脊椎动物Cyclophilin A肽基脯氨酰顺反异构酶活性及遗传变异分析

doi:10.16288/j.yczz.15-523

遗传 ›› 2016, Vol. 38 ›› Issue (8): 736-745.doi: 10.16288/j.yczz.15-523

脊椎动物Cyclophilin A肽基脯氨酰顺反异构酶活性及遗传变异分析

任丽倩^{1, 2}, 刘薇^{1, 3}, 李文博^{1, 3}, 刘文军^{1, 3}, 孙蕾^{1, 3}

1. 中国科学院微生物研究所,中国科学院病原微生物与免疫学重点实验室,北京 100101;
2. 安徽大学生命科学学院,合肥230039;
3. 中国科学院大学,北京100049

收稿日期:2016-04-07 修回日期:2016-05-26 出版日期:2016-08-20 发布日期:2016-07-22
通讯作者: 孙蕾,博士,副研究员,研究方向：病原微生物与免疫学。E-mail: sunlei362@im.ac.cn E-mail:mengxunlq@163.com
作者简介:任丽倩,硕士,专业方向：微生物学。E-mail: mengxunlq@163.com
基金资助:
国家自然科学基金项目(编号：31472178,81271849)和国家科技支撑计划项目(编号：2015BAD11B02)资助

Peptidylprolyl cis/trans isomerase activity and molecular evolution of vertebrate Cyclophilin A

Liqian Ren^{1, 2}, Wei Liu^{1, 3}, Wenbo Li^{1, 3}, Wenjun Liu^{1, 3}, Lei Sun^{1, 3}

1. CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;
2. School of Life Sciences, Anhui University, Hefei 230039, China 3. Universty of Chinese Academy of Sciences, Beijing 100049, China

Received:2016-04-07 Revised:2016-05-26 Online:2016-08-20 Published:2016-07-22
Supported by:
Supported by the National Natural Science Foundation of China (Nos; 31472178, 81271849) and the National Key Technology Support Program (No; 2015 BAD11B02)

摘要/Abstract

摘要： Cyclophilin A (简称CypA)由PPIA(Peptidylprolyl isomerase A)基因编码,是典型的Cyclophilin家族蛋白,具有肽基脯氨酰顺反异构酶活性,在蛋白质的折叠和转运、信号转导、炎症、免疫调节、细胞凋亡及病毒复制等生物学过程中发挥着重要作用。本研究重点探讨了不同脊椎动物CypA的肽基脯氨酰顺反异构酶活性及其遗传变异。根据GenBank数据库PPIA基因的序列信息,克隆了脊椎动物的PPIA基因并构建其原核表达载体,其中鼠耳蝠(Myotis davidi)和绿头鸭(Anas platyrhynchos)的PPIA基因序列为首次报道。利用大肠杆菌表达GST-CypA融合蛋白并进行亲和层析,切除GST标签后再进行分子筛层析,获得纯化的CypA蛋白。利用胰糜蛋白酶偶联法测定CypA的肽基脯氨酰顺反异构酶活性,发现12种脊椎动物CypA的肽基脯氨酰顺反异构酶活性无显著差异。同时,通过一系列遗传变异和分子进化分析,发现12种脊椎动物CypA的酶活位点、CsA结合位点等重要功能域的氨基酸序列完全一致,而且其结构和在染色体中的基因定位也非常保守。研究结果表明,脊椎动物CypA的关键功能域高度保守,这是CypA维持其酶活及生物学功能的有力保障。

关键词: 脊椎动物, Cyclophilin A, 肽基脯氨酰顺反异构酶, 分子进化

Abstract: Peptidylprolyl isomerases (PPIase) cyclophilin A (CypA, encoded by PPIA) is a typical member of the Cyclophilin family and is involved in protein folding/translocation, signal transduction, inflammation, immune system regulation, apoptosis and virus replication. In the present study, we investigated the PPIase activity and genetic variation of vertebrate CypA. According to the GenBank reference sequences, vertebrate PPIA genes were cloned, among which the bat (Myotis davidi) and duck (Anas platyrhynchos) PPIA genes were reported for the first time. Then PPIA genes were sub-cloned into the expression vector pGEX-6p-1 and expressed in Escherichia coli. Recombinant CypA proteins were purified by using sepharose 4B affinity chromatography and the GST tag was cleaved, followed by gel filtration. The PPIase activity assay indicated that there was no significant difference in the catalytic activity of prolyl peptide bond isomerization among 12 different vertebrate CypA proteins. In addition, the genetic variation and molecular evolution analysis showed that these vertebrate CypA proteins had the same CsA binding site and the PPIase active sites. Furthermore, the predicted structure and gene localization were remarkable conserved. Our data suggested that the important residues of CypA were highly conserved, which is crucial for its PPIase activity and cellular functions.

