人细胞外超氧化物歧化酶的分子改造
摘要
人细胞外超氧化物歧化酶(EC-SOD)是四亚基糖蛋白,每个亚基含有一个Cu原子和一个Zn原子,对肝素和硫酸乙酰肝素有强亲和力。它可以清除体内超氧阴离子自由基,维持NO的生物活性,具有重要的生理意义。我们针对其活性较低且存在两种结构的问题,作了改进。鉴于EC-SOD和CuZnSOD的同源性,我们将EC-SOD的活性中心和人细胞内超氧化物歧化酶(CuZnSOD)的活性中心进行互换,减少天然人EC-SOD活性中心的巯基和二硫键,以期获得较高活性的EC-SOD。经过实验,我们获得的重组SOD,在pichia酵母中获得表达,并对重组酶的部分性质进行了研究。
There are three kinds of superoxide dismutase: intracellularsuperoxide dismutase(CuZnSOD), extracellular superoxide dismutase(EC-SOD) and Mn superoxide dismutase(Mn-SOD). EC-SOD is firstisolated from mammal by Stefan L. Marklund. It is the mainSOD-isoenzyme in extracellular matrix. EC-SOD is a tetramericsecretory glycoprotein with high affinity for heparin sulfateproteoglycans. Each subunit has one coper and one zinc atom. Theprimiry structure of EC-SOD contains three parts. The N-terminalregion supports the quaternary structure and contains the consensusfor N-linked glycosylation at Asn89. The C-terminal region mediatesthe binding of EC-SOD to components in extracellular matrix. Thecentral region of human EC-SOD has the activity site and isapproximately 50% identical to the final two-thirds of Cu/Zn-SODand contains all the ligands essential for the coordination of the activesite Cu(II) and Zn(II) ions. The protein is too large for structuredetermination by NMR. Indeed difficulties in crystallization ofEC-SODcaused probably by glycosylation lead to the failure indetermination of EC-SOD's three-dimensional structure by X-raycrystallography.
With a wealth of information provided in this field over the lastfew years, the significance of EC-SOD in biology and pathologybegins to be understood. EC-SOD preserves nitric oxide bioactivityand protect against extracellular oxidative damage throughscavenging superoxide,. We can detected EC-SOD activity in plasma,lymph and synovial fluid. It prevents and cure cardiovascular andpulmonary diseases, osteoarthritis and inflammation. In clinic
treatment it can be used in operation to cure ischemia-reperfusioninjury, traumatic brain injury and acute respiratory distress syndrome.And it also can protect cells from death or apoptosis which areassociated with aging and some diseases such as Huntingdon's diseaseand Parkinson's disease.Homologous genes recombination is an important mechanism ofgene evolution,and an important method of getting new enzymes inmolecular biology. We reported here the production of a new protein,which is a modified human EC-SOD at the activation site usinghomologous genes recombination technology.Because of their high lecleof homology at the activation sitebetween EC-SOD and CuZnSOD, we replace the activation site ofEC-SOD with that of CuZnSOD's. The mutant protein compromisesthe N-terminal and C-terminal domains of hEC-SOD with activationsite of hCuZnSOD. We constructed the fusion protein using stick-feetmutagenesis technique.During the past 15 years, the methylotrophic yeast Pichia pastorishas been developed into a highly successful system for the productionof a variety of heterologous proteins. All expression vectors have beendesigned as Escherichia coli/P. pastoris shuttle vectors, containing anorigin of replication for plasmid maintenance in E. coli. We selectedvector pPIC9 which undergoes secretery expression of our gene usingthe α-factor secretion signal. The constructed gene was cloned intothe vector, which was introduced into pichia pastoris by yeasttransformation kit. The results indicated that the constructed gene wasexpressed in pichia pastoris successfully.The sample containing fusion protein was applied to ananion-exchange chromatographic column, DEAE-Sepharose, and the
fusion protein was eluted in a pulse of 0.5 M NaCl in the buffer. ThenThe sample containing fusion protein was applied to Sephacryl-S200.The fusion protein eluted was indicaded to be designed protein bySDS-PAGE.We also measured the activity of the SOD. The activity is higherthan human EC-SOD, and the protein binds to heparin-sepharose withapproximately the same affinity as hEC-SOD.
