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Angiopep-2-Mediated Delivery of Human Manganese Superoxide Dismutase in Brain Endothelial Cells and its Protective Effect Against Oxidative Stress

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Abstract

Oxidative stress has been considered as the leading cause of blood–brain barrier disruption which implicates many neurological disorders. Manganese superoxide dismutase (MnSOD or SOD2) is one of the crucial antioxidant enzymes that can provide substantial protection against oxidative damage. However, the therapeutic effect of the enzyme in such neurological diseases is limited due to poor transduction into brain microvascular endothelial cells. In the present study, a fusion protein of human SOD2 and a brain targeting peptide, Angiopep-2 (AP-2), was generated by genetic engineering. The SOD2-AP-2 and control SOD2 were successfully expressed in Escherichia coli and purified using immobilized metal affinity chromatography. Purified SOD2-AP-2 exhibited 1,090 μ/mg of specific SOD activity, which retained a significant activity in the same order of magnitude as that of native SOD2. The in vitro transduction demonstrated that 1 µM of SOD2-AP-2 delivered efficiently to immortalized mouse brain endothelial cell line within 30 min whereas, control SOD2 did not. Moreover, pretreatment with 50 units of SOD2-AP-2 for 1 h could significantly protect cells against paraquat up to 2 mM but control SOD2 pretreatment did not show a protective effect. Taken together, our findings pave the way for SOD2-AP-2 to be a potential therapeutic candidate for neurological diseases.

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Abbreviations

AP-2:

Angiopep-2

TAT:

HIV-1 TAT protein transduction domain

References

  • Abbott NJ, Revest PA, Romero IA (1992) Astrocyte–endothelial interaction: physiology and pathology. Neuropathol Appl Neurobiol 18:424–433

    Article  CAS  PubMed  Google Scholar 

  • Baret A, Jadot G, Michelson AM (1984) Pharmacokinetic and anti-inflammatory properties in the rat of superoxide dismutases (CuSODs and MnSOD) from various species. Biochem Pharmacol 33:2755–2760

    Article  CAS  PubMed  Google Scholar 

  • Becker-Hapak M, McAllister SS, Dowdy SF (2001) TAT-mediated protein transduction into mammalian cells. Methods 24:247–256

    Article  CAS  PubMed  Google Scholar 

  • Bertrand Y, Currie JC, Demeule M, Régina A, Ché C, Abulrob A, Fatehi D, Sartelet H, Gabathuler R, Castaigne JP, Stanimirovic D, Béliveau R (2010) Transport characteristics of a novel peptide platform for CNS therapeutics. J Cell Mol Med 14:2827–2839

    Article  CAS  PubMed  Google Scholar 

  • Bertrand Y, Currie JC, Poirier J, Demeule M, Abulrob A, Fatehi D, Stanimirovic D, Sartelet H, Castaigne JP, Béliveau R (2011) Influence of glioma tumour microenvironment on the transport. Br J Cancer 105:1697–1707

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Betz AL (1993) Oxygen free radicals and the brain microvasculature. In: Pardridge WM (ed) The blood–brain barrier. Raven Place, New York, pp 303–321

    Google Scholar 

  • Borgstahl GE, Parge HE, Hickey MJ, Beyer WF Jr, Hallewell RA, Tainer JA (1992) The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles. Cell 71:107–118

    Article  CAS  PubMed  Google Scholar 

  • Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chrissobolis S, Miller AA, Drummond GR, Kemp-Harper BK, Sobey CG (2011) Oxidative stress and endothelial dysfunction in cerebrovascular disease. Front Biosci 16:1733–1745

    Article  CAS  Google Scholar 

  • Demeule M, Régina A, Ché C, Poirier J, Nguyen T, Gabathuler R, Castaigne JP, Béliveau R (2008a) Identification and design of peptides as a new drug delivery system for the brain. J Pharmacol Exp Ther 324:1064–1072

