家蚕微孢子虫极管蛋白1(NbPTP1)在果蝇S2细胞中的表达与糖基化修饰
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摘要
极管蛋白(Polar tube protein)是极管的主要成分,能特异性定位于微孢子虫极管,在微孢子虫侵染宿主过程中发挥重要作用。文中分析了家蚕微孢子虫极管蛋白1中潜在的O-、N-糖基化修饰位点,克隆了家蚕微孢子虫极管蛋白1全基因序列,并将其插入带有V5和His标签的真核表达载体pMT/Bip/V5-His A中,成功构建了pMT/Bip/V5-His A-NbPTP1重组质粒,经转染果蝇S2细胞后,发现NbPTP1基因能在果蝇细胞中高效表达。此外,Lectin blotting和β-消除反应分析结果表明:果蝇S2细胞内表达的NbPTP1具有O-糖基化修饰特征。以上结果为研究NbPTP1的糖基化修饰特征与其功能之间的关系提供了基础,有助于揭示微孢子虫侵染机制,建立可行有效的微孢子虫病诊断和防治措施。
The polar tube protein is the major component of polar tube, and can specifically locate on the polar tube of microsporidia and plays an important role in invasion host cell. In this study, we analyzed the potential O-and Nglycosylation sites in polar tube protein 1 from Nosema bombycis. NbPTP1 was successfully cloned to eukaryotic expression vector pMT/Bip/V5-His A, involved V5 and His tags. After transfection, NbPTP1 gene could be efficiently expressed in Drosophila S2 cells. In addition, Lectin blotting and beta elimination analysis showed that NbPTP1 expressed in Drosophila S2 cells was O-glycosylation. These studies provided a basis for understanding the relationship between glycosylation and function of NbPTP1, helped us to reveal the infection mechanism of microsporidia and established effective diagnosis and prevention methods for microsporidia.
The polar tube protein is the major component of polar tube, and can specifically locate on the polar tube of microsporidia and plays an important role in invasion host cell. In this study, we analyzed the potential O-and Nglycosylation sites in polar tube protein 1 from Nosema bombycis. NbPTP1 was successfully cloned to eukaryotic expression vector pMT/Bip/V5-His A, involved V5 and His tags. After transfection, NbPTP1 gene could be efficiently expressed in Drosophila S2 cells. In addition, Lectin blotting and beta elimination analysis showed that NbPTP1 expressed in Drosophila S2 cells was O-glycosylation. These studies provided a basis for understanding the relationship between glycosylation and function of NbPTP1, helped us to reveal the infection mechanism of microsporidia and established effective diagnosis and prevention methods for microsporidia.
引文
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[18]Han B,Polonais V,Sugi T,et al.The role of microsporidian polar tube protein 4(PTP4)in host cell infection.PLo S Pathog,2017,13(4):e1006341.
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[20]Bouzahzah B,Weiss LM.Glycosylation of the major polar tube protein of Encephalitozoon cuniculi.Parasitol Res,2010,107(3):761-764.
[21]Xu YJ,Takvorian P,Cali A,et al.Lectin binding of the major polar tube protein(PTP1)and its role in invasion.J Eukaryot Microbiol,2003,50(6):600-601.
[22]Taupin V,Garenaux E,Mazet M,et al.Major O-glycans in the spores of two microsporidian parasites are represented by unbranched manno-oligosaccharides containingα-1,2 linkages.Glycobiology,2007,17(1):56-67.
[23]Wu YJ,Long MX,Chen J,et al.Cloning and prokaryotic expression of Nosema bombycis polar tube protein 1(Nb PTP1).Sci Sericult,2014,40(2):258-264(in Chinese).吴玉娇,龙梦娴,陈洁,等.家蚕微孢子虫极管蛋白1(Nb PTP1)的基因克隆及原核表达.蚕业科学,2014,40(2):258-264.
[24]Pan GQ,Xu JS,Li T,et al.Comparative genomics of parasitic silkworm microsporidia reveal an association between genome expansion and host adaptation.BMCGenomics,2013,14:186.
[25]Katinka MD,Duprat S,Cornillot E,et al.Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi.Nature,2000,414(6862):450-453.
[26]Varki A.Biological roles of oligosaccharides:all of the theories are correct.Glycobiology,1993,3(2):97-130.
[27]Aronson M,Medalia O,Schori L,et al.Prevention of colonization of the urinary tract of mice with Escherichia coli by blocking of bacterial adherence with methylα-D-mannopyranoside.J Infect Dis,1979,139(3):329-332.
[28]Chen XM,La Russo NF.Mechanisms of attachment and internalization of Cryptosporidium parvum to biliary and intestinal epithelial cells.Gastroenterology,2000,118(2):368-379.
