日本七鳃鳗非受体酪氨酸激酶BTK的全长cDNA克隆和生物信息学分析
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摘要
内容摘要:酪氨酸激酶BTK是非受体酪氨酸家族的成员,它由PH结构域、TH结构域、SH3结构域、SH2结构域和催化结构域五部分组成。BTK参与多种信号通路,在免疫系统中发挥重要作用。近年来BTK参与B细胞信号通路、TLR信号通路和肥大细胞脱颗粒等信号通路的具体机制问题是科学研究的热点。BTK对细胞的增殖、分化和凋亡起着重要的调控作用,其突变可导致X连锁无丙种球蛋白血症,临床表现为反复的细菌感染和B细胞数量的显著下降。本文收集了日本七鳃鳗的白细胞并提取总RNA,利用紫外光分光光度计和琼脂糖凝胶电泳的方法检测提取的总RNA质量。通过RACE的实验方法从日本七鳃鳗白细胞中成功扩增BTK基因并克隆后到pET32a(+)载体。从一级结构比对、高级结构预测、保守基序的分布、构建进化树等方面分析了这段序列。
主要的研究结果如下:
1一级结构比对结果表明日本七鳃鳗白细胞的BTK基因的氨基酸序列与黑猩猩、人类、马、牛、小家鼠、犬属、鸭嘴兽、原鸡、斑马鱼、盲鳗和果蝇等其他物种的BTK基因的氨基酸序列同源性很高。
2利用软件MEME预测日本七鳃鳗BTK氨基酸序列的高级结构,包括预测PH结构域的高级结构、SH3结构域的高级结构和激酶结构域的高级结构。预测结果表明日本七鳃鳗BTK的高级结构与模板BTK的高级结构十分相似。
3利用MEME在线软件分析在不同物种的BTK氨基酸序列中发现的保守基序的类型和分布,结果表明日本七鳃鳗BTK的氨基酸序列拥有全部的10个保守基序。
4基于邻接法构建的氨基酸序列进化树表明,所有的BTK氨基酸序列被分为两簇,一簇包括哺乳动物、鸟类、鱼类和七鳃鳗,另一簇包括盲鳗和昆虫类。
综合上述的研究结果得出:在最原始的脊椎动物七鳃鳗中存在非受体酪氨酸激酶BTK,它的具体的免疫机制尚不明确。
Content: Bruton’s agammaglobulinaemia tyrosine kinase belongs to a subfamily of non-receptor protein tyrosine kinases.It is characterized by five structural modules: Pleckstrin homology domain,Tec homology domain, Src homology3 domain,Src homology2 domain and Tyrosine kinase domain.BTK is thought to be a type of key signal molecules in various signal pathways, it plays a crucial role in immune system. Recently mechanism of how BTK plays an crucial role in B cell signaling pathway,TLR signaling pathway and mast cells degranulation is a hotspot.BTK plays important role in regulating cell proliferation, differentiation and apoptosis. Mutation of BTK could cause X-linked agammaglobulinemia, which clinically characterized by recurrent bacterial infections and markedly reduced numbers of B cells .In this work,we collected the leucocytes of lamprey, extracted the total RNA, measure concentration of RNA by ultraviolet spectrophotometer and agarose gel electrophoresis.We have successfully cloned BTK gene using RACE method and BTK gene was constructed into pET32a(+) vector. Then we study on primary structure alignment, advanced structure prediction,reconstructing a phylogenetic tree and conserved motifs distribution of this sequence.
These mainly results were as follow:
1 The intercomparison results of primary structure shows that its amino acid sequences are very conservative with other species,such as chimpanzee,human,horse,cattle,mouse,dog, ornithorhynchus,chicken,zebra fish,hagfish and drosophila and so on.
2 We predicted the advanced structure of BTK amino acid from lamprey using the MEME system.It included the prediction result of PH domain advanced structure, the prediction result of SH3 domain advanced structure and the prediction result of kinase domain advanced structure.The prediction results show that advanced structure of Lamprey BTK is similar to Temple BTK.
3 The MEME system shows that amino acid sequences of Lamprey BTK contains 10 conserved motifs totally.
4 The phylogenetic tree contains two clusters:one including mammals, birds, fishes and lamprey,the other including hagfish and insects. These above results demonstrate that non-receptor protein tyrosine kinase BTK may exist in lampreys, the precise immune mechanism of lamprey remained to be elucidated.