There are three kinds of superoxide dismutase: intracellularsuperoxide dismutase(CuZnSOD), extracellular superoxide dismutase(EC-SOD) and Mn superoxide dismutase(Mn-SOD). EC-SOD is firstisolated from mammal by Stefan L. Marklund. It is the mainSOD-isoenzyme in extracellular matrix. EC-SOD is a tetramericsecretory glycoprotein with high affinity for heparin sulfateproteoglycans. Each subunit has one coper and one zinc atom. Theprimiry structure of EC-SOD contains three parts. The N-terminalregion supports the quaternary structure and contains the consensusfor N-linked glycosylation at Asn89. The C-terminal region mediatesthe binding of EC-SOD to components in extracellular matrix. Thecentral region of human EC-SOD has the activity site and isapproximately 50% identical to the final two-thirds of Cu/Zn-SODand contains all the ligands essential for the coordination of the activesite Cu(II) and Zn(II) ions. The protein is too large for structuredetermination by NMR. Indeed difficulties in crystallization ofEC-SODcaused probably by glycosylation lead to the failure indetermination of EC-SOD's three-dimensional structure by X-raycrystallography.
With a wealth of information provided in this field over the lastfew years, the significance of EC-SOD in biology and pathologybegins to be understood. EC-SOD preserves nitric oxide bioactivityand protect against extracellular oxidative damage throughscavenging superoxide,. We can detected EC-SOD activity in plasma,lymph and synovial fluid. It prevents and cure cardiovascular andpulmonary diseases, osteoarthritis and inflammation. In clinic
treatment it can be used in operation to cure ischemia-reperfusioninjury, traumatic brain injury and acute respiratory distress syndrome.And it also can protect cells from death or apoptosis which areassociated with aging and some diseases such as Huntingdon's diseaseand Parkinson's disease.Homologous genes recombination is an important mechanism ofgene evolution,and an important method of getting new enzymes inmolecular biology. We reported here the production of a new protein,which is a modified human EC-SOD at the activation site usinghomologous genes recombination technology.Because of their high lecleof homology at the activation sitebetween EC-SOD and CuZnSOD, we replace the activation site ofEC-SOD with that of CuZnSOD's. The mutant protein compromisesthe N-terminal and C-terminal domains of hEC-SOD with activationsite of hCuZnSOD. We constructed the fusion protein using stick-feetmutagenesis technique.During the past 15 years, the methylotrophic yeast Pichia pastorishas been developed into a highly successful system for the productionof a variety of heterologous proteins. All expression vectors have beendesigned as Escherichia coli/P. pastoris shuttle vectors, containing anorigin of replication for plasmid maintenance in E. coli. We selectedvector pPIC9 which undergoes secretery expression of our gene usingthe α-factor secretion signal. The constructed gene was cloned intothe vector, which was introduced into pichia pastoris by yeasttransformation kit. The results indicated that the constructed gene wasexpressed in pichia pastoris successfully.The sample containing fusion protein was applied to ananion-exchange chromatographic column, DEAE-Sepharose, and the
fusion protein was eluted in a pulse of 0.5 M NaCl in the buffer. ThenThe sample containing fusion protein was applied to Sephacryl-S200.The fusion protein eluted was indicaded to be designed protein bySDS-PAGE.We also measured the activity of the SOD. The activity is higherthan human EC-SOD, and the protein binds to heparin-sepharose withapproximately the same affinity as hEC-SOD.
引文
1. Mc.Cord, J.M. et.al. The utility of superoxide dismutase in studying free radical reaction. J. Biol. Chem. 1969;244:6049-6053
2. Igor N. Zelko, Thomas J. Mariani, and Rodney J. Folz Superoxide dismutase multigene family: A comparison of the CuZnSOD (SOD1), MnSOD (SOD2),and EC-SOD (SOD3) gene,structures, evolution, and expression. Free Radical Biology & Medicine, 2002;33: 337–349
3. Marklund, S.L. Human copper-containing superoxide dismutase of high molecular weight. Proc. Natl. Acad. Sci. USA 1982;79:7634-7638
4. Tibell L, Hjalmarsson K, Edlund T, et al. Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product. Proc Natl Acad Sci USA 1987;84:6634–8.
5. Hendrickson D.J., Fisher J.H., Jones C., et al. Regional localization of human extracellular superoxide dismutase gene to 4pter-q21. Genomics. 1990;8:736–738.
6. Folz R.J., Crapo J.D. Extracellular superoxide dismutase (sod3): tissue-specific expression, genomic characterization, and computer-assisted sequence analysis of the human EC-SOD gene. Genomics 1994;22:162–171.