[29]Zhang Z,Duchêne M,Stanley SL Jr.A monoclonal antibody to the amebic lipophosphoglycanproteophosphoglycan antigens can prevent disease in human intestinal xenografts infected with Entamoeba histolytica.Infect Immun,2002,70(10):5873-5876.
[30]Peek R,Delbac F,Speijer D,et al.Carbohydrate moieties of microsporidian polar tube proteins are targeted by immunoglobulin G in immunocompetent individuals.Infect Immun,2005,73(12):7906-7913.
[31]Polonais V,Prensier G,Méténier G,et al.Microsporidian polar tube proteins:highly divergent but closely linked genes encode PTP1 and PTP2 in members of the evolutionarily distant Antonospora and Encephalitozoon groups.Fungal Genet Biol,2005,42(9):791-803.
[32]Lin LP,Pan GQ,Li T,et al.The protein import pore Tom40 in the microsporidian Nosema bombycis.JEukaryot Microbiol,2012,59(3):251-257.
[33]Yamashita A,Nango E,Ashikawa Y.A large-scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand-binding domains of taste receptor.Protein Sci,2017,26(11):2291-2301.
[34]Ailor E,Pathmanathan J,Jongbloed JDH,et al.Abacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells.Biochem Biophys Res Commun,1999,255:444-450.
[35]Tessier DC,Thomas DY,Khouri HE,et al.Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide.Gene,1991,98(2):177-183.
[36]Jarvis DL,Summers MD,Garcia A Jr,et al.Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system.J Biol Chem,1993,268(22):16754-16762.
[37]Shen X,Dojcinovic D,Baldi L,et al.Improved process conditions for increasing expression of MHC class IIprotein from a stable Drosophila S2 cell line.Biotechnol Lett,2018,40(1):85-92.
[38]Zitzmann J,Weidner T,Czermak P.Optimized expression of the antimicrobial protein Gloverin from Galleria mellonella using stably transformed Drosophila melanogaster S2 cells.Cytotechnology,2017,69(2):371-389.
[2]Liu H,Jiang ZH,Yuan ZY,et al.Infection by and genotype characteristics of Enterocytozoon bieneusi in HIV/AIDS patients from Guangxi Zhuang autonomous region,China.BMC Infect Dis,2017,17(1):684.
[3]Long MX,Wu YJ,Chen J,et al.Research progress in polar tube proteins from microsporidia.Sci Sericult,2014,40(5):917-923(in Chinese).龙梦娴,吴玉娇,陈洁,等.微孢子虫极管蛋白的研究进展.蚕业科学,2014,40(5):917-923.
[4]Han B,Weiss LM.Microsporidia:obligate intracellular pathogens within the fungal kingdom.Microbiol Spectr,2017,5(2):FUNK-0018-2016.
[5]Weiss LM,Becnel JJ.Microsporidia:Pathogens of Opportunity.Oxford:Wiley-Blackwell,2014.
[6]Ohshima K.On the function of the polar filament of Nosema bombycis.Parasitology,1937,29(2):220-224.
[7]Weidner E.Ultrastructural study of microsporidian invasion into cells.Z Parasitenkd,1972,40(3):227-242.
[8]Frixione E,Ruiz L,Santillán M,et al.Dynamics of polar filament discharge and sporoplasm expulsion by microsporidian spores.Cell Motil Cytoskel,1992,22(1):38-50.
[9]Delbac F,Peuvel I,Metenier G,et al.Microsporidian invasion apparatus:identification of a novel polar tube protein and evidence for clustering of ptp1 and ptp2genes in three Encephalitozoon species.Infect Immun,2001,69(2):10160-1024.
[10]Peuvel I,Peyret P,Méténier G,et al.The microsporidian polar tube:evidence for a third polar tube protein(PTP3)in Encephalitozoon cuniculi.Mol Biochem Parasitol,2002,122(1):69-80.
[11]Delbac F,Peyret P,Metenier G,et al.On proteins of the microsporidian invasive apparatus:complete sequence of a polar tube protein of Encephalitozoon cuniculi.Mol Microbiol,1998,29(3):825-834.
[12]Keohane EM,Orr GA,Zhang HS,et al.The molecular characterization of the major polar tube protein gene from Encephalitozoon hellem,a microsporidian parasite of humans.Mol Biochem Parasitol,1998,94(2):227-236.
[13]Weidner E.The microsporidian spore invasion tube.The ultrastructure,isolation,and characterization of the protein comprising the tube.J Cell Biol,1976,71(1):23-34.
[14]Keohane EM,Orr GA,Takvorian PM,et al.Purification and characterization of a microsporidian polar tube protein.Mol Biochem Parasitol,1996,79(2):255-259.