主要的研究结果如下:
1一级结构比对结果表明日本七鳃鳗白细胞的BTK基因的氨基酸序列与黑猩猩、人类、马、牛、小家鼠、犬属、鸭嘴兽、原鸡、斑马鱼、盲鳗和果蝇等其他物种的BTK基因的氨基酸序列同源性很高。
2利用软件MEME预测日本七鳃鳗BTK氨基酸序列的高级结构,包括预测PH结构域的高级结构、SH3结构域的高级结构和激酶结构域的高级结构。预测结果表明日本七鳃鳗BTK的高级结构与模板BTK的高级结构十分相似。
3利用MEME在线软件分析在不同物种的BTK氨基酸序列中发现的保守基序的类型和分布,结果表明日本七鳃鳗BTK的氨基酸序列拥有全部的10个保守基序。
4基于邻接法构建的氨基酸序列进化树表明,所有的BTK氨基酸序列被分为两簇,一簇包括哺乳动物、鸟类、鱼类和七鳃鳗,另一簇包括盲鳗和昆虫类。
综合上述的研究结果得出:在最原始的脊椎动物七鳃鳗中存在非受体酪氨酸激酶BTK,它的具体的免疫机制尚不明确。
Content: Bruton’s agammaglobulinaemia tyrosine kinase belongs to a subfamily of non-receptor protein tyrosine kinases.It is characterized by five structural modules: Pleckstrin homology domain,Tec homology domain, Src homology3 domain,Src homology2 domain and Tyrosine kinase domain.BTK is thought to be a type of key signal molecules in various signal pathways, it plays a crucial role in immune system. Recently mechanism of how BTK plays an crucial role in B cell signaling pathway,TLR signaling pathway and mast cells degranulation is a hotspot.BTK plays important role in regulating cell proliferation, differentiation and apoptosis. Mutation of BTK could cause X-linked agammaglobulinemia, which clinically characterized by recurrent bacterial infections and markedly reduced numbers of B cells .In this work,we collected the leucocytes of lamprey, extracted the total RNA, measure concentration of RNA by ultraviolet spectrophotometer and agarose gel electrophoresis.We have successfully cloned BTK gene using RACE method and BTK gene was constructed into pET32a(+) vector. Then we study on primary structure alignment, advanced structure prediction,reconstructing a phylogenetic tree and conserved motifs distribution of this sequence.
These mainly results were as follow:
1 The intercomparison results of primary structure shows that its amino acid sequences are very conservative with other species,such as chimpanzee,human,horse,cattle,mouse,dog, ornithorhynchus,chicken,zebra fish,hagfish and drosophila and so on.
2 We predicted the advanced structure of BTK amino acid from lamprey using the MEME system.It included the prediction result of PH domain advanced structure, the prediction result of SH3 domain advanced structure and the prediction result of kinase domain advanced structure.The prediction results show that advanced structure of Lamprey BTK is similar to Temple BTK.
3 The MEME system shows that amino acid sequences of Lamprey BTK contains 10 conserved motifs totally.
4 The phylogenetic tree contains two clusters:one including mammals, birds, fishes and lamprey,the other including hagfish and insects. These above results demonstrate that non-receptor protein tyrosine kinase BTK may exist in lampreys, the precise immune mechanism of lamprey remained to be elucidated.
引文
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[18]BABA Y, NONOYAMA S, MATSUSH ITA M, et al1. Involvement of Wiskott Aldrich syndrome protein in B Cell cytop lasmic tyrosine kinase pathway[ J ].Blood, 1999, 93 (6) :2003-2012.
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[30]Taku Kambayashi,Gary A. Koretzky.Proximal signaling events in FceRI-mediated mast cell activation.ALLERGY CLIN IMMUNOL,2007,10.1016.
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[34]Pessach IM,Notarangelo LD.X-linked primary immunodeficiencies as a bridge to better understanding X-chromosome related autoimmunity.J Autoimmun,2009,33(1):17-24.
[35]Howard V,Greene JM,Pahwa S, et al.The health status and quality of life of adults withX-linked agammaglobulinaemia.Clin Immunol,2006, 118(2-3):201-8.
[36]Tóth B,Volokha A,Mihas A, et al.Genetic and demographic features of X-linked agammaglobulinaemia in Eastern and Central Europe:A cohort study.Mol Immunol,2009, 46(10):2140-6.
[37]Hunter HL,McKenna KE,Edgar JD.Eczema and X-linked agammaglobulinaemia.Clin Exp Dermatol, 2008,33(2):148-50.
[38]Graziani S,Di Matteo G,Benini L, et al.Identification of a BTK mutation in a dysgammaglobulinemic patient with reduced B cells:XLA diagnosis or not.Clin Immunol,2008, 128(3):322-8.
[39]Kristufek D, Aspalter RM, Eibl MM, Wolf HM. Characterization of novel Bruton’s tyrosine kinase gene mutations in Central European patients with agammaglobulinaemia ,MolImmunol,2007,4:1639–43.
