摘要
Properdin蛋白即P因子,是补体替代激活途径中的正向调节因子,起着稳定替代途径中C3转化酶(C3bBb)的作用。为了进一步阐明Properdin的结构和功能之间的关系,探索Properdin与C3b及B因子的结合位点,我们从斑马鱼中克隆并表达了该基因,以鉴定其功能特性。
Properdin基因的开放阅读框全长为1314bp,编码一条含有437个氨基酸的多肽,其前20位氨基酸为信号肽,预测分子量大小为48.2KDa,也是由6个Ⅰ型凝血酶敏感蛋白重复序列构成。Properdin成熟蛋白中有40个半胱氨酸,预测有15个二硫键形成,31和58位处氨基酸可能为二个潜在的糖基化位点。对不同物种间Properdin的蛋白序列建进化树分析,结果显示斑马鱼Properdin蛋白和其它硬骨鱼Properdin聚成一簇。不同物种的Properdin蛋白序列及结构域进行多序列比对,发现斑马鱼properdin蛋白中存在保守的氨基酸基序WXXWXXW和RXR。与人、鼠、爪蟾、虹鳟(cfp1、cfp2、cfp3)的比较发现,同源性分别为42%、41%、43%、48%、48%和49%。实时荧光定量PCR结果显示:斑马鱼Properdin直到胚胎发育到30%下包时有母源性表达,且表达量随发育进行逐渐降低,但从20体节开始,合子基因开始表达。不同组织的实时荧光定量PCR结果表明,肝脏、肠、心脏、脑、眼睛、精巢、卵巢等组织中均有表达,在肠、鳍、肌肉的表达量相对较低,精巢、肝脏、眼睛、心脏、鳍的表达量较高,其中精巢中表达最多。
综上所述,斑马鱼Properdin的结构域特征与哺乳动物中该蛋白的结构域特征一样,与其在系统中的发育地位是相符的。预测的一个糖基化位点位于Cys45和Cys63之间,而这两个半胱氨酸预测会形成二硫键,因此推测此二硫键为与糖类的结合位点。Properdin有母源性表达,可能提示其在发育早期起作用。不同物种间Properdin表达部位不同是否提示其功能有差异,还有待于深入研究。这是关于斑马鱼中Properdin基因表达及其表达模式的首次报道,我们已经纯化得到了Properdin重组蛋白,为以后利用免疫结合等生物学技术探索Properdin蛋白的结构、功能及作用机制奠定了基础。深入了解其功能,有助于我们阐明人类补体缺乏病理机制,也促进我们对鱼类补体系统的功能有更深入的理解。
Properdin, also known as P factor, is an upregulator of the alternative complement pathway and acts by stabilizing the alternative pathway convertase (C3bBb). To elucidate the relationship between structure and function of properdin, and find the binding sites for C3b and factor B, we cloned and expressed the properdin gene in zebrafish to identify its functional properties.
The open reading frame cDNA of properdin is 1314bp, coding a polypeptide of 437 amino acids with a calculated molecular weight of about 48.2 kDa, composed of six thrombospondin repeat typeⅠdomains(TSR-1–TSR-6) too. There were forty Cys in the mature Properdin, predicted fifteen possible disulfide bonds, and two potential N-linked glycosylation site located at the residual position 31 and 58. Phylogenetic tree analysis of different Properdin protein sequences showed that zebrafish Properdin clustered in bony fish clade. On aligning the zebrafish properdin and TSR with other species, zebrafish properdins protein were found to have consensus sequence motifs WXXWXXW and RXR, The amino acid identity between zebrafish and that of humans, mouse, xenopus and trout pfc1, pfc2 and pfc3 was 42%、41%、43%、48%、48% and 49%, respectively. Zebrafish Properdin gene expression pattern at different developmental stages and in different tissues was analyzed by qRT-PCR. Results show Properdin to have maternal expression with a tendency to decrease gradually as the development continues. No expression was recorded at 30%-epiboly stage. However, from the 20-somite stage, the zygotic expression of Properdin commenced again. In adult zebrafish, Properdin was found to be expressed in all the tissues. Relatively lower expression was found in the muscles, fins, intestine, ovaries and brain; higher expression was found in the heart, testis, liver, eyes and heart.
