版纳微型猪近交系PPP2CA克隆、表达及蛋白质功能生物信息学分析
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摘要
蛋白磷酸酶2A(protein phosphatases of the type 2A, PP2A)是一种广泛表达的丝氨酸/苏氨酸磷酸酶,介导蛋白质的去磷酸化从而调控多种关键信号通路的活性。该酶由催化亚基、结构亚基、调节亚基形成异源三聚体复合物发挥作用。本研究借助GenBank中猪(Sus scrofa)及其他物种的蛋白磷酸酶2A催化亚基α(protein phosphatase 2A catalytic subunitα, PPP2CA) mRNA序列,设计特异引物扩增版纳微型猪近交系(Banna mini-pig inbred line, BMI)PPP2CA基因编码区序列,并对其进行表达和功能特性分析。应用qRT-PCR分析组织mRNA表达谱,并对编码的蛋白质序列进行生物信息学分析。扩增得到了BMI PPP2CA 930 bp (GenBank No. KU705627)的完整CDS序列,共编码309个氨基酸。与其他组织相比PPP2CA基因在尿道球腺和精囊腺中极显著高表达(P<0.01),在睾丸中表达量也相对较高,在肝、结肠、脾、肺、十二指肠、前列腺、肾、附睾及脑中中度表达;在胃、心、肌肉中表达水平极显著低于其他组织。PPP2CA蛋白质功能预测表明其存在1个保守结构域MPP_PP2A_PP4_PP6,存在4类功能活性位点,无跨膜螺旋结构,无信号肽序列,N末端和C末端均亲水,位于细胞质的概率是94.1%。二级结构预测表明该蛋白α-螺旋含量最高,是组成N端的主要结构,C端有一段无规则卷曲,多物种氨基酸序列比对结果显示,PPP2CA序列在不同物种间相似度很高,推测其在进化中高度保守,为深入研究PPP2CA基因在猪精子获能方面作用机制提供了资料基础。
Protein phosphatase 2 A(PP2A) is a ubiquitously expressed serine-threonine phosphatase mediating protein dephosphorylation to regulate the activities of many key pathways. PP2A is a heterotrimeric including catalytic subunit, structural subunit and regulatory subunit. To clone the coding sequence and analyze the character of expression and function of protein phosphatase 2 A catalytic subunit α gene(PPP2 CA), the specific primers of PPP2 CA gene of Banna mini-pig(Sus scrofa) inbred line(BMI) was designed and the coding sequence was amplified using the PPP2 CA mRNA sequences of pig and other species from GenBank as reference sequences. qRT-PCR was used to detect expression profiles of tissues mRNA. At the same time, the amino acid sequences were analysed by functional bioinformatics. A complete coding sequence of 930 bp of BMI PPP2 CA was obtained, which encoded 309 amino acids. Comparing with other tissues, the PPP2 CA gene expression in the urethral ball glands and seminal vesicle appeared extremely significance(P<0.01). The expression in the testis was higher than other tissues. It was expressed moderately in the liver, colon, spleen, lung, duodenum, prostate, kidney, epididymis and brain, weakly in the stomach,heart and muscle. Functional prediction of PPP2 CA protein indicated that the PPP2 CA contained one conserved domain MPP_PP2A_PP4_PP6 and four kinds functional active sites, without transmembrane helix and signal peptide. The N-terminal and C-terminal were hydrophilic and located in cytoplasmic with 94.1%. It was predicted that the α helices content of the second structure were the highest which were the major structure of the N terminal, while the random coils were the major structure of the C terminal. The high similarity of PPP2 CA amino acids alignment between BMI and other species indicated that this gene was highly conservative in evolution. This research can lay a further foundation for clarifying mechanism of the PPP2 CA gene on sperm capacitation.