    Article  CAS  PubMed  Google Scholar 

  • Demeule M, Currie JC, Bertrand Y, Ché C, Nguyen T, Régina A, Gabathuler R, Castaigne JP, Béliveau R (2008b) Involvement of the low-density lipoprotein receptor-related protein in the transcytosis of the brain delivery vector angiopep-2. J Neurochem 106:1534–1544

    Article  CAS  PubMed  Google Scholar 

  • Eum WS, Choung IS, Li MZ, Kang JH, Kim DW, Park J, Kwon HY, Choi SY (2004) HIV-1 Tat-mediated protein transduction of Cu, Zn-superoxide dismutase into pancreatic beta cells in vitro and in vivo. Free Radic Biol Med 37:339–349

    Article  CAS  PubMed  Google Scholar 

  • Faraci FM, Didion SP (2004) Vascular protection: superoxide dismutase isoforms in the vessel wall. Artherioscler Throm Vasc Biol 24:1367–1373

    Article  CAS  Google Scholar 

  • Fennell JP, Brosnan MJ, Frater AJ, Hamilton CA, Alexander MY, Nicklin SA, Heistad DD, Baker AH, Dominiczak AF (2002) Adenovirus-mediated overexpression of extracellular superoxide dismutase improves endothelial dysfunction in a rat model of hypertension. Gene Ther 9:110–117

    Article  CAS  PubMed  Google Scholar 

  • Fukai T, Ushio-Fukai M (2011) Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal 15:1583–1606

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gorecki M, Beck Y, Hartman JR, Fischer M, Weiss L, Tochner Z, Slavin S, Nimrod A (1991) Recombinant human superoxide dismutases: production and potential therapeutical uses. Free Radic Res Commun 12–13:401–410

    Article  PubMed  Google Scholar 

  • Grey M, Yainoy S, Prachayasittikul V, Bülow L (2009) A superoxide dismutase-human hemoglobin fusion protein showing enhanced antioxidative properties. FEBS J 276:6195–6203

    Article  CAS  PubMed  Google Scholar 

  • Hawkins BT, Davis TP (2005) The blood–brain barrier/neurovascular unit in health and disease. Pharmacol Rev 57:173–185

    Article  CAS  PubMed  Google Scholar 

  • He SQ, Zhang YH, Venugopal SK, Dicus CW, Perez RV, Ramsamooj R, Nantz MH, Zern MA, Wu J (2006) Delivery of antioxidative enzyme genes protects against ischemic/reperfusion-induced liver injury in mice. Liver Transpl 12:1869–1879

    Article  PubMed  Google Scholar 

  • Herve F, Ghinea N, Scherrmann JM (2008) CNS delivery via adsorptive transcytosis. AAPS J 10:455–472

    Article  PubMed Central  PubMed  Google Scholar 

  • Hood ED, Chorny M, Greineder CF, Alferiev IS, Levy RJ, Muzykantov VR (2014) Endothelial targeting of nanocarriers loaded with antioxidant enzymes for protection against vascular oxidative stress and inflammation. Biomaterials 35:3708–3715

    Article  CAS  PubMed  Google Scholar 

  • Huang HF, Guo F, Cao YZ, Shi W, Xia Q (2012) Neuroprotection by manganese superoxide dismutase (MnSOD) mimics: antioxidant effect and oxidative stress regulation in acute experimental stroke. CNS Neurosci Ther 18:811–818

    Article  CAS  PubMed  Google Scholar 

  • Jin LH, Bahn JH, Eum WS, Kwon HY, Jang SH, Han KH, Kang TC, Won MH, Kang JH, Cho SW, Park J, Choi SY (2001) Transduction of human catalase mediated by an HIV-1 TAT protein basic domain and arginine-rich peptides into mammalian cells. Free Radic Biol Med 31:1509–1519

    Article  CAS  PubMed  Google Scholar 

  • Jung JE, Kim GS, Narasimhan P, Song YS, Chan PH (2009) Regulation of Mn-superoxide dismutase activity and neuroprotection by STAT3 in mice after cerebral ischemia. J Neurosci 29:7003–7014