7. Marklund SL. Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species. Biochem J 1984;222: 649–655.
8. Marklund SL. Extracellular superoxide dismutase in human tissues and human cell lines. J Clin Invest 1984;74: 1398–403.
9. Marklund SL, Holme E, Hellner L. Superoxide dismutase in extracellular fluids. Clin Chim Acta 1982;126:41–51.
10. Hjalmarsson K, Marklund SL, Engstrom A, et al. Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product. Proc Natl Acad Sci USA 1987;84:6340–4.
11. Stenlund P, Andersson D, Tibell LA. Subunit interaction in extracellular superoxide dismutase: effects of mutations in the N-terminal domain. Protein Sci 1997;6:2350–8.
12. Carlsson L. M., Marklund S. L., and Edlund T. The rat extracellular superoxide dismutase dimer is converted to a tetramer by the exchange of a single amino acid. Proc. Natl. Acad. Sci. U.S.A. 1996;93: 5219-5222
13. Stromqvist M, Holgersson J, Samuelsson B. Glycosylation of extracellular superoxide dismutase studied by high-performance liquid chromatography and mass spectrometry. J Chromatogr 1991;548:293–301.
14. Edlund A, Edlund T, Hjalmarsson K, et al. A non-glycosylated extracellular superoxide dismutase variant. Biochem J 1992;288:451–6.
15. Tainer JA, Getzoff ED, Beem KM, et al. Determination and analysis of the 2A-structure of copper, zinc superoxide dismutase. J Mol Biol 1982;160:181–217.
16. Parge HE, Getzoff ED, Scandella CS, et al. Crystallographic characterization of recombinant human CuZn superoxide dismutase. J Biol Chem 1986;261:16215–8.
17. Marklund S.L. Properties of extracellular superoxide dismutase from human lung. Biochem J 1984;220:269–72.
18. Tibell L., Aasa R., Marklund S.L. Spectral and physical properties of human extracellular superoxide dismutase: a comparison with Cu,Zn superoxide dismutase. Arch. Biochem. Biophys. 1993;304:429–433.
19. Jeney V, Itoh S, Wendt M, et.al. Role of antioxidant-1 in extracellular superoxide dismutase function and expression. Circ Res. 2005;96(7),723-729
20. Sandstrom J, Carlsson L, Marklund SL, et al. The heparin-binding domain of extracellular superoxide dismutase C and formation of variants with reduced heparin affinity. J Biol Chem 1992;267:18205–9.
21. Tibell LA, Sethson I, Buevich AV. Characterization of the heparin-binding domain of human extracellular superoxide dismutase. Biochim Biophys Acta 1997;1340:21–32.
22. Karlsson K, Edlund A, Sandstrom J, et al. Proteolytic modification of the heparin-binding affinity of extracellular superoxide dismutase. Biochem J 1993;290: 623–6.
23. Sandstrom J, Karlsson K, Edlund T, et al. Heparin-affinity patterns and composition of extracellular superoxide dismutase in human plasma and tissues. Biochem J 1993;294: 853–7.
24. Ookawara T, Kizaki T, Ohishi S, et al. Purification and subunit structure of extracellular superoxide dismutase from mouse lung tissue. Arch Biochem Biophys 1997;340:299–304.
25. Bowler RP, Nicks M, Olsen DA, et al. Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase J Biol Chem 2002;277:16505–11.
26. Olsen DA, Petersen SV, Oury TD, et al. The intracellular proteolytic processing of extracellular superoxide dismutase (EC-SOD) is a two-step event. J Biol Chem 2004;279:22152–7.
27. Karlsson K, Lindahl U, Marklund SL. Binding of human extracellular superoxide dismutase C to sulphated glycosaminoglycans. Biochem J 1988;256:29–33.
28. Adachi T, Marklund SL. Interactions between human extracellular superoxide dismutase C and sulfated polysaccharides. J Biol Chem 1989;264:8537–41.
29. Adachi T, Kodera T, Ohta H, et al. The heparin binding site of human extracellular-superoxide dismutase. Arch Biochem Biophys 1992;297:155–61.
30. Stenlund P, Lindberg MJ, Tibell LA. Structural requirements for high-affinity heparin binding: alanine scanning analysis of charged residues in the C-terminal domain of human extracellular superoxide dismutase. Biochemistry 2002;41:3168–75.
