鲫葡萄糖调节蛋白78基因部分序列的克隆、鉴定及功能预测
|
摘要
【目的】分析和预测鲫(Carassius auratus)葡萄糖调节蛋白78(Glucose regulated protein 78,GRP78)的功能,为研究GRP78在鱼类受细胞外刺激过程中的作用机制奠定基础。【方法】通过分子克隆技术获得鲫GRP78基因的部分序列,并利用生物信息学方法分析了该基因片段编码的蛋白质序列的二级结构、亲水性、可塑性、表面可及性和抗原表位。【结果】获得了长度为617bp的鲫GRP78基因片段序列,共编码113个氨基酸。这些氨基酸所构成序列的相对分子质量约为1.3kDa,等电点为5.82,无信号肽;可塑性区域位于第7～10位、第18～21位、第28～42位、第51～62位、第68～78位和第94～109位氨基酸残基;表面可及性区域位于第29～30位、第38～43位、第50～65位、第68～79位、第82～86位、第97～99位、第102～104位和第106～111位氨基酸残基;有1个B细胞抗原表位区域,位于第28～45位氨基酸残基。【结论】所获的鲫GRP78序列中含有ATPase保守结构域区段,该区段定位于内质网,具有较强亲水性。
[Purposes]To analyze and predict the function of glucose regulated protein 78(GRP78)in Carassius auratus and provide a foundation for studying the mechanism of GRP78 during the process of extracellular stress in fish.[Methods]A partial sequence of GRP78 gene in C.auratus was cloned,whose physicochemical property including secondary structures,hydrophilicity,flexibility,surface probability,antigen epitope,was analyzed by bioinformatics methods.[Findings]The GRP78 gene fragment sequence with long 617 bp was obtained.The sequence encodes a total of 113 amino acids with molecular weight about 1.3 kDa and pI 5.82.None signal peptide was existed.The flexibility was located at 7 th～10 th,18 th～21 st,28 th～42 nd,51 st～62 nd,68 th～78 th,and 94 th～109 th amino acid residues;the surface probability was placed at 29 th～30 th,38 th～43 rd,50 th～65 th,68 th～79 th,82 nd～86 th,97 th～99 th,102 nd～104 th,and 106 th～111 th amino acid residues.Additionally,one B cell epitope was predicted as 28 th～45 th amino acids residues.[Conclusions]ATPase domain was obtained in the C.auratus GRP78 gene fragment with higher hydrophilicity,which is located in ER.
[Purposes]To analyze and predict the function of glucose regulated protein 78(GRP78)in Carassius auratus and provide a foundation for studying the mechanism of GRP78 during the process of extracellular stress in fish.[Methods]A partial sequence of GRP78 gene in C.auratus was cloned,whose physicochemical property including secondary structures,hydrophilicity,flexibility,surface probability,antigen epitope,was analyzed by bioinformatics methods.[Findings]The GRP78 gene fragment sequence with long 617 bp was obtained.The sequence encodes a total of 113 amino acids with molecular weight about 1.3 kDa and pI 5.82.None signal peptide was existed.The flexibility was located at 7 th～10 th,18 th～21 st,28 th～42 nd,51 st～62 nd,68 th～78 th,and 94 th～109 th amino acid residues;the surface probability was placed at 29 th～30 th,38 th～43 rd,50 th～65 th,68 th～79 th,82 nd～86 th,97 th～99 th,102 nd～104 th,and 106 th～111 th amino acid residues.Additionally,one B cell epitope was predicted as 28 th～45 th amino acids residues.[Conclusions]ATPase domain was obtained in the C.auratus GRP78 gene fragment with higher hydrophilicity,which is located in ER.
引文
[1]LEE A S.The glucose-regulated proteins:stress induction and clinical applications[J].Trends in Biochemical Sciences,2001,26(8):504-510.
[2]JI C,KAPLOWITZ N,LAU M Y,et al.Liver-specific loss of glucose-regulated protein 78perturbs the unfolded protein response and exacerbates a spectrum of liver diseases in mice[J].Hepatology,2011,54(1):229-239.