In Summary, zebrafish properdin resemble its mammalian counterpart, which coincides with the phylogenetic status of zebrafish. One of the predicted glycosylation sites located between Cys45 and Cys63, which were predicted to form a disulfide bond, possibly serving to bind saccharide. Zebrafish Properdin has maternal expression, indicating its role in the early development. This is the first report of properdin in zebrafish. We have purified the recombinant Properdin protein thus making it possible to conduct immunoassays and other biological investigations in order to study the structure, function and mechanism of the action of Properdin. Further characterization of Properdin will help us clarify the pathological mechanism of the human complement defect and help us understand the functioning of the complement system.
引文
[1]于学慧,张世栋,张承梅,等.发育生物学的理想动物模型-斑马鱼.中国试验动物学杂志, 2001, 11(3): 172-175
[2] Thomas DN, Dieckmann GS. Antarctic sea ice-a habitat forextremophiles. Science, 2002, 295: 641-644
[3] MedzhitovR, Janeway CA Jr. Innate immunity:the virtues of a nonclonal system of recognition. Cell, 1997, 91: 295-298
[4] Cooper MD, Alder MN. The evolution of adaptive immune systems. Cell, 2006, 24, 124: 815-1822
[5] Kasamatsu J,Suzuki T,Ishijima J,Matsuda Y,Kasahara M.Two variable lymphocyte receptor genes of the inshore hagfish are located far apart on the same cllromosome.Immunogenetics,2007,59:329-331
[6] Zhang S C, Wang C F, Wang YJ ,et al.Presence and characterization of complement2like activity in the amphioxus Branchiostoma belcheri tsingtauense[J].Zoological Science,2003,20:1207-1214
[7]王长法,张士璀,王勇军,等.补体系统的进化.海洋科学,2004,28(8):55-58
[8]赵修竹主编.补体学.长沙:湖北科学技术出版社,1998
[9] Perrin-Cocon LA, Villiers CL, Salamero J, et al. B cell receptors and complement receptors target the antigen to distinct intracellular compartments. J Immunol, 2004, 172:3564-3572
[10] C. Kemper, D.E. Hourcade, et al. Properdin: New roles in pattern recognition and targetclearance. Molecular Immunology, 2008, 45: 4048–4056
[11]赵修竹. Pillemer分子-备解素.《免疫学杂志》,1996,12(3):208-213
[12] Lachman PJ. The control of momologous lysis. Immunology Today, 1992, 12: 312-315
[13] Kerekes K, Cooper PD, Prech1 J, et al. Adjuvant effect of gamma-inulin is mediated by C3 fragments deposited on antigen-presenting cells. J leukoc Biol, 2001, 69: 69-74
[14] Lange S, Bambir S, Dodds AW, Magnadottir B. An immunohistochemical study on complement component C3 in juvenile Atlantic halibut (Hippoglossus hippoglossus L.). Dev Comp Immunol, 2004, 28: 593-601
[15] Lee Y, Haas KM, Gor DO. Complement component C3d-antigen complexes can either augment or inhibit B lymphocyte activation and humoral immunity in mice depending on the degree of CD21/CD19 complex engagement. J Immunol, 2005, 175: 8011-8023
[16] Villiers MB, Villiers CL, Laharie AM, et al. Different stimulating effects of complement C3b and complete Freund’s adjuvant on antibody response. Immunopharmacology, 1999, 42: 151-157
[17] Michael V. Perdikoulis, Uday Kishore, Kenneth B.M. Reid, et al. Expression and characterisation of the thrombospondin type I repeats of human properdin. Biochimica et Biophysica Acta, 2001, 1548: 265-277
[18]金伯泉.细胞和分子免疫学.科学出版社,北京, 2001, 16
[19]邹雄,张利宁.分子免疫学与临床.山东科学技术出版社,济南, 2003
[20]安云庆,高晓朋.医学免疫学.北京:北京大学医学出版社,2004.33~40