[40]Doris Kristufek, Rosa Maria Aspalter, Martha Marianne Eibl, Hermann Maximilian Wolf,Characterization of novel Bruton’s tyrosine kinase gene mutations in Central European patients with agammaglobulinaemia,Molecular Immunology ,2007,44 :1639–1643.
[41]Thomas Marron, Kaileen Rohr, Monica Martinez-Gallo, Joyce Yu, Charlotte Cunningham-Rundles. New York,Toll-like Receptor Signaling Dependence of BTK: Assessing Innate Immunity in BTK Deficiency,Mount Sinai School of Medicine, 2009,10.1016:03.185.
[42]Arnon Broides,Wenjian Yang, Mary Ellen Conley,Genotype/phenotype correlations in X-linked agammaglobulinaemia,Clinical Immunology ,2006,118:195– 200.
[43]A. Plebani, A. Soresina, R. Rondelli, G. Amato, C. Azzari, F. Cardinale,G. Cazzola, R. Consolini, D. De Mattia,G. Dell’Erba, M. Duse, M. Fiorini, S. Martino, B. Martire, M. Masi, V. Monafo, V. Moschese, L. Notarangelo, P.Orlandi, P. Panei, A. Pession, M. Pietrogrande, C. Pignata,Quinti, V. Ragno, P. Rossi, A. Sciotto, A. Stabile, A.Ugaziothe ItalianPediatric Group for XLA-AIEOP,Clinical,immunological, and molecular analysis in a large cohortof patients with X-linked agammaglobulinaemia:an Italian multicenter study, Clin. Immunol,2002,104 :221– 230.
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[2] Janvier P.Early Vertebrates.Oxford Scientific Publication,1996,554-559.
[3] Neidert A H,Virupannavar V,et al.Lamprey Dlx genes and early vertebrate evolution.Proc .Natl Acad.Sci USA,2001,98:1665-1670.
[4] Forey P,Janvier P. Evolution of the early vertebrates.Am Sci,1994,82:554-565.
[5] Kuraku S,et al.Monophyly of Lamprey and Hagfishes Supported by Nuclear DNA-Coded Genes.Molecular evolution,1999,49:729-735.
[6] Holland P W H,Fernandez G J.Hox Genes and Chordate Evolution.Delopmental Biology,1996,173:382-395.
[7] Osório J, Rétaux S. The lamprey in evolutionary studies. Dev. Genes Evol., 2008, 218:221-235.
[8] Kuratani S, Kuraku S, Murakami Y. Lamprey as an evo-devo model: lessons from comparative embryology and molecular phylogenetics. Genesis, 2002, 34:175-183.
[9] Vetrie D,Vorechovsky I,Sideras P,et al.The gene involved in X-linked agammaglobuli- naemia is a member of the src family of protein tyrosine kinases.Nature,1993, 361(6409):226-33.
[10]V?liaho J,Smith CI,Vihinen M.BTKbase:the mutation database for X-linked agammaglobulinaemia.Hum Mutat,2006,27(12):1209-17.
[11]Tsukada S,Saffran DC,Rawlings DJ, et al.Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinaemia. Cell,1993,29;72(2):279-90.
[12]Huang KC,Cheng HT,Pai MT, et al.Solution structure and phosphopeptide binding of the SH2 domain from the human Bruton’s tyrosine kinase.J Biomol NMR,2006,36(1):73-8.
[13]苏珊,酪氨酸激酶Btk家族的结构和功能,医学分子生物学杂志, 2006, 3 (6) : 442-445.
[14]Mihara S,Suzuki N.Role of Txk,a member of the Tec family of tyrosine kinases,in immuneinflammatory diseases.Int Rev Immunol,2007,26(5-6):333-48.
[15]Lopez-Herrera G,Berron-Ruiz L,Mogica-Martinez D,et al. Characterization of Bruton’s tyrosine kinase mutations in Mexican patients with X-linked agammaglobulinaemia.Mol Immunol,2008,45(4):1094-8.
[16]DiNitto JP,Lambright DG.Membrane and juxtamembrane targeting by PH and PTB domains.Biochim Biophys Acta,2006,1761(8):850-67.
[17]GLASSFORD J, SOEIRO I, SKARELL SM, et al1.BCR targets cyclin D via Btk and the p85 apha subunit of PI-3K to induce cell cycle p rogression in p rimarymouse B cells[ J ].Oncogene, 2003,22 (15) : 2248222591.
[18]BABA Y, NONOYAMA S, MATSUSH ITA M, et al1. Involvement of Wiskott Aldrich syndrome protein in B Cell cytop lasmic tyrosine kinase pathway[ J ].Blood, 1999, 93 (6) :2003-2012.