[3]HENDERSHOT L M.The ER function BiP is a master regulator of ER function[J].Mount Sinai Journal of Medicine,2004,71(5):289-297.
[4]QUINONES Q J,de RIDDER G G,PIZZO S V.GRP78:a chaperone with diverse roles beyond the endoplasmic reticulum[J].Histology&Histopathology,2008,23(11):1409-1416.
[5]DAISUKE M,KAZUHIRO N.ER stress proteins in autoimmune and inflammatory diseases[EB/OL].(2012-03-15)[2018-07-09].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342303/.
[6]ZHANG Y,LIU R,NI M,et a1.Cell surface relocalization of the endoplasmic reticulum chaperone and unfolded protein response regulator GRP78/BiP[J].Journal of Biological Chemistry,2010,285(20):15065-15075.
[7]YU Z,LUO H,FU W,et al.The endoplasmic reticulum stress-responsive protein GRP78protects neurons against excitotoxicity and apoptosis:suppression of oxidative stress and stabilization of calcium homeostasis[J].Experimental Neurology,1999,155(2):302-314.
[8]PFAFFENBACH K T,LEEAS.ThecriticalroleofGRP78in physiologic and pathologic stress[J].Current Opinion in Cell Biology,2011,23(2):150-156.
[9]DEY A,KESSOVA I G,CEDERBAUM A I.Decreased protein and mRNA expression of ER stress proteins GRP78and GRP94in HepG2cells over-expressing CYP2E1[J].Archives of Biochemistry Biophysics,2006,447(2):155-166.
[10]WU C T,WANG W C,CHEN M F,et al.Glucose-regulated protein 78 mediates hormone-independent prostate cancer progression and metastasis through maspin and COX-2expression[J].Tumor Biology,2014,35(1):195-204.
[11]GONICK H C.Lead-binding proteins:a review[J].Journal of Toxicology,2011,2011(2011):686050.
[12]ZHU Y,FAN Q,MAO H,et al.GRP78from grass carp(Ctenopharyngodon idella)provides cytoplasm protection against thermal and Pb2+stress[J].Fish&Shellfish Immunology,2013,34(2):617-622.
[13]OJIMA N,YAMASHITA M,WATABE S.Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells[J].BiochemicalBiophysical Research Communications,2005,329(1):51-57.
[14]吴初新,刘毅,马梅生,等.热休克、Poly I:C上调草鱼GRP78基因表达[J].应用与环境生物学报,2009,15(6):814-818.WU C X,LIU Y,MA M S,et al.Up-regulation of grass carp GRP78 gene expression under heat shock and poly I:C stress[J].Chinese Journal of Applied&Environmental Biology,2009,15(6):814-818.
[15]DAS S,MOHAPATRA A,SAHOO P K.Expression analysis of heat shock protein genes during Aeromonas hydrophilainfection in rohu,Labeo rohita,with special reference to molecular characterization of Grp78[J].Cell Stress and Chaperones,2015,20(1):73-84.
[16]ZHONG B,WANG X,MAO H,et al.A mechanism underlies fish GRP78protection against Pb2+toxicity[J].Fish&Shellfish Immunology,2017,66:185-188.
[17]ZHONG B,MAO H,FAN Q,et al.SiRNA-mediated knockdown of CiGRP78 gene expression leads cell susceptibility to heavy metal cytotoxicity[J].Gene,2014,552(2):219-224.
[18]SONG Y F,LUO Z,HUANG C,et al.Endoplasmic reticulum stress-related genes in yellow catfish Pelteobagrus fulvidraco:molecular characterization,tissue expression,and expression responses to dietary copper deficiency and excess[J].G3Genesgenetics,2015,5(10):2091-2104.
[19]WHIPPS C M,KENT M L.Phylogeography of the cosmopolitan marine parasite Kudoa thyrsites(Myxozoa:Myxosporea)[J].JournalofEukaryotic Microbiology,2006,53(5):364-373.