[21]王志平.斑马鱼(Danio rerio)母源性补体因子的传递和免疫功能以及补体系统的个体发育:[中国海洋大学博士学位论文].青岛:中国海洋大学,2008
[22] Sire RB and Tsiftsoglou SA.Proteases of the complement system.Biochemical Society Transactions.2004,32:21-27
[23] Dirk Spitzer, Lynne M. Mitchell, John P. Atkinson, et al. Properdin Can Initiate Complement Activation by Binding Specific Target Surfaces and Providing a Platform for DeNovo Convertase Assembly. The Journal of Immunology, 2007;179;2600-2608
[24] Volanakis, J.E. Overview of the complement system. In The Human Complement System in Health and Disease (Volanakis JE, Frank M, eds), Marcel Dekker Inc. 1998: 9-32
[25]王长法.文昌鱼体液和鱼类血清补体溶血系统比较研究.博士毕业论文.中国海洋大学, 2005
[26] Sunyer JO, Lambris JD. Evolution and diversity of the complement system of poikilothermic vertebrates. Immunol Rev, 1998, 166: 39-57
[27] Ruddy S. The immunobiology of complement: Chemistry and biologic activity of the complement system. Trans Proc, 1974, 6: 1-7
[28] Koppenheffer TL, Spong KD, and Falvo HM. The complement system of the marsupial Monodelphis domestica. Developmental and Comparative Immunology, 1998, 22: 231-327
[29] Villiers CL, Lefebvre N., Villiers MB, et al. Role of C3 fragments in the control of the intracellular antigen processing. Mol Immunol, 1996, 33: 1
[30]杨汉春.动物免疫学.北京:中国农业大学出版社,2003.140~159
[31] Meri T, Murgia R, Stefanel P, et al. Regulation of complement activation at the C3-level by serum resistant. leptospires. Microb Pathog, 2005, 39: 139-47
[32] Thiel, S., Vorup-Jensen, T., Stover, C.M., Schwaeble, W., Laursen, S.B., Poulsen, K., Willis, A.C., Eggleton, P., Hansen, S., Holmskov, U., Reid, K.B.M., Jensenius, J.C. A second serine protease associated with mannan-binding lectin that activates complement. Nature. 1997, 386: 506-510
[33] FujitaT. Evolution of lecfin - complement pathway and its role in innate immunity. Nature, 2002, 2: 347-353
[34] Naughton MA, Walport MJ, Wtkrzner R Carter MJ, et al. Organ-specific contribution to circulating C7 levels by the bone. Eur J Immunol, 1996, 26: 2108-2112
[35] Hosgan AK, Wtlrzner RAbrahamsen TG, Dierich MP. Human polymorphonuclear leukoycytes Store large amounts of.J Immunol, 1995, 154: 4734-4740
[36] Andrews PA, Zhou W, Sacks SH. Tissue synthesis of complement as an immune regulator. Mol Med Today, 1995, 1: 202-207
[37] Morgan BP, Gasgue P. Extrahepatic complement biosynthesis: where, when and why? Clin Exp lmmunol, 1997, 107: 1-7
[38] Morgan BP, Gasque P. Expression of complement in the brain: role in health and disease. Immunol Today, 1996, 17: 461-466
[39] Volanakis JE. Transcriptional regulation ofcomplement genes. Annu Rev Immunol, 1995, 13: 277-305
[40]满孝勇,李学平,冒长峙,等. C1q与系统性红斑狼疮.国外医学皮肤性病学册, 2001, 27(3):141-144
[41]安云庆,高晓朋.医学免疫学.北京:北京大学医学出版社,2004.33~40
[42]李建国,肖慧捷,杨霁云,等.补体H因子研究进展.国外医学儿科学分册, 2005, 32(3): 143-146
[43]华春珍,综述,俞惠民,尚世强,等. MBL遗传缺陷与疾病相关性.杭州师范学院学报, 2007, 27(4): 252-255
[44]刘艳英,刘永铭,杨京港,等.高脂血症患者血清补体C3、C4、备解素和白细胞补体调节蛋白CD35、CD55、CD59的表达.中国循环杂志, 2005, 20(3): 176-179
[45] Nonaka M, Yamaguehi N, Natsuume-Sakai S, Takahashi M. The complement system of rainbow trout(Salmo gairdneri). I. Identification of the serunl lyric system homologous to mammalian complement. J Immunol, 1981, 126: 1489-1494
[46] Nikolakopoulou k, Zarkadis IK. Molecular cloning and characterisation of two homologues of Mannose-Binding Lectin in rainbow trout. Fish Shellfish Immunol, 2006, 21: 305—314
