鼠隐藏管状线虫烯醇化酶基因的克隆与序列分析
|
摘要
为获得鼠隐藏管状线虫(Syphacia obvelata)烯醇化酶结构及其功能,采用同源克隆方法结合RACE技术克隆获得了烯醇化酶基因c DNA序列,并对其进行了序列分析。测序结果显示,扩增获得的鼠隐藏管状线虫烯醇化酶基因大小序列为1603 bp,包含全长为1311 bp的开放阅读框(open reading frame,ORF)序列,共编码436个氨基酸,推导分子量为47.428 k Da。序列分析结果显示,鼠隐藏管状线虫烯醇化酶含有10个丝氨酸激酶磷酸化位点、3个苏氨酸激酶磷酸化位点和4个酪氨酸激酶磷酸化位点,1个潜在的跨膜螺旋结构,无信号肽剪切位点;在亚细胞水平,预测其主要定位于胞浆;二级结构主要以α-螺旋和无规则卷曲为主,其中α-螺旋占34.86%,无规则卷曲占30.50%,延伸链占24.08%,β-转角占10.55%。Swiss-Model模建的3D结构显示,鼠隐藏管状线虫烯醇化酶为一"哑铃"样结构,包含氨基端和羧基端两个结构域,结构域之间为Linker区。每个结构域由多个α-螺旋、β-折叠和卷曲所构成桶形结构,催化中心和Mg~(2+)结合位点位于桶形结构的中心。本研究结果为鼠隐藏管状线虫烯醇化酶基因的功能研究奠定基础。
To learn the structure and function of the enolase gene of Syphacia obvelata, we first obtained the full-length c DNA of enolase by using homology-based cloning together with RACE technology. The c DNA sequence was then analyzed by bioinformatics databases and software. The full-length c DNA of enolase was 1611 bp in length, with a coding region of 1311 bp. This sequence encoded a protein of 436 amino acids with a molecular weight of 47.428 k Da. The enolase contained ten serine phosphorylation sites,three threonine phosphorylation sites,four tyrosine phosphorylation sites and a potential transmembrane helices region, but no signal peptide was found in the protein. At the subcellular level, the enolase protein was mainly localized to the cytoplasm, peroxisome, mitochondrial matrix space and lysosome. The secondary structure of enolase was mainly composed of alpha helix, consisting of 34.86% helix, 30.50% random coil, 24.08% extended strand, and 10.55% beta turn. Swiss-Model modulated predicted that 3D structure was like dumbbell with a linker in the middle, each substrate unit was comprised of alternatively arranged α helix-β sheet and forms a barrel, the active sites, Mg~(2+) binding sites closely located in the center of barrel. By cloning the enolase gene and predicting its structure, character and functions,we have obtained valuable information which can be used to study the relationship between parasite and host in the future.
To learn the structure and function of the enolase gene of Syphacia obvelata, we first obtained the full-length c DNA of enolase by using homology-based cloning together with RACE technology. The c DNA sequence was then analyzed by bioinformatics databases and software. The full-length c DNA of enolase was 1611 bp in length, with a coding region of 1311 bp. This sequence encoded a protein of 436 amino acids with a molecular weight of 47.428 k Da. The enolase contained ten serine phosphorylation sites,three threonine phosphorylation sites,four tyrosine phosphorylation sites and a potential transmembrane helices region, but no signal peptide was found in the protein. At the subcellular level, the enolase protein was mainly localized to the cytoplasm, peroxisome, mitochondrial matrix space and lysosome. The secondary structure of enolase was mainly composed of alpha helix, consisting of 34.86% helix, 30.50% random coil, 24.08% extended strand, and 10.55% beta turn. Swiss-Model modulated predicted that 3D structure was like dumbbell with a linker in the middle, each substrate unit was comprised of alternatively arranged α helix-β sheet and forms a barrel, the active sites, Mg~(2+) binding sites closely located in the center of barrel. By cloning the enolase gene and predicting its structure, character and functions,we have obtained valuable information which can be used to study the relationship between parasite and host in the future.
引文
[1]Bazzano T,Restel T I,Pinto R M,et al.Patterns of infection with the nematodes Syphacia obvelata and Aspicularis tetraptera in conventionaly maintained laboratory mice[J].Mem Inst Oswaldo Cruz,2002,97(6):847-853.