[20]刘晓聪,赵元莙,张金叶.瓶囊碘泡虫HSP70基因的克隆、鉴定及功能预测[J].重庆师范大学学报(自然科学版),2016,33(2):37-42.LIU X C,ZHAO Y J,ZHANG J Y.Cloning,identification and functional prediction of heat shock protein 70gene in Myxobolus ampullicapsulatus[J].Journal of Chongqing Normal University(Natural Science),2016,33(2):37-42.
[21]陈四妙,陈晓峰,杨成东等.稻瘟病菌分泌复合物的生物信息学分析及MoSec15定位研究[J].福建农林大学学报(自然科学版),2014,43(3):282-288.CHEN S M,CHEN X F,YANG C D,et al.Bioinformatics analysis of exocyst complex and the subcellular localization of MoSec15in Magnaporthe oryzae[J].JournalofFujian Agriculture and Forestry University(Natural Science Edition),2014,43(3):282-288.
[22]GARNIER J,OSGUTHORPE D J,ROBSON B.Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins[J].Journal of Molecular Biology,1978,120(1):97-120.
[23]CHOU P Y,FASMAN G D.Prediction of the secondary structure of protein conformation[M].New York:Plenum Press,1990:549-586.
[24]KYTE J,DOOLITTLE R F.A simple method for displaying the hydropathic character of a protein[J].Journal of Molecular Biology,1982,157(1):105-132.
[25]KARPLUS P A,SCHULZ G E.Prediction of chain flexibility in proteins[J].Naturwissenschaften,1985,72(4):212-213.
[26]JAMESON B A,WOLF H.The antigenic index:a novel algorithm for predicting antigenic determinants[J].Computer Applications in the Biosciences Cabios,1988,4(1):181.
[27]EMINI E A,HUGHES J V,PERLOW D S,et al.Induction of hepatitis A virus-neutralizing antibody by a virusspecific synthetic peptide[J].Journal of Virology,1985,55(3):836-839.
[28]Reddy R K,Mao C,Baumeister P,et al.Endoplasmic reticulum chaperone protein GRP78protects cells from apoptosis induced by topoisomerase inhibitors:role of ATPbinding site in suppression of caspase-7activation[J].Journal of Biological Chemistry,2003,278(23):20915-20924.
[29]李波,陈誉华.内质网分子伴侣GRP78在小鼠脑发育过程中的时空表达[J].实验生物学报,2003,36(2):137-144.LI B,CHEN Y H.The expression of glucose-regulated protein 78(GRP78)in mouse brain during early development[J].Acta Biologiae Experimentalis Sinica,2003,36(2):137-144.
[30]GAUT J R,HENDERSHOT L M.Mutations within the nucleotide binding site of immunoglobulin-binding protein inhibit ATPase activity and interfere with release of immunoglobulin heavy chain[J].Journal of Biological Chemistry,1993,268(10):7248-7255.
[31]LU M W,NGOU F H,CHAO Y M,et al.Transcriptome characterization and gene expression of Epinephelus spp in endoplasmic reticulum stress-related pathway during betanodavirus infection in vitro[J].Bmc Genomics,2012,13(1):651-651.
[32]CHA I S,KWON J,PARK S B,et al.Heat shock protein profiles on the protein and gene expression levels in olive flounder kidney infected with Streptococcus parauberis[J].Fish&ShellfishImmunology,2013,34(6):1455-1462.
[33]XI X Z,MA K S.Molecular cloning and expression analysis of glucose-regulated protein 78(GRP78)gene in silkworm Bombyx mori[J].Biologia,2013,68(3):559-564.
[34]高玒,余传信,宋丽君,等.日本血吸虫热休克蛋白70(Sj HSP70)的抗体反应特征及免疫诊断价值[J].中国病原生物学杂志,2014,9(8):699-705.GAO W,YU C X,SONG L J,et al.Characterization of the antibody response to heat shock protein 70(Sj HSP70)of Schistosoma japonicum and the immunological diagnostic value of that protein[J].Journal of Pathogen Biology,2014,9(8):699-705.