Key words: vertebrate, Cyclophilin A, peptidylprolyl cis/trans isomerase, molecular evolutiontext

任丽倩, 刘薇, 李文博, 刘文军, 孙蕾. 脊椎动物Cyclophilin A肽基脯氨酰顺反异构酶活性及遗传变异分析[J]. 遗传, 2016, 38(8): 736-745.

Liqian Ren, Wei Liu, Wenbo Li, Wenjun Liu, Lei Sun. Peptidylprolyl cis/trans isomerase activity and molecular evolution of vertebrate Cyclophilin A[J]. Hereditas(Beijing), 2016, 38(8): 736-745.

参考文献

[1] Liu J, Farmer JD, Lane WS, Friedman J, Weissman I, Schreiber SL. Calcineurin is a common target of cyclophilin-cyclosporine A and Fkbp-Fk506 complexes. Cell , 1991, 66(4): 807-815.
[2] Galat A. Peptidylproline cis -trans-isomerases: immunophilins. Eur J Biochem , 1993, 216(3): 689-707.
[3] Colgan J, Asmal M, Neagu M, Yu B, Schneidkraut J, Lee Y, Sokolskaja E, Andreotti A, Luban J. Cyclophilin A regulates TCR signal strength in CD4 + T cells via a proline- directed conformational switch in Itk. Immunity , 2004, 21(2): 189-201.
[4] Doti N, Reuther C, Scognamiglio PL, Dolga AM, Plesnila N, Ruvo M, Culmsee C. Inhibition of the AIF/CypA complex protects against intrinsic death pathways induced by oxidative stress. Cell Death Dis , 2014, 5(1): e993.
[5] Bonfils C, Bec N, Larroque C, Del Rio M, Gongora C, Pugnière M, Martineau P. Cyclophilin A as negative regulator of apoptosis by sequestering cytochrome c . Biochem Biophys Res Commun , 2010, 393(2): 325-330.
[6] Colgan J, Yuan HE, Franke EK, Luban J. Binding of the human immunodeficiency virus type 1 Gag polyprotein to cyclophilin A is mediated by the central region of capsid and requires Gag dimerization. J Virol , 1996, 70(7): 4299-4310.
[7] Zander K, Sherman MP, Tessmer U, Bruns K, Wray V, Prechtel AT, Schubert E, Henklein P, Luban J, Neidleman J, Greene WC, Schubert U. Cyclophilin A interacts with HIV-1 Vpr and is required for its functional expression. J Biol Chem , 2003, 278(44): 43202-43213.
[8] Yang F, Robotham JM, Nelson HB, Irsigler A, Kenworthy R, Tang HL. Cyclophilin A is an essential cofactor for hepatitis C virus infection and the principal mediator of cyclosporine resistance in vitro . J Virol , 2008, 82(11): 5269-5278.
[9] Chatterji U, Bobardt M, Selvarajah S, Yang F, Tang HL, Sakamoto N, Vuagniaux G, Parkinson T, Gallay P. The isomerase active site of cyclophilin A is critical for hepatitis C virus replication. J Biol Chem , 2009, 284(25): 16998-17005.
[10] Bienkowska-Haba M, Williams C, Kim SM, Garcea RL, Sapp M. Cyclophilins facilitate dissociation of the human papillomavirus type 16 capsid protein L1 from the L2/ DNA complex following virus entry. J Virol , 2012, 86(18): 9875-9887.
[11] Liu XL, Sun L, Yu MR, Wang ZF, Xu CF, Xue QH, Zhang K, Ye X, Kitamura Y, Liu WJ. Cyclophilin A interacts with influenza A virus M1 protein and impairs the early stage of the viral replication. Cell Microbiol , 2009, 11(5): 730-741.
[12] Liu XL, Zhao ZD, Xu CF, Sun L, Chen JL, Zhang LF, Liu WJ. Cyclophilin A restricts influenza A virus replication through degradation of the M1 protein. PLoS One , 2012, 7(2): e31063.
[13] Xu CF, Meng SS, Liu XL, Sun L, Liu WJ. Chicken cyclophilin A is an inhibitory factor to influenza virus replication. Virol J , 2010, 7: 372.
[14] He HY, Zhou DJ, Fan WP, Fu XL, Zhang J, Shen ZG, Li J, Li JT, Wu YZ. Cyclophilin A inhibits rotavirus replication by facilitating host IFN-I production. Biochem Biophys Res Commun , 2012, 422(4): 664-669.
[15] Qing J, Wang YX, Sun YN, Huang JY, Yan WZ, Wang JL, Su D, Ni C, Li J, Rao ZH, Liu L, Lou ZY. Cyclophilin A associates with enterovirus-71 virus capsid and plays an essential role in viral infection as an uncoating regulator. PLoS Pathog , 2014, 10(10): e1004422.
[16] Wang N, Zhang LZ, Chen YM, Lu Z, Gao L, Wang YQ, Gao YL, Gao HL, Cui HY, Li K, Liu CJ, Zhang YP, Qi XL, Wang XM. Cyclophilin A interacts with viral VP4 and inhibits the replication of infectious bursal disease virus. BioMed Res Int , 2015, 2015: 719454.
[17] Fischer G, Bang H, Mech C. Determination of enzymatic catalysis for the cis -trans-isomerization of peptide binding in proline-containing peptides. Biomed Biochim Acta , 1984, 43(10): 1101-1111.
[18] Fischer G, Wittmann-Liebold B, Lang K, Kiefhaber T, Schmid FX. Cyclophilin and peptidyl-prolyl cis - trans isomerase are probably identical proteins. Nature , 1989, 337(6206): 476-478.
[19] Takahashi N, Hayano T, Suz