[2]Carneiro C R,Postol E,Nomizo R,et al.Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus[J].Microbes Infec,2004,6(6):604-608.
[3]Pérez Sánchez R,Valero M L,Ramajo Hernández A,et al.A proteomic approach to the identification of tegumental proteins of male and female Schistosoma bovis worms[J].Mol Biochem Parasitol,2008,161(2):112-123.
[4]Wang X,Chen W,Hu F,et al.Clonorchis sinensisenolase:identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products[J].Mol Biochem Parasitol,2011,177(2):135-142.
[5]Wang X,Chen W,Tian Y,et al.RNAi-mediated silencing of enolase confirms its biological importance in Clonorchis sinensis[J].Parasitol Res,2014,113(4):1451-1458.
[6]Chen N,Yuan Z G,Xu M J,et al.Ascaris suum Enolase is a potential vaccine candidate against Ascarisis in mice[J].Vaccine,2012,30(23):3478-3482.
[7]Ghosh A K,Marcelo J L.Surface-expressed enolases of Plasmodium and other pathogens[J].Mem Inst Oswaldo Cruz,2011,106(01):85-90.
[8]Pal-Bhowmick I,Mehta M,Coppens I,et al.Protective properties and surface localization of Plasmodium falciparum enolase[J].Infect Immun,2007,75(11):5500-8.
[9]Ruan J,Mouveaux T,Light S H,et al.The structure of bradyzoite-specific enolase from Toxoplasma gondiireveals insights into its dual cytoplasmic and nuclear functions[J].Acta Crystallogr D Biol Crystallogr,2015,71(Pt 3):417-426.
[10]Avilán L,Gualdrón-López M,Qui?ones W,et al.Enolase:a key player in the metabolism and a probable virulence factor of trypanosomatid parasites-perspectives for its use as a therapeutic target[J].Enzyme Res,2011,932549:1-14.
[11]Zhang J,Yu C,Wang Y,et al.Enolase of Angiostrongylus cantonensis:more likely a structural component[J]?Parasitol Res,2014,113(11):3927-3934.
[12]Han K,Xu L,Yan R,et al.Molecular cloning,expression and characterization of enolase from adult Haemonchus contortus[J].Res Vet Sci,2012,92(2):259-265.
[13]Gan W,Zhao G,Xu H,et al.Reverse vaccinology approach identify an Echinococcus granulosus tegumental membrane protein enolase as vaccine candidate[J].Parasitol Res,2010,106(4):873-882.
[14]戴佳琳,黄江,廖兴江.生物信息学分析牛带绦虫烯醇酶基因及其蛋白的结构和特性[J].热带医学杂志,2010,10(10):1169-1172.
[15]黄艳,黄江,胡旭初,等.亚洲带绦虫烯醇酶基因及其蛋白质的结构与功能[J].中山大学学报(医学科学版),2008,29(5):575-580.
[16]邱春辉.日本血吸虫重组烯醇化酶研究[D].福建:福建农林大学,2009.
[17]杜春娟,朱云平,贺福初,等.蛋白质家族模体(motif)的评价策略[J].北京生物医学工程,2005(2):19-24
[18]Salzillo M,Vastano V,Capri U,et al.Identification and characterization of enolase as a collagen-binding protein in Lactobacillus plantarum[J].J Basic Microbiol,2015,55(7):890-897.
[19]Agarwal S,Kulshreshtha P,Bambah Mukku D,et al.alpha-Enolase binds to human plasminogen on the surface of Bacillus anthracis[J].Biochim Biophys Acta,2008,1784(7-8):986-994.
[20]Qui?ones W,Pe?a P,Domingo-Sananes M,et al.Leishmania mexicana:molecular cloning and characterization of enolase[J].Exp Parasitol,2007,116(3):241-251.
[21]Andrade M A,Siles-Lucas M,López-Abán J,et al.Antigens from Ascaris suum trigger in vitro macrophage NO production[J].Parasite Immunol,2005,27(6):235-242.