[47] Jackson AN, McLure CA, Dawkins RL, et a1. Mannose binding lectin(MBL)copy number Polymorphism in Zcbrafish(D. redo)and identification of haplotypes resistant to L. anguillarum. Immunogenetics, 2007, 59: 861-872
[48] Jensen JA, Festa E, Smith DS, and Cayer M. The complemem system of the nulse shark: Hemolytic and comparative characteristics. Science, 1981, 214: 566-569
[49] Matsushita M, Matsushita A, Endo Y, Nakata M, Kojima N, Mizuoehi T, Fujita T. Origin of the classical complement pathway: Lamprey orthologue ofmammalian Clq acts as a lectin. Proc Natl Acad Sci USA, 2004, 101: 10127-10131
[50] Takahashi M, Iwaki D, Matsushita A, Nakata M, Matsushita M, Endo Y, Fujita T. Cloning and characterization of mannose-binding lectin from lamprey(Agnathans). J Immunol, 2006, 176: 4861-4868
[51] Nonaka M, Takahashi M, Sasaki M. Molecular cloning of a lamprey homologue of themammalian MHC class III gene, complement factor B J Immunol, 1994, 152: 2263-2269
[52] Nonaka M, Fujii T, Kaidoh T, Natsuume-Sakai S, Nonaka M, Yamaguchi N, Takahashi M. Purification ofa lamprey complement protein homologous to the third component ofthemammalian complement system. J lmmunol, 1984, 133: 3242-3249
[53] Sakai D K. Heat inactivation of complements and immune hemolysis reactions in rainbow trout, masu salmon, coho salmon, gold fish, and tilapia [ J ]. Bull Japan Soc Sci Fish, 1981, 47: 565-571
[54] Holland M C, Lambris J D. The complement system in teleosts[ J ]. Fish & Shellfish Immunology, 2002, 12: 399-420
[55] Nonaka M, Natsuume2Sakai S, TakahashiM. The complement system in rainbow trout (Salmo gairdneri). II Purification and characterization of the fifth component(C5) [J]. J Imm unol,1981, 126: 1495-1498
[56] Kuroda N, Wada H, Naruse K, et al. Molecular cloning and linkage analysis of the Japanese medaka fish comp lement Bf/C2 gene [J]. Immunogenetics, 1996, 44: 459-467
[57] Kuroda N, Naruse K, Shima A, et al. Molecular cloning and linkage analysis of complement C3 and C4 genes of the Japanese medakafish [ J ]. Imm unogenetics, 2000, 51: 117-128
[58] NakaoM, Mutsuro J, Obo R, et al. Molecular cloning and protein analysis of divergent forms of the comp lement component C3 from a bony fish, the common carp (Cyprinus carpio) : presence of variants lacking the catalytic histidine [ J ]. Eur J Immunol, 2000, 30: 858—866
[59] NonakaM, IrieM, Tanabe K, et al. Identification and characterization of a variant of the third component of complement(C3) in rainbow trout ( Salmo gairdneri) serum [ J ]. J BiolChem , 1985, 260: 809-814
[60] Gongora R, Figuema F, K1ein J. Independent duplications of Bf and C3 complement genes in the Zebrafish [ J ]. Scand J Immunol, l998, 48: 651-658
[61] Cooper EL, Rinkevich B, Uhlenbruck G, Valembois P. Invertebrate immunity-another viewpointeditorial. Scand J Immunol, 1992, 35: 247-266
[62] Ratcliffe NA, Leonard C,Rowley AF. Prophenoloxidase activation, non-self recognition and cell co-opermion in insect immunity. Science, 1984, 226: 557-559
[63] Magor KE, Vasta GR. Ancestral immunity comes ofage. Immtmol Today, 1998, 19: 54-56
[64] Vasta GR, Ahmed A, Fink NE, Elola MT, Marsh AG, Snowden A, Odom EW. Animal lectins as self/non-self recognition molecules-biochemical and genetic approaches to understanding their biological roles and evolution. Primodial immunity: foundations for the vertebrate immune system. New York: Annals ofthe New York Academy of Sciences. 1994, 55-73