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

脊椎动物Cyclophilin A肽基脯氨酰顺反异构酶活性及遗传变异分析

Peptidylprolyl cis/trans isomerase activity and molecular evolution of vertebrate Cyclophilin A

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	孟玉,杨若林. 基于基因家族大小的比较研究脊椎动物的适应性进化[J]. 遗传, 2019, 41(2): 158-174.
[2]	苏鹏冯少姝李庆伟. NF-кB和IκB在不同动物类群中的结构及功能研究进展[J]. 遗传, 2016, 38(6): 523-531.
[3]	浦懋懋, 姚俊, 曹新. 基因组学：揭秘人类特有遗传变异对大脑皮层进化与发育的影响[J]. 遗传, 2016, 38(11): 957-970.
[4]	刘志祥曾超珍谭晓风. 杨树MIR169基因家族分子进化分析[J]. 遗传, 2013, 35(11): 1307-1316.
[5]	危金普，潘学峰，李红权，段斐. 简单重复DNA序列在哺乳动物mtDNA D-loop区的分布及进化特征[J]. 遗传, 2011, 33(1): 67-74.
[6]	张永科，陈争，范安，张亚男，武艳平，赵倩君，周璨林，毛新民，藏玉亮，叶尔哈孜·扎尔胡马尔，哈森别克·马力克，哈布德力·达拉拜依，卡马力亚·托塔哈孜，梁玲，古力努尔·叶尔肯，马月辉，饶绍奇. 新疆阿勒泰地区图瓦人与邻近人群遗传关系初探[J]. 遗传, 2009, 31(8): 818-824.
[7]	徐怀亮,姚永芳,朱庆. 猪獾苦味受体T2R2基因的分子克隆与进化分析[J]. 遗传, 2009, 31(11): 1113-1120.
[8]	梁佼，刘欣，吴芬芳，李庆伟. 无颌类脊椎动物适应性免疫系统的研究进展[J]. 遗传, 2009, 31(10): 969-976.
[9]	聂庆华，刘清神，方梅霞，谢亮，张细权. 犬MC1R基因的分子进化分析[J]. 遗传, 2008, 30(4): 469-474.
[10]	谢飞，高波，宋成义，陈国宏. “睡美人”转座子的研究进展[J]. 遗传, 2007, 29(7): 785-792.
[11]	于海龙，肖云，艾静，李霞，宫滨生. 毒蕈乙酰胆碱受体亚型关系的研究[J]. 遗传, 2007, 29(10): 1280-1288.
[12]	王云生，黄宏文，王瑛. 植物分子群体遗传学研究动态[J]. 遗传, 2007, 29(10): 1191-1191―1198.
[13]	贾晓晖，王宝维，王雷，李桢，张名爱，吴晓平，刘光磊，杨志刚，龙芳羽，张旭晖. 五龙鹅MHC ClassⅠ基因克隆及同源建模研究[J]. 遗传, 2006, 28(9): 1087-1092.
[14]	熊爱生，姚泉洪，章镇，彭日荷，庄静，徐芳，刘金戈，朱宏. 基因的推理设计与改造—体外分子进化的捷径[J]. 遗传, 2006, 28(1): 92-96.
[15]	朱新宇，谢晓玲，陈佩林. 新编分子进化实验一组A[J]. 遗传, 2004, 26(4): 505-508.