Protein phosphatase 2 A(PP2A) is a ubiquitously expressed serine-threonine phosphatase mediating protein dephosphorylation to regulate the activities of many key pathways. PP2A is a heterotrimeric including catalytic subunit, structural subunit and regulatory subunit. To clone the coding sequence and analyze the character of expression and function of protein phosphatase 2 A catalytic subunit α gene(PPP2 CA), the specific primers of PPP2 CA gene of Banna mini-pig(Sus scrofa) inbred line(BMI) was designed and the coding sequence was amplified using the PPP2 CA mRNA sequences of pig and other species from GenBank as reference sequences. qRT-PCR was used to detect expression profiles of tissues mRNA. At the same time, the amino acid sequences were analysed by functional bioinformatics. A complete coding sequence of 930 bp of BMI PPP2 CA was obtained, which encoded 309 amino acids. Comparing with other tissues, the PPP2 CA gene expression in the urethral ball glands and seminal vesicle appeared extremely significance(P<0.01). The expression in the testis was higher than other tissues. It was expressed moderately in the liver, colon, spleen, lung, duodenum, prostate, kidney, epididymis and brain, weakly in the stomach,heart and muscle. Functional prediction of PPP2 CA protein indicated that the PPP2 CA contained one conserved domain MPP_PP2A_PP4_PP6 and four kinds functional active sites, without transmembrane helix and signal peptide. The N-terminal and C-terminal were hydrophilic and located in cytoplasmic with 94.1%. It was predicted that the α helices content of the second structure were the highest which were the major structure of the N terminal, while the random coils were the major structure of the C terminal. The high similarity of PPP2 CA amino acids alignment between BMI and other species indicated that this gene was highly conservative in evolution. This research can lay a further foundation for clarifying mechanism of the PPP2 CA gene on sperm capacitation.
引文
任亮,付伟,金素钰,等.2017.牦牛和雄性不育犏牛睾丸PPP2CA、PME-1基因mRNA水平的比较[J].四川动物,36(3):306-310.(Ren L,Fu W,Jin S Y,et al.2017.Comparison of m rna levels of PPP2CA and PME-1genes in the testes of yaks and sterile cattle-yaks[J].Sichuan Journal of Zoology,36(3):306-310.)
王镜岩,朱圣庚,徐长法.2002.生物化学[M].第三版.北京:高等教育出版社,207-220.(Wang J Y,Zhu S G,Xu C F.2002.Biochemistry[M].Higher Education Press,Beijing.207-220.)
张霞,曾日彬,霍金龙,等.2017.版纳微型猪近交系RNF4基因克隆、多组织qPCR表达及功能生物信息学分析[J].东北农业大学学报,48(3):24-30.(Zhang X,Zeng R B,Huo J L,et al.2017.Cloning,muti-tissues qPCRexpression and functional bioinformatics analysis of Banna mini-pig inbred RNF4[J].Journal of Northeast Agricultural University,48(3):24-30.)
张赟恺,周庆卿,陈祥,等.2017.蛋白磷酸酶在抗病毒天然免疫应答中的调控作用[J].第二军医大学学报,38(6):769-773.(Zhang Y K,Zhou Q Q,Chen X,et al.2017.Role of protein phosphatase in regulating antiviral innate immune response[J].Academic Journal of Second Military Medical University,38(6):769-773.)
曾日彬,张霞,霍金龙,等.2017.版纳微型猪近交系PRPS2基因克隆、表达及生物信息学分析[J].农业生物技术学报,25(3):425-433.(Zeng R B,Zhang X,Huo J L,et al.2017.Cloning,Expression and bioinformatics analysis of PRPS2 gene of Banna Mini-pig(Sus scrofa)inbred[J].Journal of Agricultural Biotechnology,25(3):425-433.).
Andjelkovi?N,Zolnierowicz S,Van Hoof C,et al.1996.The catalytic subunit of protein phosphatase 2A associates with the translation termination factor e RF1[J].Embo Journal,15(24):7156-7167.
Cai X,Yu S,Mipam T,et al.2017.Comparative analysis of testis transcriptomes associated with male infertility in cattleyak[J].Theriogenology,88:28-42.
Campion S,Catlin N,Heger N,et al.2012.Male reprotoxicity and endocrine disruption[J].Experientia Supplementum,101(101):315-360.
Dai X N,Liu S,Shao L,et al.2014.Expression of the SETprotein in testes of mice at different developmental stages[J].Asian Journal of Andrology,16(5):689-693.
Fang C,Li L,Li J.2016.Conditional knockout in mice reveals the critical roles of Ppp2ca in epidermis development[J].International Journal of Molecular Sciences,17(5):756.
González-Alonso P,Cristóbal I,Manso R,et al.2015.PP2Ainhibition as a novel therapeutic target in castration-resistant prostate cancer[J].Tumor Biology,36(8):5753-5755.
Gu P,Qi X,Zhou Y,et al.2012.Generation of Ppp2Ca and Ppp2Cb conditional null alleles in mouse[J].Genesis,50(5):429-436.
