微隐孢子虫中含铁相关蛋白的表达、纯化与功能研究
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摘要
微小隐孢子虫(Category B agent)是一种会导致人类和动物急性腹泻的寄生虫。如今在美国只有一种用于治疗对感染了隐孢子虫病艾滋病患者的药物(i.e., nitazoxanide),由于对其抗药性的担心,新药的开发迫在眉睫。本研究者尝试表达的铁氧还蛋白,正是处于微小隐孢子虫的线粒体样细胞器中具有硫铁簇结构的一种功能蛋白(Ferredoxin)、硫铁簇结构在细胞中是普遍存在且必不可少的组成部分,参与各种细胞功能的运作。近30多年对植物性铁氧还蛋白和脊椎动物中的铁氧还蛋白的结构和功能研究都有了一定的基础。
在本文中,首次成功表达与纯化CpmtFd和CpmtFNR蛋白,同时对它们进行了表征与生化性质研究。EPR,UV,HPLC和EPR研究表明这两个纯化蛋白分别具有[2Fe-S]簇和FAD辅基,且具有生物功能。CpmtFNR在细胞色素c参与下可以将电子从NADPH传递给CpmtFd。顶复门生物的mtFd和mtFNR从进化角度分析,是与人和其他动物的mtFd和mtFNR存在较大差异的一类。因此,它们可能在将来作为潜在的药物靶点帮助我们攻克隐孢子虫病和其它顶复门寄生虫疾病。
Cryptosporidium parvum (Category B agent) is a protozoan parasite that can cause severe watery diarrhea in humans and animals. Currently, only a single drug (i.e., nita-zoxanide [NTZ]) has been approved for treating cryptosporidiosis in immunocompe-tent (but not immunocompromized) patients in the United States. We have successfully expressed recombinant mitochondrial-type ferredoxin (mtFd) and ferredoxin:NADP+ reductase (mtFNR) from Cryptosporidium parvum and characterized their biochemical features for the first time. Both CpmtFd and CpmtFNR were obtained and purified as holo-proteins, in which the correct assembly of [2Fe-2S] cluster in Fd and that of FAD in FNR were confirmed and characterized by UV/Vis and EPR. These proteins were fully functional and CpmtFNR was capable of transferring electrons from NADPH to CpmtFd in a cytochrome c-coupled assay that followed a typical Michaelis-Menten ki-netics. Apicomplexan mtFd and mtFNR proteins were evolutionarily divergent from their counterparts in humans and animals, and could be explored to be acted as poten-tial drug targets in Cryptosporidium and other apicomplexans.
在本文中,首次成功表达与纯化CpmtFd和CpmtFNR蛋白,同时对它们进行了表征与生化性质研究。EPR,UV,HPLC和EPR研究表明这两个纯化蛋白分别具有[2Fe-S]簇和FAD辅基,且具有生物功能。CpmtFNR在细胞色素c参与下可以将电子从NADPH传递给CpmtFd。顶复门生物的mtFd和mtFNR从进化角度分析,是与人和其他动物的mtFd和mtFNR存在较大差异的一类。因此,它们可能在将来作为潜在的药物靶点帮助我们攻克隐孢子虫病和其它顶复门寄生虫疾病。
Cryptosporidium parvum (Category B agent) is a protozoan parasite that can cause severe watery diarrhea in humans and animals. Currently, only a single drug (i.e., nita-zoxanide [NTZ]) has been approved for treating cryptosporidiosis in immunocompe-tent (but not immunocompromized) patients in the United States. We have successfully expressed recombinant mitochondrial-type ferredoxin (mtFd) and ferredoxin:NADP+ reductase (mtFNR) from Cryptosporidium parvum and characterized their biochemical features for the first time. Both CpmtFd and CpmtFNR were obtained and purified as holo-proteins, in which the correct assembly of [2Fe-2S] cluster in Fd and that of FAD in FNR were confirmed and characterized by UV/Vis and EPR. These proteins were fully functional and CpmtFNR was capable of transferring electrons from NADPH to CpmtFd in a cytochrome c-coupled assay that followed a typical Michaelis-Menten ki-netics. Apicomplexan mtFd and mtFNR proteins were evolutionarily divergent from their counterparts in humans and animals, and could be explored to be acted as poten-tial drug targets in Cryptosporidium and other apicomplexans.