31. Petersen SV, Oury T, Ostergaard L, et al. Extracellular superoxide dismutase (EC-SOD) binds to type i collagen and protects against oxidative fragmentation. J Biol Chem 2004;279:13705–10.
32. Sandstrom J, Nilsson P, Karlsson K, et al. 10-fold increase in human plasma extracellular superoxide dismutase content caused by a mutation in heparin-binding domain. J Biol Chem 1994;269:19163–6.
33. Folz RJ, Peno-Green L, Crapo JD. Identification of a homozygous missense mutation (Arg to Gly) in the critical binding region of the human EC-SOD gene (SOD3) and its association with dramatically increased serum enzyme levels. Hum Mol Genet 1994;3:2251–4.
34. Yamada H, Yamada Y, Adachi T, et al. Molecular analysis of extracellular-superoxide dismutase gene associated with high level in serum. Jpn J Hum Genet 1995;40:177–84.
35. Petersen SV, Olsen DA, Kenney JM, et al. The high concentration of Arg213-->Gly extracellular superoxide dismutase (EC-SOD) in plasma is caused by a reduction of both heparin and collagen affinities. Biochem J. 2005;15(385):427-32.
36. Ookawara T, Kizaki T, Takayama E, et al. Nuclear translocation of extracellular superoxide dismutase. Biochem Biophys Res Commun 2002;296:54–61.
37. Karlsson K, Marklund SL. Heparin-induced release of extracellular superoxide dismutase to human blood plasma BiochemJ 1987;242:55–9.
38. Karlsson K, Marklund SL. Plasma clearance of human extracellular-superoxide dismutase C in rabbits. J Clin Invest 1988;82:762–6.
39. Petersen SV, Oury TD, Valnickova Z, et al. The dual nature of human extracellular superoxide dismutase: one sequence and two structures. Proc Natl Acad Sci USA 2003;100:13875–80.
40. Heistad DD. Oxidative stress and vascular disease: 2005 Duff lecture. Arterioscler Thromb Vasc Biol. 2006;26(4):689-95.
41. Beckman JS, Beckman TW, Chen J, et al. Apparent hydroxyl radical production by peroxynitrite: implicationsfor endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 1990;87:1620–1624.
42. Steinberg D, Parthasarathy S, Carew TE, et al. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. New Engl J Med 1989;320:915~924
43. Stralin P., Karlsson K., Johansson B.O., et al. The interstitium of the human arterial wall contains very large amounts of extracellular superoxide dismutase. Arterioscler. Thromb. Vasc. Biol. 15:2032–2036;1995.
44. Jay L. Zweier, Barry K. Rayburn, John T. Flaherty et al. Recombinant superoxide dismutase reduces oxygen free radical concentrations in reperfused myocardium. J.Clin. Inverst. 1987;80:1728-1734
45. Gallagher K.P., Buda A.J., Pace D., et al. Failure of superoxide dismutase and catalase to alter size of infarction in conscious dogs after 3 hours of occlusion followed by reperfusion. Circulation. 1987;75(6):1237-48
46. Karlsson K, Sandstrom J, Edlund A, et al. Pharmacokinetics of extracellular superoxide dismutase in the vassuscular system. Free Rad Biol Med 1993;14:185–190.
47. Levin E.D, Christopher NC, Lateef S, et.al. Memory decline of aging reduced by extracellular superoxide dismutase overexpression. Behav Genet. 2002;32(2):119-25.
48. Levin E.D Extracellular superoxide dismutase (EC-SOD) quenches free radicals and attenuates age-related cognitive decline: opportunities for novel drug development in aging. Curr Alzheimer Res. 2005;2(2):191-6.
49. Thiels E. , Urban N. N. , Gonzalez-BurgosG. R., et al. Impairment of long-term potentiation and associative memory in mice that overexpress extracellular superoxide dismutase. J. Neurosci. 2000;20:7631–7639.
50. Crow J. P.;Beckman J. S. The importance of superoxide innitric oxide-dependent toxicity: evidence for peroxynitrite-mediated injury. Adv. Exp. Med. Biol. 1996;387:147–161.
51. Pineda J.A., Aono M., Sheng H., et al. Extracellular superoxide dismutase overexpression improves behavioral outcome from closed head injury in the mouse. J. Neurotrauma 2001;18:625–634.