Janghorban M,Langer E M,Wang X,et al.2017.The tumor suppressor phosphatase PP2A-B56αregulates stemness and promotes the initiation of malignancies in a novel murine model[J].PLoS One,12(11):e0188910.
Janssens V,Goris J.2001.Protein phosphatase 2A:A highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling[J].Biochemical Journal,353(Pt 3):417-439.
Janssens V,Longin S,Goris J.2008.PP2A holoenzyme assembly:In cauda venenum(the sting is in the tail)[J].Trends in Biochemical Sciences,33(3):113-121.
Lambrecht C,Haesen D,Sents W,et al.2013.Structure,regulation,and pharmacological modulation of PP2A phosphatases[J].Methods in Molecular Biology,1053:283-305.
Marchler-Bauer A,Derbyshire M K,Gonzales N R,et al.2015.CDD:NCBI's conserved domain database[J].Nucleic Acids Research,43(D1):D222-226.
Moorhead G B,De Wever V,Templeton G,et al.2009.Evolution of protein phosphatases in plants and animals[J].Biochemical Journal,417(2):401-409.
Pan X,Chen X,Tong X,et al.2015.Ppp2ca knockout in mice spermatogenesis[J].Reproduction,149(4):385-391.
Sents W,Ivanova E,Lambrecht C,et al.2013.The biogenesis of active protein phosphatase 2A holoenzymes:A tightly regulated process creating phosphatase specificity[J].Febs Journal,280(2):644-661.
Tahmasbpour E,Balasubramanian D,Agarwal A.2014.Amulti-faceted approach to understanding male infertility:Gene mutations,molecular defects and assisted reproductive techniques(ART)[J].Journal of Assisted Reproduction and Genetics,31(9):1115-1137.
Tan X,Chen M.2014.MYLK and MYL9 expression in nonsmall cell lung cancer identified by bioinformatics analysis of public expression data[J].Tumor Biology,35(12)12189-12200.
Wang P,Huo H L,Wang S Y,et al.2015.Cloning,sequence characterization,and expression patterns of members of the porcine TSSK family[J].Genetics and Molecular Research,14(4):14908-14919.
Wang Y,Xia Y,Kuang D,et al.2017.PP2A regulates SCF-induced cardiac stem cell migration through interaction with p38 MAPK[J].Life Science,191:59-67.
Yan Z,Fedorov S A,Mumby M C,et al.2000.PR48,a novel regulatory subunit of protein phosphatase 2A,interacts with Cdc6 and modulates DNA replication in human cells[J].Molecular Cell Biology,20(3):1021-1029.
Yang C C,Kuai X X,Gao W B,et al.2016.Morroniside-induced PP2A activation antagonizes tau hyperphosphorylation in a cellular model of neurodegeneration[J].Jalzheimersdis Journal of Alzheimers Disease,51(1):33-44.
Zeng R,Zeng Y Z.2005.Molecular cloning and characterization of SLA-DR genes in the 133-family of the Banna mini-pig inbred line Family 133[J].Animal Genetics,36(3):267-269.
王镜岩,朱圣庚,徐长法.2002.生物化学[M].第三版.北京:高等教育出版社,207-220.(Wang J Y,Zhu S G,Xu C F.2002.Biochemistry[M].Higher Education Press,Beijing.207-220.)
张霞,曾日彬,霍金龙,等.2017.版纳微型猪近交系RNF4基因克隆、多组织qPCR表达及功能生物信息学分析[J].东北农业大学学报,48(3):24-30.(Zhang X,Zeng R B,Huo J L,et al.2017.Cloning,muti-tissues qPCRexpression and functional bioinformatics analysis of Banna mini-pig inbred RNF4[J].Journal of Northeast Agricultural University,48(3):24-30.)
张赟恺,周庆卿,陈祥,等.2017.蛋白磷酸酶在抗病毒天然免疫应答中的调控作用[J].第二军医大学学报,38(6):769-773.(Zhang Y K,Zhou Q Q,Chen X,et al.2017.Role of protein phosphatase in regulating antiviral innate immune response[J].Academic Journal of Second Military Medical University,38(6):769-773.)
曾日彬,张霞,霍金龙,等.2017.版纳微型猪近交系PRPS2基因克隆、表达及生物信息学分析[J].农业生物技术学报,25(3):425-433.(Zeng R B,Zhang X,Huo J L,et al.2017.Cloning,Expression and bioinformatics analysis of PRPS2 gene of Banna Mini-pig(Sus scrofa)inbred[J].Journal of Agricultural Biotechnology,25(3):425-433.).