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kaur J, Arora S, Singh B, Thakur LC, Gambhir J, Prabhu KM (2011) Role of oxidative stress in pathophysiology of transient ischemic attack and stroke. Int J Biol Med Res 2:611–615

    Google Scholar 

  • Kim GW, Kondo T, Chan PH (2002) Manganese superoxide dismutase deficiency exacerbates cerebral infarction after focal cerebral ischemia/reperfusion in mice: implications for the production and role of superoxide anions. Stroke 33:809–815

    Article  CAS  PubMed  Google Scholar 

  • Kortekaas R, Leenders KL, Van Oostrom JC, Vaalburg W, Bart J, Willemsen AT, Hendrikse NH (2005) Blood–brain barrier dysfunction in parkinsonian midbrain in vivo. Ann Neurol 57:176–179

    Article  CAS  PubMed  Google Scholar 

  • Lebovitz RM, Zhang H, Vogel H, Cartwright J Jr, Dionne L, Lu NF, Huang S, Matzuk MM (1996) Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice. Proc Natl Acad Sci USA 93:9782–9787

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Li G, Simon MJ, Cancel LM, Shi ZD, Ji X, Tarbell JM, Morrison B 3rd, Fu BM (2010) Permeability of endothelial and astrocyte cocultures: in vitro blood–brain barrier models for drug delivery studies. Ann Biomed Eng 38:2499–2511

    Article  PubMed Central  PubMed  Google Scholar 

  • Lum H, Roebuck KA (2001) Oxidant stress and endothelial cell dysfunction. Am J Physiol Cell Physiol 280:C719–C741

    CAS  PubMed  Google Scholar 

  • McCord JM, Boyle JA, Day ED, Rizzolo LJ, Salin ML (1977) Superoxide dismutase: a manganese-containing superoxide dismutase from human liver. Academic Press, London

    Google Scholar 

  • Minagar A, Alexander JS (2003) Blood–brain barrier disruption in multiple sclerosis. Mult Scler 9:540–549

    Article  CAS  PubMed  Google Scholar 

  • Pan J, Konstas AA, Bateman B, Ortolano GA, Pile-Spellman J (2007) Reperfusion injury following cerebral ischemia: pathophysiology, MR imaging, and potential therapies. Neuroradiology 49:93–102

    Article  PubMed Central  PubMed  Google Scholar 

  • Pardridge WM (1999) Blood–brain barrier biology and methodology. J Neurovirol 5:556–569

    Article  CAS  PubMed  Google Scholar 

  • Petito CK, Cash KS (1992) Blood–brain barrier abnormalities in the acquired immune deficiency syndrome: immunohistochemical localization of serum proteins in postmortem brain. Ann Neurol 32:658–666

    Article  CAS  PubMed  Google Scholar 

  • Sano Y, Shimizu F, Abe M, Maeda T, Kashiwamura Y, Ohtsuki S, Terasaki T, Obinata M, Kajiwara K, Fujii M, Suzuki M, Kanda T (2010) Establishment of a new conditionally immortalized human brain microvascular endothelial cell line retaining an in vivo blood–brain barrier function. J Cell Physiol 225:519–528

    Article  CAS  PubMed  Google Scholar 

  • Schwarze SR, Dowdy SF (2000) In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol Sci 21:45–48

    Article  CAS  PubMed  Google Scholar 

  • Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF (1999) In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285:1569–1572

    Article  CAS  PubMed  Google Scholar 

  • Sheng Y, Durazo A, Schumacher M, Gralla EB, Cascio D, Cabelli DE, Valentine JS (2013) Tetramerization reinforces the dimer interface of MnSOD. PLoS ONE 8(5):e62446

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tayarani I, Cloez I, Clement M, Bourre JM (1989) Antioxidant enzymes and related trace elements in aging brain capillaries and choroid plexus. J Neurochem 53:817–824

    Article  CAS  PubMed  Google Scholar 

  • Trehin R, Merkle HP (2004) Chances and pitfalls of cell penetrating peptides for cellular drug delivery. Eur J Pharm Biopharm 58:209–223

