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.

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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

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