52. Tate R.M., Repine J.E. Neutrophils and the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 1983;128:552–559.
53. Folz R. J., Abushamaa A. M., Suliman H. B. Extracellularsuperoxide dismutase in the airways of transgenic mice reducesin flammation and attenuates lung toxicity following hyperoxia. J. Clin. Invest. 1999;103:1055–1066.
54. Bowler R.P., Nicks M., Warnick K.,et al. Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 2002;282:L719–L726.
55. Cheryl L.Fattman, Roderick J.Tan, Jacob M. Tobolewski, et al. Increased sensitivity to asbestos-induced lung injury in mice lacking extracellular superoxide dismutase. Free Radical Biology & Medicine 2006;40 : 601 – 607.
56. Parizada B., Werber M.M., Nimrod A. Protective effects of human recombinant MnSOD in adjuvant arthritis and bleomycin-induced lung fibrosis. Free Radic. Res. Commun. 1991;15:297–301.
57. Monboisse J.C., Bellon G., Randoux A., et al. Activation of human neutrophils by typeⅠcollagen. Requirement of two different sequences. Biochem. J. 1990;270:459–462. 58. Elizabeth Regan, Joanne Flannelly, Russell Bowler, et al. Extracellular superoxide dismutase and oxidant damage in osteoarthritis. Arthritis Rheum. 2005;52(11):3479-3491.
59. Ames B.N. , Shigenaga M.K., Hagen T.M. Oxidants, antioxidants,and the degenerative diseases of aging. Proc. Natl.Acad. Sci. USA 1993;90:7915–7922.
60. Boggess K. A., Kay H.H., Crapo J. D., et al. Differential localization of placental extracellular superoxide dismutase as pregnancy progresses. Am. J. Obstet. Gynecol. 2000;83:199–205.
61. Adachi T., Yamamoto M., Hara H. et al. Extracellular superoxide dismutase incerebrospinal fluid from infants/children. Clin. Chim. Acta.2001;308:191–193.
62. Adachi T., Wang J., Wang X.L. Age-related change of plasma extracellular superoxide dismutase. Clin. Chim. Acta. 2000;290:169–178.
63. Huajun He, Qingsheng Yuan, Guanzheng Yang et al. High-Level Expression of Human Extracellular Superoxide Dismutase in Escherichia coli and Insect Cells. Protein Expression and Purification 2002;24:13–17
64. Petersen SV, Due AV, Valnickova Z, et al. The structure of rabbit extracellular superoxide dismutase differs from the human protein Biochemistry 2004;43:14275–81
65. Parge HE, Hallewell RA, Tainer JA. Atomic structures of wild-type and thermostable mutant recombinant human Cu,Zn superoxide dismutase. Proc Natl Acad Sci U S A. 1992;89(13):6109-13.
2. Igor N. Zelko, Thomas J. Mariani, and Rodney J. Folz Superoxide dismutase multigene family: A comparison of the CuZnSOD (SOD1), MnSOD (SOD2),and EC-SOD (SOD3) gene,structures, evolution, and expression. Free Radical Biology & Medicine, 2002;33: 337–349
3. Marklund, S.L. Human copper-containing superoxide dismutase of high molecular weight. Proc. Natl. Acad. Sci. USA 1982;79:7634-7638
4. Tibell L, Hjalmarsson K, Edlund T, et al. Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product. Proc Natl Acad Sci USA 1987;84:6634–8.
5. Hendrickson D.J., Fisher J.H., Jones C., et al. Regional localization of human extracellular superoxide dismutase gene to 4pter-q21. Genomics. 1990;8:736–738.
6. Folz R.J., Crapo J.D. Extracellular superoxide dismutase (sod3): tissue-specific expression, genomic characterization, and computer-assisted sequence analysis of the human EC-SOD gene. Genomics 1994;22:162–171.
7. Marklund SL. Extracellular superoxide dismutase and other superoxide dismutase isoenzymes in tissues from nine mammalian species. Biochem J 1984;222: 649–655.
8. Marklund SL. Extracellular superoxide dismutase in human tissues and human cell lines. J Clin Invest 1984;74: 1398–403.
9. Marklund SL, Holme E, Hellner L. Superoxide dismutase in extracellular fluids. Clin Chim Acta 1982;126:41–51.
10. Hjalmarsson K, Marklund SL, Engstrom A, et al. Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product. Proc Natl Acad Sci USA 1987;84:6340–4.
11. Stenlund P, Andersson D, Tibell LA. Subunit interaction in extracellular superoxide dismutase: effects of mutations in the N-terminal domain. Protein Sci 1997;6:2350–8.