[2]JI C,KAPLOWITZ N,LAU M Y,et al.Liver-specific loss of glucose-regulated protein 78perturbs the unfolded protein response and exacerbates a spectrum of liver diseases in mice[J].Hepatology,2011,54(1):229-239.
[3]HENDERSHOT L M.The ER function BiP is a master regulator of ER function[J].Mount Sinai Journal of Medicine,2004,71(5):289-297.
[4]QUINONES Q J,de RIDDER G G,PIZZO S V.GRP78:a chaperone with diverse roles beyond the endoplasmic reticulum[J].Histology&Histopathology,2008,23(11):1409-1416.
[5]DAISUKE M,KAZUHIRO N.ER stress proteins in autoimmune and inflammatory diseases[EB/OL].(2012-03-15)[2018-07-09].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342303/.
[6]ZHANG Y,LIU R,NI M,et a1.Cell surface relocalization of the endoplasmic reticulum chaperone and unfolded protein response regulator GRP78/BiP[J].Journal of Biological Chemistry,2010,285(20):15065-15075.
[7]YU Z,LUO H,FU W,et al.The endoplasmic reticulum stress-responsive protein GRP78protects neurons against excitotoxicity and apoptosis:suppression of oxidative stress and stabilization of calcium homeostasis[J].Experimental Neurology,1999,155(2):302-314.
[8]PFAFFENBACH K T,LEEAS.ThecriticalroleofGRP78in physiologic and pathologic stress[J].Current Opinion in Cell Biology,2011,23(2):150-156.
[9]DEY A,KESSOVA I G,CEDERBAUM A I.Decreased protein and mRNA expression of ER stress proteins GRP78and GRP94in HepG2cells over-expressing CYP2E1[J].Archives of Biochemistry Biophysics,2006,447(2):155-166.
[10]WU C T,WANG W C,CHEN M F,et al.Glucose-regulated protein 78 mediates hormone-independent prostate cancer progression and metastasis through maspin and COX-2expression[J].Tumor Biology,2014,35(1):195-204.
[11]GONICK H C.Lead-binding proteins:a review[J].Journal of Toxicology,2011,2011(2011):686050.
[12]ZHU Y,FAN Q,MAO H,et al.GRP78from grass carp(Ctenopharyngodon idella)provides cytoplasm protection against thermal and Pb2+stress[J].Fish&Shellfish Immunology,2013,34(2):617-622.
[13]OJIMA N,YAMASHITA M,WATABE S.Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells[J].BiochemicalBiophysical Research Communications,2005,329(1):51-57.
[14]吴初新,刘毅,马梅生,等.热休克、Poly I:C上调草鱼GRP78基因表达[J].应用与环境生物学报,2009,15(6):814-818.WU C X,LIU Y,MA M S,et al.Up-regulation of grass carp GRP78 gene expression under heat shock and poly I:C stress[J].Chinese Journal of Applied&Environmental Biology,2009,15(6):814-818.
[15]DAS S,MOHAPATRA A,SAHOO P K.Expression analysis of heat shock protein genes during Aeromonas hydrophilainfection in rohu,Labeo rohita,with special reference to molecular characterization of Grp78[J].Cell Stress and Chaperones,2015,20(1):73-84.
[16]ZHONG B,WANG X,MAO H,et al.A mechanism underlies fish GRP78protection against Pb2+toxicity[J].Fish&Shellfish Immunology,2017,66:185-188.
[17]ZHONG B,MAO H,FAN Q,et al.SiRNA-mediated knockdown of CiGRP78 gene expression leads cell susceptibility to heavy metal cytotoxicity[J].Gene,2014,552(2):219-224.
[18]SONG Y F,LUO Z,HUANG C,et al.Endoplasmic reticulum stress-related genes in yellow catfish Pelteobagrus fulvidraco:molecular characterization,tissue expression,and expression responses to dietary copper deficiency and excess[J].G3Genesgenetics,2015,5(10):2091-2104.
[19]WHIPPS C M,KENT M L.Phylogeography of the cosmopolitan marine parasite Kudoa thyrsites(Myxozoa:Myxosporea)[J].JournalofEukaryotic Microbiology,2006,53(5):364-373.
