Expression of Human SOD1 and Mutant SOD1 (G93A) in E. coli and Identification of SOD1 as a Substrate of HtrA2 Serine Protease

대장균에서의 human SOD1과 mutant SOD1 (G93A) 단백질의 발현과 HtrA2의 기질 여부 확인에 관한 연구

  • Kim, Goo-Young (Department of Biomedical Sciences,Research Institute of Molecular Genetics, College of Medicine, the Catholic University of Korea) ;
  • Kim, Sang-Soo (Department of Biomedical Sciences,Research Institute of Molecular Genetics, College of Medicine, the Catholic University of Korea) ;
  • Park, Hyo-Jin (Research Institute of Molecular Genetics, College of Medicine, the Catholic University of Korea,School of Life Science and Biotechnology, Korea University) ;
  • Rhim, Hyang-Shuk (Department of Biomedical Sciences,Research Institute of Molecular Genetics, College of Medicine, the Catholic University of Korea)
  • 김구영 (가톨릭대학교 생명의과학과,분자유전학연구소) ;
  • 김상수 (가톨릭대학교 생명의과학과,분자유전학연구소) ;
  • 박효진 (가톨릭대학교 분자유전학연구소,고려대학교 생명과학대학 생명과학부) ;
  • 임향숙 (가톨릭대학교 생명의과학과,분자유전학연구소)
  • Published : 2006.08.30


Superoxide dismutase (SOD) is physiologically important in regulating cellular homeostasis and apoptotic cell death, and its mutations are the cause of familial amyotrophic lateral sclerosis (FALS). Mitochondrial serine protease HtrA2 has a pro-apoptotic function and has known to be associated with neurodegenerative disorders. To investigate the relationship between genes associated with apoptotic cell death, such as HtrA2 and SOD1, we utilized the pGEX expression system to develop a simple and rapid method for purifying wild-type and ALS-associated mutant SOD1 proteins in a suitable form for biochemical studies. We purified SOD1 and SOD1 (G93A) proteins to approximately 90% purity with relatively high yields (3 mg per liter of culture). Consistent with the result in mammalian cells, SOD1 (G93A) was more insoluble than wild-type SOD1 in E. coli, indicating that research on the aggregate formation of SOD1 may be possible using this pGEX expression system in E. coli. We investigated the HtrA2 serine protease activity on SOD1 to assess the relationship between two proteins. Not only wild-type SOD1 but also ALS-associated mutant SOD1 (G93A) were cleaved by HtrA2, resulting in the production of the 19 kDa and 21 kDa fragments that were specific for anti-SOD1 antibody. Using protein gel electrophoresis and immunoblot assay, we compared the relative molecular masses of thrombin-cleaved GST-SOD1 and HtrA2-cleaved SOD1 fragments and can predict that the HtrA2-cleavage sites within SOD1 are the peptide bonds between leucine 9-lysine 10 (L9-K10) and glutamine 23-lysine 24 (Q23-K24). Our study indicates that SOD1 is one of the substrate for HtrA2, suggesting that both HtrA2 and SOD1 may be important for modulating the HtrA2-SOD1-mediated apopotic cell death that is associated with the pathogenesis of neurodegenerative disorder.