引文
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[18]微小隐抱子虫表面抗原CP23基因、cP15/60-23复合基因载体的构建、表达及其保护性的研究,专业,病原生物学,宰德富,刘克义(研究员)
[19]微小隐抱子虫致病相关蛋白的研究进展,陶艳琳,综述,程训佳,审校,中国寄生虫学与寄生虫病杂志,2004年8月,第22卷,第4期
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[25]隐孢子虫线粒体研究进展王进产张龙现宁长申菅复春邵朝霞石团员中国寄生虫学与寄生虫病杂志2006年12月第24卷第6期文章编号：1000-7423(2006)-06-0461-05
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[3]Tyzzer EE. Crytosoridium parvum (sp.nov),acoccidium found in the small intestine of the common mouse.Arch Protistenk,1912,26:394-421
[4]隐孢子虫病的流行与诊断研究进展,吴亮,陈盛霞综述,曹建平审校,国际医学寄生虫病杂志,2006年9月,第33卷,第5期
[5]Satin M,Trout JM,Xiao L,et al. Prevalence and age-related variation of Crytosorid-ium species and genotypes in dairy calves. Vet Parasitol,2004,122(2):103-117
[6]Yu JR,Seo M. Infection status of pigs with Crytosoridium parvum.Korean J Parasitol,2004,42(1):45-47
[7]Morgan UM,Xiao L,Monis P. et al. Crytosoridium spp. In domestic dogs:the "dog" genotype.Appl Environ Microbiol,2000,66(s):2220-2223
[8]Fayer R,Trout JM,Xiao L et al. Crytosoridium canis n. sp. From clomestic dogs.Parasitology,2001,87(6):1415-1422
[9]Glaser CA,Safrin S, Reingold A. et al. Association between Crytosoridium infec-tion and animal exposure in HIV infected individuals.Acquir Immune Defic syndr Hum Retrovirol,1998,17(1):79-82
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[11]Mackonzie WR,Hoxie NJ,Proctor ME,et al. A massive outbreak in Wilwaukee of Crytosoridium infection transmitted through the public water supply. N Engl J Med,1994,33(3):161-167
[12]Cranendonk RJ,Kodde CJ,Chipeta D,et al. Crytosoridium parvum and Isospora belli infections among patients with and without diarrhoea.East Afr Med,2003,80(8):398-401
[13]Banwat EB,Egah DZ,Onil BA. Prevalence of Crytosoridium infection among un-dernourished children in jos,Central Nigeria,Niger. Postgrad Med,2003,10(2):84-87
[14]Carcamo C,Hooton T,Wener MH,et al. Etiologies and manifestations of persis-tent diarrhea in adults with HIV-1 infection:a casecontrol study in Lima.Peru Infect Dis,2005,19(1):11-19
[15]安氏隐孢子虫热休克蛋白编码基因的克隆,表达和分析,刘海鹏,曹建平,李小红,卢潍媛,沈玉娟,徐馀信臧炜,刘述先,中国寄生虫学与寄生虫病杂志,2006年6月,第25卷第3期,文章编号1000-7423(2007-03-0163-08
[16]陈有贵,栗海思,戴梅秀,等。人体隐孢子虫病流行学调查。中国寄生虫学与寄生虫病杂志。1993,11(3)：207-210.