[20]刘晓聪,赵元莙,张金叶.瓶囊碘泡虫HSP70基因的克隆、鉴定及功能预测[J].重庆师范大学学报(自然科学版),2016,33(2):37-42.LIU X C,ZHAO Y J,ZHANG J Y.Cloning,identification and functional prediction of heat shock protein 70gene in Myxobolus ampullicapsulatus[J].Journal of Chongqing Normal University(Natural Science),2016,33(2):37-42.
[21]陈四妙,陈晓峰,杨成东等.稻瘟病菌分泌复合物的生物信息学分析及MoSec15定位研究[J].福建农林大学学报(自然科学版),2014,43(3):282-288.CHEN S M,CHEN X F,YANG C D,et al.Bioinformatics analysis of exocyst complex and the subcellular localization of MoSec15in Magnaporthe oryzae[J].JournalofFujian Agriculture and Forestry University(Natural Science Edition),2014,43(3):282-288.
[22]GARNIER J,OSGUTHORPE D J,ROBSON B.Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins[J].Journal of Molecular Biology,1978,120(1):97-120.
[23]CHOU P Y,FASMAN G D.Prediction of the secondary structure of protein conformation[M].New York:Plenum Press,1990:549-586.
[24]KYTE J,DOOLITTLE R F.A simple method for displaying the hydropathic character of a protein[J].Journal of Molecular Biology,1982,157(1):105-132.
[25]KARPLUS P A,SCHULZ G E.Prediction of chain flexibility in proteins[J].Naturwissenschaften,1985,72(4):212-213.
[26]JAMESON B A,WOLF H.The antigenic index:a novel algorithm for predicting antigenic determinants[J].Computer Applications in the Biosciences Cabios,1988,4(1):181.
[27]EMINI E A,HUGHES J V,PERLOW D S,et al.Induction of hepatitis A virus-neutralizing antibody by a virusspecific synthetic peptide[J].Journal of Virology,1985,55(3):836-839.
[28]Reddy R K,Mao C,Baumeister P,et al.Endoplasmic reticulum chaperone protein GRP78protects cells from apoptosis induced by topoisomerase inhibitors:role of ATPbinding site in suppression of caspase-7activation[J].Journal of Biological Chemistry,2003,278(23):20915-20924.
[29]李波,陈誉华.内质网分子伴侣GRP78在小鼠脑发育过程中的时空表达[J].实验生物学报,2003,36(2):137-144.LI B,CHEN Y H.The expression of glucose-regulated protein 78(GRP78)in mouse brain during early development[J].Acta Biologiae Experimentalis Sinica,2003,36(2):137-144.
[30]GAUT J R,HENDERSHOT L M.Mutations within the nucleotide binding site of immunoglobulin-binding protein inhibit ATPase activity and interfere with release of immunoglobulin heavy chain[J].Journal of Biological Chemistry,1993,268(10):7248-7255.
[31]LU M W,NGOU F H,CHAO Y M,et al.Transcriptome characterization and gene expression of Epinephelus spp in endoplasmic reticulum stress-related pathway during betanodavirus infection in vitro[J].Bmc Genomics,2012,13(1):651-651.
[32]CHA I S,KWON J,PARK S B,et al.Heat shock protein profiles on the protein and gene expression levels in olive flounder kidney infected with Streptococcus parauberis[J].Fish&ShellfishImmunology,2013,34(6):1455-1462.
[33]XI X Z,MA K S.Molecular cloning and expression analysis of glucose-regulated protein 78(GRP78)gene in silkworm Bombyx mori[J].Biologia,2013,68(3):559-564.
[34]高玒,余传信,宋丽君,等.日本血吸虫热休克蛋白70(Sj HSP70)的抗体反应特征及免疫诊断价值[J].中国病原生物学杂志,2014,9(8):699-705.GAO W,YU C X,SONG L J,et al.Characterization of the antibody response to heat shock protein 70(Sj HSP70)of Schistosoma japonicum and the immunological diagnostic value of that protein[J].Journal of Pathogen Biology,2014,9(8):699-705.