[15]Keohane EM,Weiss LM.Characterization and function of the microsporidian polar tube:a review.Folia Parasitol(Praha),1998,45(2):117-127.
[16]Xu YJ,Weiss LM.The microsporidian polar tube:a highly specialised invasion organelle.Int J Parasitol,2005,35(9):941-953.
[17]Lom J.On the structure of the extruded microsporidian polar filament.Z Parasitenkd,1972,38(3):200-213.
[18]Han B,Polonais V,Sugi T,et al.The role of microsporidian polar tube protein 4(PTP4)in host cell infection.PLo S Pathog,2017,13(4):e1006341.
[19]Xu YJ,Takvorian P M,Cali A,et al.Glycosylation of the major polar tube protein of Encephalitozoon hellem,a microsporidian parasite that infects humans.Infect Immun,2004,72(11):6341-6350.
[20]Bouzahzah B,Weiss LM.Glycosylation of the major polar tube protein of Encephalitozoon cuniculi.Parasitol Res,2010,107(3):761-764.
[21]Xu YJ,Takvorian P,Cali A,et al.Lectin binding of the major polar tube protein(PTP1)and its role in invasion.J Eukaryot Microbiol,2003,50(6):600-601.
[22]Taupin V,Garenaux E,Mazet M,et al.Major O-glycans in the spores of two microsporidian parasites are represented by unbranched manno-oligosaccharides containingα-1,2 linkages.Glycobiology,2007,17(1):56-67.
[23]Wu YJ,Long MX,Chen J,et al.Cloning and prokaryotic expression of Nosema bombycis polar tube protein 1(Nb PTP1).Sci Sericult,2014,40(2):258-264(in Chinese).吴玉娇,龙梦娴,陈洁,等.家蚕微孢子虫极管蛋白1(Nb PTP1)的基因克隆及原核表达.蚕业科学,2014,40(2):258-264.
[24]Pan GQ,Xu JS,Li T,et al.Comparative genomics of parasitic silkworm microsporidia reveal an association between genome expansion and host adaptation.BMCGenomics,2013,14:186.
[25]Katinka MD,Duprat S,Cornillot E,et al.Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi.Nature,2000,414(6862):450-453.
[26]Varki A.Biological roles of oligosaccharides:all of the theories are correct.Glycobiology,1993,3(2):97-130.
[27]Aronson M,Medalia O,Schori L,et al.Prevention of colonization of the urinary tract of mice with Escherichia coli by blocking of bacterial adherence with methylα-D-mannopyranoside.J Infect Dis,1979,139(3):329-332.
[28]Chen XM,La Russo NF.Mechanisms of attachment and internalization of Cryptosporidium parvum to biliary and intestinal epithelial cells.Gastroenterology,2000,118(2):368-379.
[29]Zhang Z,Duchêne M,Stanley SL Jr.A monoclonal antibody to the amebic lipophosphoglycanproteophosphoglycan antigens can prevent disease in human intestinal xenografts infected with Entamoeba histolytica.Infect Immun,2002,70(10):5873-5876.
[30]Peek R,Delbac F,Speijer D,et al.Carbohydrate moieties of microsporidian polar tube proteins are targeted by immunoglobulin G in immunocompetent individuals.Infect Immun,2005,73(12):7906-7913.
[31]Polonais V,Prensier G,Méténier G,et al.Microsporidian polar tube proteins:highly divergent but closely linked genes encode PTP1 and PTP2 in members of the evolutionarily distant Antonospora and Encephalitozoon groups.Fungal Genet Biol,2005,42(9):791-803.
[32]Lin LP,Pan GQ,Li T,et al.The protein import pore Tom40 in the microsporidian Nosema bombycis.JEukaryot Microbiol,2012,59(3):251-257.
[33]Yamashita A,Nango E,Ashikawa Y.A large-scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand-binding domains of taste receptor.Protein Sci,2017,26(11):2291-2301.
[34]Ailor E,Pathmanathan J,Jongbloed JDH,et al.Abacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells.Biochem Biophys Res Commun,1999,255:444-450.
[35]Tessier DC,Thomas DY,Khouri HE,et al.Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide.Gene,1991,98(2):177-183.
[36]Jarvis DL,Summers MD,Garcia A Jr,et al.Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system.J Biol Chem,1993,268(22):16754-16762.
[37]Shen X,Dojcinovic D,Baldi L,et al.Improved process conditions for increasing expression of MHC class IIprotein from a stable Drosophila S2 cell line.Biotechnol Lett,2018,40(1):85-92.
[38]Zitzmann J,Weidner T,Czermak P.Optimized expression of the antimicrobial protein Gloverin from Galleria mellonella using stably transformed Drosophila melanogaster S2 cells.Cytotechnology,2017,69(2):371-389.