  1. Andersen J. K. 2004. Oxidative stress in neurodegeneration: cause or consequence? Nat Med. 10, Suppl:S18-25
  2. Cilenti L., M. M. Soundarapandian, G. A. Kyriazis, V. Stratico, S. Singh, S. Gupta, J. V. Bonventre, E. S. Alnemri, A. S. Zervos. 2004. Regulation of HAX-1 anti- apoptotic protein by Omi/HtrA2 protease during cell death. J Biol Chem. 279, 50295-301
  3. Dal Canto M. C., M. E. Gurney . 1995. Neuropathological changes in two lines of mice carrying a transgene for mutant human Cu,Zn SOD, and in mice overexpressing wild type human SOD: a model of familial amyotrophic lateral sclerosis (FALS). Brain Res. 676, 25-40
  4. Dawson V. L. 2004. Maiming mitochondria in familial ALS. Nat Med. 10, 905-6
  5. Higgins C. M., C. Jung, Z. Xu. 2003. ALS-associated mutant SOD1G93A causes mitochondrial vacuolation by expansion of the intermembrane space and by involvement of SOD1 aggregation and peroxisomes. BMC Neurosci. 4, 16
  6. Jones J. M., P. Datta, S. M. Srinivasula, W. Ji, S. Gupta, Z. Zhang, E. Davies, G. Hajnoczky, T. L. Saunders, M. L. Van Keuren, T. Fernandes-Alnemri, M. H. Meisler, E. S. Alnemri. 2003. Loss of Omi mitochondrial protease activity causes the neuromuscular disorder of mnd2 mutant mice. Nature. 425, 721-7
  7. Kim S. S., K. H. Kim, H. J. Park, E. H. Hur, H. Rhim. 2005. Inhibitory effect of the N-termnal GST on the Tautomerase activity of marcrophage migration inhibitory factor. J. Life Science. 15, 961-967
  8. Li W, S. M. Srinivasula, J. Chai, P. Li, J. W. Wu, Z. Zhang, E. S. Alnemri, Y. Shi. 2002. Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi. Nat Struct Biol. 9, 436-41
  9. Maier C. M., P. H. Chan. 2002. Role of superoxide dismutases in oxidative damage and neurodegenerative disorders. Neuroscientist. 8, 323-34
  10. Martins L. M., B. E. Turk, V. Cowling, A. Borg, E. T. Jarrell, L. C. Cantley, J. Downward. 2003. Binding specificity and regulation of the serine protease and PDZ domains of HtrA2/Omi. J Biol Chem. 278, 49417-27
  11. Martins L. M., A. Morrison, K. Klupsch, V. Fedele, N. Moisoi, P. Teismann, A. Abuin, E. Grau, M. Geppert, G. P. Livi, C. L. Creasy, A. Martin, I. Hargreaves, S. J. Heales, H. Okada, S. Brandner, J. B. Schulz, T. Mak, J. Downward. 2004. Neuroprotective role of the Reaper-related serine protease HtrA2/Omi revealed by targeted deletion in mice. Mol Cell Biol. 24, 9848-62
  12. Miller A. F. 2004. Superoxide dismutases: active sites that save, but a protein that kills. Curr Opin Chem Biol. 8, 162-8
  13. Nam M. K., H. M. Park, J. Y. Choi, H. J. Park, K. C. Chung, S. Kang, H. Rhim, 2005. The expression patterns of Human parkin in E. coli and mammalian cells. J. Life Science. 15, 916-922
  14. Noor R., S. Mittal, J. Iqbal. 2002. Superoxide dismutase- applications and relevance to human diseases. Med Sci Monit. 8, 210-5
  15. Pasinelli P., M. E. Belford, N. Lennon, B. J. Bacskai, B. T. Hyman, D. Trotti , R. H. Jr. Brown. 2004. Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2 in spinal cord mitochondria. Neuron. 43, 19-30
  16. Potter S. Z., J. S. Valentine. 2003. The perplexing role of copper-zinc superoxide dismutase in amyotrophic lateral sclerosis (Lou Gehrig's disease). J Biol Inorg Chem. 8, 373-80
  17. Saito A., T. Hayashi, S. Okuno, T. Nishi, P. H. Chan. 2004. Modulation of the Omi/HtrA2 signaling pathway after transient focal cerebral ischemia in mouse brains that overexpress SOD1. Brain Res Mol Brain Res. 127, 89-95
  18. Sekine K, Y. Hao, Y. Suzuki, R. Takahashi, T. Tsuruo, M. Naito. 2005. HtrA2 cleaves Apollon and induces cell death by IAP-binding motif in Apollon-deficient cells. Biochem Biophys Res Commun. 330, 279-85
  19. Selverstone Valentine J., P. A. Doucette, S. Zittin Potter. 2005. Copper-zinc superoxide dismutase and amyotrophic lateral sclerosis. Annu Rev Biochem. 74, 563-93
  20. Seong Y. M., J. Y. Choi, H. J. Park, K. J. Kim, S. G. Ahn, G. H. Seong, I. K. Kim, S. Kang, H. Rhim. 2004. Autocatalytic processing of HtrA2/Omi is essential for induction of caspase-dependent cell death through antagonizing XIAP. J Biol Chem. 279, 37588-96
  21. Seong Y. M., H. J. Park, G. H. Seong, J. Y. Choi, S. J. Yoon, B. R. Min, S. Kang, H. Rhim. 2004. N-terminal truncation circumvents proteolytic degradation of the human HtrA2/Omi serine protease in Escherichia coli: rapid purification of a proteolytically active HtrA2/Omi. Protein Expr Purif. 33, 200-8
  22. Suzuki Y., Y. Imai, H. Nakayama, K. Takahashi, K. Takio, R. Takahashi. 2001. A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol Cell. 8, 613-21
  23. Takeuchi H, Y. Kobayashi, S. Ishigaki, M. Doyu, G. Sobue. 2002. Mitochondrial localization of mutant superoxide dismutase 1 triggers caspase-dependent cell death in a cellular model of familial amyotrophic lateral sclerosis. J Biol Chem. 277, 50966-72
  24. van Gurp M., N. Festjens, G. van Loo, X. Saelens, P. Vandenabeele. 2003. Mitochondrial intermembrane proteins in cell death. Biochem Biophys Res Commun. 304, 487-97
  25. Verhagen A. M., J. Silke, P. G. Ekert, M. Pakusch, H. Kaufmann, L. M. Connolly, C. L. Day, A. Tikoo, R. Burke, C. Wrobel, R. L. Moritz, R. J. Simpson, D. L. Vaux. 2002. HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins. J Biol Chem. 277, 445-454

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