[65] Muller WE, Blumbaeh B, Miiller IM. Evolution ofthe innate and adaptive immune systems: relationships between potential immune molecules in the lowest metazoan phylum(Porifera) and those in vertebrates. Transplantation, 1999, 68: 1215-1227
[66] Ratios DA, Nair SV, Robbins J, et a1. A complement component C3-like protein from the tunicate, Styela plicata. Dev Comp Immunol, 2002, 26: 307-312
[67] Marino R, Kimura Y, De Santis R, et a1. Complement in urochordates: cloning and characterization oftwo C3-like genes in the ascidian Ciona intestinalis. Immunogenetics, 2002, 53: 1055-1064
[68] Nonaka M, Azumi K, Ji X, et a1. Opsonic complement component C3 in the solitary ascidian, Haiocynthia roretzi. J Immunol. 1999, 162: 387-391
[69] Azumi K, De SantisR, De Tomaso A, et a1. Genomic analysis of immunity in a Urochordate and the emergence ofthe vertebrate immune system:“waiting for Godot.”Immunogeneties, 2003, 55: 570-581
[70] Yoshizaki FY, Ikawa S, Satake M, et a1. Structure and the evolutionary implication of the triDlicated complement factor B genes of a uroehordate ascidian, Ciona intestinalis. Immunogenetics, 2005, 56: 930-942
[71] Dehal P, Satou Y, Campbell RK, et a1. The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science, 2002, 298: 2157-2167
[72] Kenjo A, Takahashi M, Matsushita M, et a1. Cloning and characterization ofnovel ficolins from the solitary ascidian,Haloeynthia roretzi. J Biol Chem, 2001, 276: 19959-19965
[73] Ji X, Azumi K, Sasaki M, et a1. Ancient origin of the complemem leetin pathway revealed bymolecular cloning of mannan binding protein-associated serine protease from a urochordate, the Japanese ascidian, Halocynthia roretzi. Proc Natl Acad Sci USA, 1997, 94: 6340-6345
[74] Miyazawa S, Nonaka M. Characterization of novel ascidian beta integrias as primitive complement receptor subunits. Immunogeneties, 2004, 55: 836-844
[75] Miyazawa S, Azumi k, Nonaka M. Cloning and characterization of integrins alpha subunit from the solitary ascidian, Halocynthia roretzi. J Immunol, 2001, 166: 1710-1715
[76]臧人杰,叶天星,程松高.备解素系统.见:谢少文,余贺主编.免疫学进展.上海科学技术出版社, 1962: 15-43
[77] Maves KK, Weil er JM. Properd in: approaching four decades of research. Immunol Res, 1993, 12: 233-237
[78] Lynne M, John P. Atkinson, Dennis E, et al. Properdin Can Initiate Complement Activation by Binding Specific Target Surfaces and Providing a Platform for De Novo Convertase Assembly. J. Immunol, 2007, 179: 2600-2608
[79] Maria Chondroua, Anastasios D. Papanastasioua, Georgios A. Spyrouliasb, et al. Three isoforms of complement properdin factor P in trout: Cloning, expression, gene organization and constrained modeling. Developmental and Comparative Immunology, 2008(32): 1454–1466
[80] Nolan KF, Schwaeble W, Kaluz S, et al . Molecular cloning of the cDNA coding for properdin, a positive regulator of the alternative pathway of h uman complement. Eur J Immu nol, 1991, 21: 771-775
[81] Schwaeble W, Huemer H, Most J , et al . Ex pres sion of properdin in h uman monocyt es. Eur J Immunol, 1994,219: 759
[82] Whaley K. Biosynthesis of th e complement components and the regulatory proteins of th ealternative pathwayby human peripheral blood monocytes. J Exp Med,1980; 151: 50