Andjelkovi?N,Zolnierowicz S,Van Hoof C,et al.1996.The catalytic subunit of protein phosphatase 2A associates with the translation termination factor e RF1[J].Embo Journal,15(24):7156-7167.
Cai X,Yu S,Mipam T,et al.2017.Comparative analysis of testis transcriptomes associated with male infertility in cattleyak[J].Theriogenology,88:28-42.
Campion S,Catlin N,Heger N,et al.2012.Male reprotoxicity and endocrine disruption[J].Experientia Supplementum,101(101):315-360.
Dai X N,Liu S,Shao L,et al.2014.Expression of the SETprotein in testes of mice at different developmental stages[J].Asian Journal of Andrology,16(5):689-693.
Fang C,Li L,Li J.2016.Conditional knockout in mice reveals the critical roles of Ppp2ca in epidermis development[J].International Journal of Molecular Sciences,17(5):756.
González-Alonso P,Cristóbal I,Manso R,et al.2015.PP2Ainhibition as a novel therapeutic target in castration-resistant prostate cancer[J].Tumor Biology,36(8):5753-5755.
Gu P,Qi X,Zhou Y,et al.2012.Generation of Ppp2Ca and Ppp2Cb conditional null alleles in mouse[J].Genesis,50(5):429-436.
Janghorban M,Langer E M,Wang X,et al.2017.The tumor suppressor phosphatase PP2A-B56αregulates stemness and promotes the initiation of malignancies in a novel murine model[J].PLoS One,12(11):e0188910.
Janssens V,Goris J.2001.Protein phosphatase 2A:A highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling[J].Biochemical Journal,353(Pt 3):417-439.
Janssens V,Longin S,Goris J.2008.PP2A holoenzyme assembly:In cauda venenum(the sting is in the tail)[J].Trends in Biochemical Sciences,33(3):113-121.
Lambrecht C,Haesen D,Sents W,et al.2013.Structure,regulation,and pharmacological modulation of PP2A phosphatases[J].Methods in Molecular Biology,1053:283-305.
Marchler-Bauer A,Derbyshire M K,Gonzales N R,et al.2015.CDD:NCBI's conserved domain database[J].Nucleic Acids Research,43(D1):D222-226.
Moorhead G B,De Wever V,Templeton G,et al.2009.Evolution of protein phosphatases in plants and animals[J].Biochemical Journal,417(2):401-409.
Pan X,Chen X,Tong X,et al.2015.Ppp2ca knockout in mice spermatogenesis[J].Reproduction,149(4):385-391.
Sents W,Ivanova E,Lambrecht C,et al.2013.The biogenesis of active protein phosphatase 2A holoenzymes:A tightly regulated process creating phosphatase specificity[J].Febs Journal,280(2):644-661.
Tahmasbpour E,Balasubramanian D,Agarwal A.2014.Amulti-faceted approach to understanding male infertility:Gene mutations,molecular defects and assisted reproductive techniques(ART)[J].Journal of Assisted Reproduction and Genetics,31(9):1115-1137.
Tan X,Chen M.2014.MYLK and MYL9 expression in nonsmall cell lung cancer identified by bioinformatics analysis of public expression data[J].Tumor Biology,35(12)12189-12200.
Wang P,Huo H L,Wang S Y,et al.2015.Cloning,sequence characterization,and expression patterns of members of the porcine TSSK family[J].Genetics and Molecular Research,14(4):14908-14919.
Wang Y,Xia Y,Kuang D,et al.2017.PP2A regulates SCF-induced cardiac stem cell migration through interaction with p38 MAPK[J].Life Science,191:59-67.
Yan Z,Fedorov S A,Mumby M C,et al.2000.PR48,a novel regulatory subunit of protein phosphatase 2A,interacts with Cdc6 and modulates DNA replication in human cells[J].Molecular Cell Biology,20(3):1021-1029.
Yang C C,Kuai X X,Gao W B,et al.2016.Morroniside-induced PP2A activation antagonizes tau hyperphosphorylation in a cellular model of neurodegeneration[J].Jalzheimersdis Journal of Alzheimers Disease,51(1):33-44.
Zeng R,Zeng Y Z.2005.Molecular cloning and characterization of SLA-DR genes in the 133-family of the Banna mini-pig inbred line Family 133[J].Animal Genetics,36(3):267-269.