12. Carlsson L. M., Marklund S. L., and Edlund T. The rat extracellular superoxide dismutase dimer is converted to a tetramer by the exchange of a single amino acid. Proc. Natl. Acad. Sci. U.S.A. 1996;93: 5219-5222
13. Stromqvist M, Holgersson J, Samuelsson B. Glycosylation of extracellular superoxide dismutase studied by high-performance liquid chromatography and mass spectrometry. J Chromatogr 1991;548:293–301.
14. Edlund A, Edlund T, Hjalmarsson K, et al. A non-glycosylated extracellular superoxide dismutase variant. Biochem J 1992;288:451–6.
15. Tainer JA, Getzoff ED, Beem KM, et al. Determination and analysis of the 2A-structure of copper, zinc superoxide dismutase. J Mol Biol 1982;160:181–217.
16. Parge HE, Getzoff ED, Scandella CS, et al. Crystallographic characterization of recombinant human CuZn superoxide dismutase. J Biol Chem 1986;261:16215–8.
17. Marklund S.L. Properties of extracellular superoxide dismutase from human lung. Biochem J 1984;220:269–72.
18. Tibell L., Aasa R., Marklund S.L. Spectral and physical properties of human extracellular superoxide dismutase: a comparison with Cu,Zn superoxide dismutase. Arch. Biochem. Biophys. 1993;304:429–433.
19. Jeney V, Itoh S, Wendt M, et.al. Role of antioxidant-1 in extracellular superoxide dismutase function and expression. Circ Res. 2005;96(7),723-729
20. Sandstrom J, Carlsson L, Marklund SL, et al. The heparin-binding domain of extracellular superoxide dismutase C and formation of variants with reduced heparin affinity. J Biol Chem 1992;267:18205–9.
21. Tibell LA, Sethson I, Buevich AV. Characterization of the heparin-binding domain of human extracellular superoxide dismutase. Biochim Biophys Acta 1997;1340:21–32.
22. Karlsson K, Edlund A, Sandstrom J, et al. Proteolytic modification of the heparin-binding affinity of extracellular superoxide dismutase. Biochem J 1993;290: 623–6.
23. Sandstrom J, Karlsson K, Edlund T, et al. Heparin-affinity patterns and composition of extracellular superoxide dismutase in human plasma and tissues. Biochem J 1993;294: 853–7.
24. Ookawara T, Kizaki T, Ohishi S, et al. Purification and subunit structure of extracellular superoxide dismutase from mouse lung tissue. Arch Biochem Biophys 1997;340:299–304.
25. Bowler RP, Nicks M, Olsen DA, et al. Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase J Biol Chem 2002;277:16505–11.
26. Olsen DA, Petersen SV, Oury TD, et al. The intracellular proteolytic processing of extracellular superoxide dismutase (EC-SOD) is a two-step event. J Biol Chem 2004;279:22152–7.
27. Karlsson K, Lindahl U, Marklund SL. Binding of human extracellular superoxide dismutase C to sulphated glycosaminoglycans. Biochem J 1988;256:29–33.
28. Adachi T, Marklund SL. Interactions between human extracellular superoxide dismutase C and sulfated polysaccharides. J Biol Chem 1989;264:8537–41.
29. Adachi T, Kodera T, Ohta H, et al. The heparin binding site of human extracellular-superoxide dismutase. Arch Biochem Biophys 1992;297:155–61.
30. Stenlund P, Lindberg MJ, Tibell LA. Structural requirements for high-affinity heparin binding: alanine scanning analysis of charged residues in the C-terminal domain of human extracellular superoxide dismutase. Biochemistry 2002;41:3168–75.
31. Petersen SV, Oury T, Ostergaard L, et al. Extracellular superoxide dismutase (EC-SOD) binds to type i collagen and protects against oxidative fragmentation. J Biol Chem 2004;279:13705–10.
32. Sandstrom J, Nilsson P, Karlsson K, et al. 10-fold increase in human plasma extracellular superoxide dismutase content caused by a mutation in heparin-binding domain. J Biol Chem 1994;269:19163–6.
33. Folz RJ, Peno-Green L, Crapo JD. Identification of a homozygous missense mutation (Arg to Gly) in the critical binding region of the human EC-SOD gene (SOD3) and its association with dramatically increased serum enzyme levels. Hum Mol Genet 1994;3:2251–4.