    Article  CAS  PubMed  Google Scholar 

  • Trinh CH, Hunter T, Stewart EE, Phillips SE, Hunter GJ (2008) Purification, crystallization and X-ray structures of the two manganese superoxide dismutases from Caenorhabditis elegans. Acta Crystallogr, Sect F: Struct Biol Cryst Commun 64:1110–1114

    Article  CAS  Google Scholar 

  • Tyagi N, Moshal KS, Sen U, Vacek TP, Kumar M, Hughes WM Jr, Kundu S, Tyagi SC (2009) H2S protects against methionine-induced oxidative stress in brain endothelial cells. Antioxid Redox Signal 11:25–33

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Uyama O, Matsuyama T, Michishita H, Nakamura H, Sugita M (1992) Protective effects of human recombinant superoxide dismutase on transient ischemic injury of CA1 neurons in gerbils. Stroke 23:75–81

    Article  CAS  PubMed  Google Scholar 

  • Wadia JS, Dowdy SF (2002) Protein transduction technology. Curr Opin Biotechnol 13:52–56

    Article  CAS  PubMed  Google Scholar 

  • Wagner UG, Pattridge KA, Ludwig ML, Stallings WC, Werber MM, Oefner C, Frolow F, Sussman JL (1993) Comparison of the crystal structures of genetically engineered human manganese superoxide dismutase and manganese superoxide dismutase from Thermus thermophilus: differences in dimer–dimer interaction. Protein Sci 2:814–825

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Watson K, Edward RJ (1999) HIV-1-transactivating (TAT) protein: both a target and a tool in therapeutic approaches. Biochem Pharmacol 58:1521–1528

    Article  CAS  PubMed  Google Scholar 

  • Xin H, Sha X, Jiang X, Zhang W, Chen L, Fang X (2012) Anti-glioblastoma efficacy and safety of placlitaxel-loading Angiopep-conjugated dual targeting PEG-PCL nanoparticles. Biomaterials 33:8167–8176

    Article  CAS  PubMed  Google Scholar 

  • Yainoy S, Isarankura-Na-Ayudhya C, Tantimongcolwat T, Prachayasittikul V (2007) Cloning of active human manganese superoxide dismutase and its oxidative protection in Escherichia coli. Pak J Biol Sci 10:3541–3548

    Article  CAS  PubMed  Google Scholar 

  • Yang Y, Ma J, Song Z, Wu M (2002) HIV-1 TAT-mediated protein transduction and subcellular localization using novel expression vectors. FEBS Lett 532:36–44

    Article  CAS  PubMed  Google Scholar 

  • Zipser BD, Johanson CE, Gonzalez L, Berzin TM, Tavares R, Hulette CM, Vitek MP, Hovanesian V, Stopa EG (2007) Microvascular injury and blood–brain barrier leakage in Alzheimer’s disease. Neurobiol Aging 28:977–986

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative and the research grant of Mahidol University (B.E. 2551-2555).

Conflict of Interest

Warawan Eiamphungporn, Sakda Yainoy and Virapong Prachayasittikul declare that they have no conflict of interest.

Human and Animal Rights and Informed Consent

This article does not contain any studies with human or animal subjects performed by any of the authors.

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Authors and Affiliations

  1. Faculty of Medical Technology, Center for Innovation Development and Technology Transfer, Mahidol University, Bangkok, 10700, Thailand

    Warawan Eiamphungporn & Sakda Yainoy

  2. Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, 2 Prannok Rd., Bangkok-Noi, Bangkok, 10700, Thailand

    Warawan Eiamphungporn, Sakda Yainoy & Virapong Prachayasittikul

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  1. Warawan Eiamphungporn
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  2. Sakda Yainoy
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Correspondence to Virapong Prachayasittikul.

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Eiamphungporn, W., Yainoy, S. & Prachayasittikul, V. Angiopep-2-Mediated Delivery of Human Manganese Superoxide Dismutase in Brain Endothelial Cells and its Protective Effect Against Oxidative Stress. Int J Pept Res Ther 21, 63–71 (2015). https://doi.org/10.1007/s10989-014-9433-9

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