[22]Pancholi V.Multifunctional a-enolase:its role in diseases[J].Cell Mol Life Sci,2001,(58):902-920.
[2]Carneiro C R,Postol E,Nomizo R,et al.Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus[J].Microbes Infec,2004,6(6):604-608.
[3]Pérez Sánchez R,Valero M L,Ramajo Hernández A,et al.A proteomic approach to the identification of tegumental proteins of male and female Schistosoma bovis worms[J].Mol Biochem Parasitol,2008,161(2):112-123.
[4]Wang X,Chen W,Hu F,et al.Clonorchis sinensisenolase:identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products[J].Mol Biochem Parasitol,2011,177(2):135-142.
[5]Wang X,Chen W,Tian Y,et al.RNAi-mediated silencing of enolase confirms its biological importance in Clonorchis sinensis[J].Parasitol Res,2014,113(4):1451-1458.
[6]Chen N,Yuan Z G,Xu M J,et al.Ascaris suum Enolase is a potential vaccine candidate against Ascarisis in mice[J].Vaccine,2012,30(23):3478-3482.
[7]Ghosh A K,Marcelo J L.Surface-expressed enolases of Plasmodium and other pathogens[J].Mem Inst Oswaldo Cruz,2011,106(01):85-90.
[8]Pal-Bhowmick I,Mehta M,Coppens I,et al.Protective properties and surface localization of Plasmodium falciparum enolase[J].Infect Immun,2007,75(11):5500-8.
[9]Ruan J,Mouveaux T,Light S H,et al.The structure of bradyzoite-specific enolase from Toxoplasma gondiireveals insights into its dual cytoplasmic and nuclear functions[J].Acta Crystallogr D Biol Crystallogr,2015,71(Pt 3):417-426.
[10]Avilán L,Gualdrón-López M,Qui?ones W,et al.Enolase:a key player in the metabolism and a probable virulence factor of trypanosomatid parasites-perspectives for its use as a therapeutic target[J].Enzyme Res,2011,932549:1-14.
[11]Zhang J,Yu C,Wang Y,et al.Enolase of Angiostrongylus cantonensis:more likely a structural component[J]?Parasitol Res,2014,113(11):3927-3934.
[12]Han K,Xu L,Yan R,et al.Molecular cloning,expression and characterization of enolase from adult Haemonchus contortus[J].Res Vet Sci,2012,92(2):259-265.
[13]Gan W,Zhao G,Xu H,et al.Reverse vaccinology approach identify an Echinococcus granulosus tegumental membrane protein enolase as vaccine candidate[J].Parasitol Res,2010,106(4):873-882.
[14]戴佳琳,黄江,廖兴江.生物信息学分析牛带绦虫烯醇酶基因及其蛋白的结构和特性[J].热带医学杂志,2010,10(10):1169-1172.
[15]黄艳,黄江,胡旭初,等.亚洲带绦虫烯醇酶基因及其蛋白质的结构与功能[J].中山大学学报(医学科学版),2008,29(5):575-580.
[16]邱春辉.日本血吸虫重组烯醇化酶研究[D].福建:福建农林大学,2009.
[17]杜春娟,朱云平,贺福初,等.蛋白质家族模体(motif)的评价策略[J].北京生物医学工程,2005(2):19-24
[18]Salzillo M,Vastano V,Capri U,et al.Identification and characterization of enolase as a collagen-binding protein in Lactobacillus plantarum[J].J Basic Microbiol,2015,55(7):890-897.
[19]Agarwal S,Kulshreshtha P,Bambah Mukku D,et al.alpha-Enolase binds to human plasminogen on the surface of Bacillus anthracis[J].Biochim Biophys Acta,2008,1784(7-8):986-994.
[20]Qui?ones W,Pe?a P,Domingo-Sananes M,et al.Leishmania mexicana:molecular cloning and characterization of enolase[J].Exp Parasitol,2007,116(3):241-251.
[21]Andrade M A,Siles-Lucas M,López-Abán J,et al.Antigens from Ascaris suum trigger in vitro macrophage NO production[J].Parasite Immunol,2005,27(6):235-242.
[22]Pancholi V.Multifunctional a-enolase:its role in diseases[J].Cell Mol Life Sci,2001,(58):902-920.