, superoxide levels in aorta were similar in old and young rats. Gene transfer of an ECSOD with the heparin-binding domain deleted had no effect on vascular function or superoxide levels in old rats. These results suggest that 1) vascular dysfunction associated with aging is mediated in part by increased levels of superoxide, 2) gene transfer of ECSOD reduces vascular superoxide and dysfunction in old rats, and 3) beneficial effects of ECSOD in old rats require the heparin-binding domain of ECSOD.

scholarly journals Non-enzymic glycation of human extracellular superoxide dismutase

1991 ◽  
Vol 279 (1) ◽  
pp. 263-267 ◽  
Author(s):  
T Adachi ◽  
H Ohta ◽  
K Hirano ◽  
K Hayashi ◽  
S L Marklund
Keyword(s):  
Superoxide Dismutase ◽  
Heparan Sulphate ◽  
Normal Subjects ◽  
Enzymic Activity ◽  
Diabetic Patients ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Binding ◽  
A Minor ◽  
Heparin Affinity

The secretory enzyme extracellular superoxide dismutase (EC-SOD) is in plasma heterogenous with regard to heparin-affinity and can be divided into three fractions, A that lacks affinity, B with intermediate affinity and C with high affinity. The C fraction forms an equilibrium between the plasma phase and heparan sulphate proteoglycan on the surface of the endothelium. In vitro EC-SOD C could be time-dependently glycated. The enzymic activity was not affected in glycated EC-SOD, but the high heparin-affinity was lost in about half of the studied glycated fraction. Addition of heparin decreased the glycation in vitro, and EC-SOD C modified with the lysine-specific reagent trinitrobenzenesulphonic acid could not be glycated in vitro. The findings suggest that the glycation sites are localized rather far away from the active site and may occur on lysine residues in the heparin-binding domain in the C-terminal end of the enzyme. The proportion of glycated EC-SOD in serum of diabetic patients was considerably higher than in normal subjects. Of the subfractions, EC-SOD B was by far the most highly glycated, followed by EC-SOD A. EC-SOD C was glycated only to be a minor extent. The findings suggest that glycation is one of the factors that contribute to the heterogeneity in heparin-affinity of plasma EC-SOD. Since this phenomenon is increased in diabetes, the cell-surface-associated EC-SOD may be decreased in this disease, increasing the susceptibility of cells to superoxide radicals produced in the extracellular space.

Depletion of pulmonary EC-SOD after exposure to hyperoxia

2002 ◽  
Vol 283 (4) ◽  
pp. L777-L784 ◽  
Author(s):  
Tim D. Oury ◽  
Lisa M. Schaefer ◽  
Cheryl L. Fattman ◽  
Augustine Choi ◽  
Karen E. Weck ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage Fluid ◽  
Lavage Fluid ◽  
Binding Domain ◽  
Extracellular Superoxide Dismutase ◽  
Pulmonary Injury ◽  
Heparin Binding ◽  
Cell Surfaces ◽  
Heparin Binding Domain ◽  
High Concentrations ◽  
Alveolar Parenchyma

Extracellular superoxide dismutase (EC-SOD) is highly expressed in lung tissue. EC-SOD contains a heparin-binding domain that is sensitive to proteolysis. This heparin-binding domain is important in allowing EC-SOD to exist in relatively high concentrations in specific regions of the extracellular matrix and on cell surfaces. EC-SOD has been shown to protect the lung against hyperoxia in transgenic and knockout studies. This study tests the hypothesis that proteolytic clearance of EC-SOD from the lung during hyperoxia contributes to the oxidant-antioxidant imbalance that is associated with this injury. Exposure to 100% oxygen for 72 h resulted in a significant decrease in EC-SOD levels in the lungs and bronchoalveolar lavage fluid of mice. This correlated with a significant depletion of EC-SOD from the alveolar parenchyma as determined by immunofluorescence and immunohistochemistry. EC-SOD mRNA was unaffected by hyperoxia; however, there was an increase in the ratio of proteolyzed to uncut EC-SOD after hyperoxia, which suggests that hyperoxia depletes EC-SOD from the alveolar parenchyma by cutting the heparin-binding domain. This may enhance hyperoxic pulmonary injury by altering the oxidant-antioxidant balance in alveolar spaces.

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