[83] Bentey C , Freis W, Brade V. Synthes is of factors D, B and P of the alternative pathway of complement activation as well as of C3 by guinea-pig perit onealm acrophages invitro. Immunol, 1979, 35: 971
[84] Farries T C, Atkinson JP. Biosynthesis of properdin. J Immunol, 1989, 142: 842
[85] Schw aeble W, Dippold WG, S chaf er MKH, et al. Properdin, a positive regulator of complement activation, is expressed in human T cell lines and peripheral blood T cells . J Im munol, 1993, 151: 2521
[86] Whaley K. Biosynthesis of th e complement components and the regulatory proteins of th ealternative pathwayby human peripheral blood monocytes. J Exp Med,1980, 151: 501
[87] Kemper, C., Mitchell, L.M., Zhang, L., Hourcade, D.E., 2008. The complement proteinproperdin binds apoptotic T cells and promotes complement activation and phagocytosis. Proc. Natl. Acad. Sci. U.S.A. 105, 9023–9028
[88] Spitzer, D., Mitchell, L.M., Atkinson, J.P., Hourcade, D.E., 2007. Properdin can initiate complement activation by binding specific target surfaces and providing a platform for DeNovo convertase assembly. J. Immunol. 179, 2600–2608
[89] Yu, H., Munoz, E.M., Edens, R.E., Linhardt, R.J., 2005. Kinetic studies on the interactions of heparin and complement proteins using surface plasmon resonance. Biochim. Biophys. Acta 1726, 168–176
[90] Daoudaki ME, Becherer JD, Lambris JD. A 34-amino acid peptide of the third component of complement mediates properdin binding. J Immunol 1988;140:1577–1580
[91] Farries TC , Finch JT , Lachmann PJ , et al . Resolution and anaysis of‘native’and‘act ivated’properdin.Biochem J, 1987, 243: 507
[92] Farries TC, Finch JT , Lachmann PJ , et al . Resolution and anaysis of‘native’and‘act ivated’properdin.Biochem J, 1987; 243: 507
[93] Densen P, Weiler JM, Griffiss JM, et al. Familial properdin deficiency and fatal men ingococcemia. N Engl J Med, 1987; 316: 922
[94] Ellis AE. Ontogenyoftheimmunesysteminteleostfish.In:EllisAE, editor. Fishvaccination. London: AcademicPress, 1988, 20-31
[95] Lange S, Dodds AW, Gudmundsdóttir S, Bambir SH,Magnadóttir B. The on to genic transcription of complement component C3 and ApolipoproteinA-ItRNA in Atlanticcod (GadusmorhuaL.)-arolein development and homeostasis? Dev Comp Immunol. 2005, 29: 1065-1077
[96] Lange S, Bambir SH, Dodds AW, Bowden T, Bricknell I, Espelid S, Magnadóttir B. Complement component C3 transcription in Atlantichali but (HippoglossushippoglossusL.) larvae. Fish She l fish Immunology, 2006, 20: 285-294
[97] L?voll M, Johnsen H, Boshra H, B?gwald, Sunyer JO, Dalmo RA. Heontogeny and extra hepatic expression of complement factor C3 in Atlanticsalmon(Salmosalar). Fish Shell fish Immunol, 2007, 23: 542-552]
[98] Ellingsen T, Strand C, Monsen E, B? gwald J, Dalmo RA. The onto geny of complement component C3 in the spotted wol fish (AnarhichasminorOlafsen). Fish Shell fish Immunol, 2005, 18: 351-358
[99] Tang R, Dodd AW, Lai D, McNabb WC, Love DR. Validation of zebra fish (Daniorerio) reference genes for quantitative real-time RT-PCR normalization. Acta Biochim BiophysSin(Shanghai), 2007, 39: 384-390
[100] Chen JY, Chen JC, Wu JI. Molecular cloning and functional analysis of high density lipoprotein bin ding protein. Comp Biochem PhysiolB Biochem MolBiol, 2003, 36: 117-130