34. Yamada H, Yamada Y, Adachi T, et al. Molecular analysis of extracellular-superoxide dismutase gene associated with high level in serum. Jpn J Hum Genet 1995;40:177–84.
35. Petersen SV, Olsen DA, Kenney JM, et al. The high concentration of Arg213-->Gly extracellular superoxide dismutase (EC-SOD) in plasma is caused by a reduction of both heparin and collagen affinities. Biochem J. 2005;15(385):427-32.
36. Ookawara T, Kizaki T, Takayama E, et al. Nuclear translocation of extracellular superoxide dismutase. Biochem Biophys Res Commun 2002;296:54–61.
37. Karlsson K, Marklund SL. Heparin-induced release of extracellular superoxide dismutase to human blood plasma BiochemJ 1987;242:55–9.
38. Karlsson K, Marklund SL. Plasma clearance of human extracellular-superoxide dismutase C in rabbits. J Clin Invest 1988;82:762–6.
39. Petersen SV, Oury TD, Valnickova Z, et al. The dual nature of human extracellular superoxide dismutase: one sequence and two structures. Proc Natl Acad Sci USA 2003;100:13875–80.
40. Heistad DD. Oxidative stress and vascular disease: 2005 Duff lecture. Arterioscler Thromb Vasc Biol. 2006;26(4):689-95.
41. Beckman JS, Beckman TW, Chen J, et al. Apparent hydroxyl radical production by peroxynitrite: implicationsfor endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 1990;87:1620–1624.
42. Steinberg D, Parthasarathy S, Carew TE, et al. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. New Engl J Med 1989;320:915~924
43. Stralin P., Karlsson K., Johansson B.O., et al. The interstitium of the human arterial wall contains very large amounts of extracellular superoxide dismutase. Arterioscler. Thromb. Vasc. Biol. 15:2032–2036;1995.
44. Jay L. Zweier, Barry K. Rayburn, John T. Flaherty et al. Recombinant superoxide dismutase reduces oxygen free radical concentrations in reperfused myocardium. J.Clin. Inverst. 1987;80:1728-1734
45. Gallagher K.P., Buda A.J., Pace D., et al. Failure of superoxide dismutase and catalase to alter size of infarction in conscious dogs after 3 hours of occlusion followed by reperfusion. Circulation. 1987;75(6):1237-48
46. Karlsson K, Sandstrom J, Edlund A, et al. Pharmacokinetics of extracellular superoxide dismutase in the vassuscular system. Free Rad Biol Med 1993;14:185–190.
47. Levin E.D, Christopher NC, Lateef S, et.al. Memory decline of aging reduced by extracellular superoxide dismutase overexpression. Behav Genet. 2002;32(2):119-25.
48. Levin E.D Extracellular superoxide dismutase (EC-SOD) quenches free radicals and attenuates age-related cognitive decline: opportunities for novel drug development in aging. Curr Alzheimer Res. 2005;2(2):191-6.
49. Thiels E. , Urban N. N. , Gonzalez-BurgosG. R., et al. Impairment of long-term potentiation and associative memory in mice that overexpress extracellular superoxide dismutase. J. Neurosci. 2000;20:7631–7639.
50. Crow J. P.;Beckman J. S. The importance of superoxide innitric oxide-dependent toxicity: evidence for peroxynitrite-mediated injury. Adv. Exp. Med. Biol. 1996;387:147–161.
51. Pineda J.A., Aono M., Sheng H., et al. Extracellular superoxide dismutase overexpression improves behavioral outcome from closed head injury in the mouse. J. Neurotrauma 2001;18:625–634.
52. Tate R.M., Repine J.E. Neutrophils and the adult respiratory distress syndrome. Am. Rev. Respir. Dis. 1983;128:552–559.
53. Folz R. J., Abushamaa A. M., Suliman H. B. Extracellularsuperoxide dismutase in the airways of transgenic mice reducesin flammation and attenuates lung toxicity following hyperoxia. J. Clin. Invest. 1999;103:1055–1066.
54. Bowler R.P., Nicks M., Warnick K.,et al. Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 2002;282:L719–L726.
55. Cheryl L.Fattman, Roderick J.Tan, Jacob M. Tobolewski, et al. Increased sensitivity to asbestos-induced lung injury in mice lacking extracellular superoxide dismutase. Free Radical Biology & Medicine 2006;40 : 601 – 607.
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