[19]Kristufek D,Aspalter RM,Eibl MM, et al.Characterization of novel Bruton’s tyrosine kinase gene mutations in Central European patients with agammaglobulinaemia.Mol Immunol,2007, 44(7):1639-43.
[20]Murayama K,Kato-Murayama M,Mishima C, et al.Crystal structure of the Bruton’s tyrosine kinase PH domain with phosphatidylinositol.Biochem Biophys Res Commun, 2008,377(1): 23-8.
[21]Qiu Y,Kung HJ.Signaling network of the BTK family kinases. Oncogene, 2000,19(49): 5651-61.
[22]Taneichi H,Kanegane H,Sira MM,et al. Miyawaki T.Toll-like receptor signaling is impaired in dendritic cells from patients with X-linked agammaglobulinaemia.Clin Immunol,2008, 126(2):148-54.
[23]Pérezde Diego R,Bravo J,Allende LM, et al.Identification of novel non-pathogenic mutation in SH3 domain of BTK in an XLA patient.Mol Immunol,2008,45(1):301-3.
[24]Sheedy FJ,O'NeillLA.The Troll in Toll:Mal and Tram as bridges for TLR2 and TLR4 signaling. J Leukoc Biol,2007,82(2):196-203.
[25]Kawai T,Akira S.Signaling to NF-kappaB by Toll-like receptors.Trends Mol Med,2007, 13(11):460-9.
[26]Liew F Y, Xu D, Brint E K, et al. Negative regulation of toll-like receptor-mediated immune responses.Nat Rev Immunol,2005,5(6):446-458.
[27]Kaisho T, Akira S. Toll-like receptor function and signaling.J Allergy Clin Immunol, 2006,117(5):979-987.
[28]Kawakami Y,Yao L,Mirura T.Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon FceRI crosslinking.Mol Cell Biol,1994,14:5108.
[29]Rivera J,Gilfillan AM.Molecular regulation of mast cell activation.J Allergy Clin Immunol, 2006, 117(6):1214-25.
[30]Taku Kambayashi,Gary A. Koretzky.Proximal signaling events in FceRI-mediated mast cell activation.ALLERGY CLIN IMMUNOL,2007,10.1016.
[31]Hubbard KB, Hepler JR.Cell signalling diversity of the Gqa family of heterotrimeric G proteins. Cell Signal,2006,18(2):135-50.
[32]Xu C,Xu B,Huang H,et al.Preimplantation genetic diagnosis for X-linked agammaglobulinaemia:a case report.Fertil Steril,2009,91(5):1958.
[33]Winkelstein JA,Marino MC,Lederman HM, et al.X-linked agammaglobulinaemia:report on a United States registry of 201 Patients. Medicine, 2006, 85(4):193-202.
[34]Pessach IM,Notarangelo LD.X-linked primary immunodeficiencies as a bridge to better understanding X-chromosome related autoimmunity.J Autoimmun,2009,33(1):17-24.
[35]Howard V,Greene JM,Pahwa S, et al.The health status and quality of life of adults withX-linked agammaglobulinaemia.Clin Immunol,2006, 118(2-3):201-8.
[36]Tóth B,Volokha A,Mihas A, et al.Genetic and demographic features of X-linked agammaglobulinaemia in Eastern and Central Europe:A cohort study.Mol Immunol,2009, 46(10):2140-6.
[37]Hunter HL,McKenna KE,Edgar JD.Eczema and X-linked agammaglobulinaemia.Clin Exp Dermatol, 2008,33(2):148-50.
[38]Graziani S,Di Matteo G,Benini L, et al.Identification of a BTK mutation in a dysgammaglobulinemic patient with reduced B cells:XLA diagnosis or not.Clin Immunol,2008, 128(3):322-8.
[39]Kristufek D, Aspalter RM, Eibl MM, Wolf HM. Characterization of novel Bruton’s tyrosine kinase gene mutations in Central European patients with agammaglobulinaemia ,MolImmunol,2007,4:1639–43.
[40]Doris Kristufek, Rosa Maria Aspalter, Martha Marianne Eibl, Hermann Maximilian Wolf,Characterization of novel Bruton’s tyrosine kinase gene mutations in Central European patients with agammaglobulinaemia,Molecular Immunology ,2007,44 :1639–1643.
[41]Thomas Marron, Kaileen Rohr, Monica Martinez-Gallo, Joyce Yu, Charlotte Cunningham-Rundles. New York,Toll-like Receptor Signaling Dependence of BTK: Assessing Innate Immunity in BTK Deficiency,Mount Sinai School of Medicine, 2009,10.1016:03.185.
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