[17]DuPont HL,Chappell CL,Sterling CR,et al. The infection of Crytosoridium parvum in healthy volunteers. N Engl J Med 1995,332(13):855-859
[18]微小隐抱子虫表面抗原CP23基因、cP15/60-23复合基因载体的构建、表达及其保护性的研究,专业,病原生物学,宰德富,刘克义(研究员)
[19]微小隐抱子虫致病相关蛋白的研究进展,陶艳琳,综述,程训佳,审校,中国寄生虫学与寄生虫病杂志,2004年8月,第22卷,第4期
[20]Vossbrinck CR,Maddox JV,Friedman S,et al.Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes [J].Nature,1987,(326):411—414
[21]Germot A, Herve P, Guyader HL. Evidence for loss of mitochondria in Mi-crosporidia from amitochondrial-type HSP70 in nosema locustaoe[J]. Molecular and Biochemical Parasitolog,1997,87:159-168
[22]Abrahamsen, M. S., T. J. Templeton, S. Enomoto, J. E. Abrahante, G. Zhu, C. A.Lancto, M. Deng, C. Liu, G. Widmer, S. Tzipori, G. A. Buck, P. Xu, A. T. Bankier, P. H. Dear, B. A. Konfortov, H. F. Spriggs, L. Iyer, V. Anantharaman, L. Aravind, and V. Kapur.2004. Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304:441-445.
[23]Putignani, L., A. Tait, H. V. Smith, D. Homer, J. Tovar, L. Tetley, and J. M. Wastling. Characterization of a mitochondrion-like organelle in Cryptosporidium parvum.Parasitology,2004,129:1-18.
[24]Putignani, L., A. Tait, H. V. Smith, D. Horner, J. Tovar, L. Tetley, and J. M. Wastling. Characterization of a mitochondrion-like organelle in Cryptosporidium parvum. Parasitology,2004,129:1-18.,1996,13:297—305.
[25]隐孢子虫线粒体研究进展王进产张龙现宁长申菅复春邵朝霞石团员中国寄生虫学与寄生虫病杂志2006年12月第24卷第6期文章编号：1000-7423(2006)-06-0461-05
[26]Aliverti, A., V. Pandini, A. Pennati, M. de Rosa, and G. Zanetti.2008. Structural and functional diversity of ferredoxin-NADP(+) reductases. Arch Biochem Biophys 474:283-291.
[27]Beinert H, Sands RH (1960) Biochem Biophys Res Commun 3:41-46
[28]Mortenson LE, Valentine RC, Carnahan JE (1962) BiochemBiophys Res Commun 7:448-452
[29]Tagawa K, Arnon DI (1962) Nature 195:537-543
[30]Malkin R, Rabinowitz JC (1966) Biochem Biophys Res Commun 23:822-827
[31]Beinert H, Holm RH, Munck E (1997) Science 277:653-659
[32]Rao PV, Holm RH (2004) Chem Rev 104:527-559
[33]Dennis T. Ta, LarryE.VickeryS Cloning, Sequencing, and Overexpression of a [2Fe-2S] Ferredoxin Gene from Escherichia coli* THE JOURNAL OF BIOLOGICAL CHEMISTRY 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol.267, No.16, Issue of June 5, pp.11120-11125,1992 Printed in U.S.A.
[34]Asya V. Grinberg, Frank Hannemann, Burkhard Schiffler, Jurgen Muller, Udo Heinemann,and Rita Bernhardt. Adrenodoxin: Structure, Stability, and Electron Trans-fer Properties. PROTEINS: Structure, Function, and Genetics 40:590-612 (2000)
[35]F. William Studier Protein production by auto-induction in high-density shaking cultures. Protein Expression and Purification 41 (2005) 207-234
[36]Amon DI. Trends Biochem Sci,1988,13:30-33
[37]Knaff DB. In: Ort DR et al. eds. Ferredoxin and Ferredoxin Dependent Enzymes, The Netherlands Kluner: Acadenmic Press,1996,333-361
[38]Emes MJ et al. J.Exp Bot,1997,48:1995-2005
[39]Neuhaus HE st al. Annu Rev Plant Physiol Plant Biol,2000,51:111-140
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