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. 2020 Apr 18;52(2):221–226. [Article in Chinese] doi: 10.19723/j.issn.1671-167X.2020.02.005

免疫相关GTP结合蛋白2的亚细胞定位分析

Subcellular localization of GTPase of immunity-associated protein 2

Hong-quan QIN 1,2,*, You ZHENG 2,*, Man-na WANG 2, Zheng-rong ZHANG 2, Zu-biao NIU 2, Li MA 1, Qiang SUN 2, Hong-yan Huang 3,△, Xiao-ning WANG 1,△
PMCID: PMC7433440  PMID: 32306002

Abstract

Objective

To analyze the subcellular localization of GTPase of immunity-associated protein 2 (GIMAP2) for the further functional study.

Methods

In the study,we first obtained the protein sequences of GTPase of immunity-associated protein 2 (GIMAP2) from National Center for Biotechnology Information (NCBI) database, and then performed a prediction analysis of its transmembrane structure, nuclear localization signal (NLS), nuclear export signal (NES) and subcellular localization through bioinformatics online tools. GIMAP2 gene amplified by PCR was inserted into the expression vector pQCXIP-mCherry-N1 and positive clones were selected by ampicillin resistance. After using methods to extract and purify, the sequenced recombinant plasmid pQCXIP-GIMAP2-mCherry, together with the retroviral packaging plasmids VSVG and Gag/pol, was transferred into HEK293FT cells by liposomes for virus packaging. The virus supernatant was collected 48 h after transfection and directly infected the human breast cancer cell line MDA-MB-436. Immunofluorescence staining was constructed to detect the localization of endogenous and exogenous GIMAP2 in MDA-MB-436 cells. Meanwhile, green fluorescent chemical dyes were used to label mitochondria, endoplasmic reticulum, and lipid droplets in living MDA-MB-436 cells stably expressing the GIMAP2-mCherry fusion protein. Images for the three dye-labeled organelles and GIMAP2-mCherry fusion protein were captured by super-resolution microscope N-SIM.

Results

Bioinformatics analysis data showed that GIMAP2 protein composed of 337 amino acids might contain two transmembrane helix (TM) structures at the carboxyl terminus, of which TMs were estimated to contain 40-41 expected amino acids,followed by the residual protein structures toward the cytoplasmic side. NES was located at the 279-281 amino acids of the carboxyl terminus whereas NLS was not found. GIMAP2 might locate in the lumen of the endoplasmic reticulum. Sequencing results indicated that the expression vector pQCXIP-GIMAP2-mCherry was successfully constructed. Fluorescent staining confirmed that GIMAP2-mCherry fusion protein, co-localized well with endogenous GIMAP2, expressed successfully in the endoplasmic reticulum and on the surface of lipid droplets in MDA-MB-436 cells.

Conclusion

GIMAP2 localizes in the endoplasmic reticulum and on the surface of LDs, suggesting potential involvement of GIMAP2 in lipid metabolism.

Keywords: GTPase of immunity-associated protein 2, Bioinformatics, Subcellular localization

免疫相关GTP结合蛋白家族(GTPase of immunity-associated proteins,GIMAPs,别名immunity-associated nucleotide-binding proteins,IANs)是独特的GTP结合蛋白家族,与免疫系统紧密联系,广泛存在于各种动,植物细胞以及某些细菌中[1,2].目前发现的7种人源GIMAPs家族基因均分布于染色体7q36.1,其编码蛋白的相对分子质量为33 000~75 000[3].蛋白结构分析显示,GIMAPs蛋白家族主要由氨基端的鸟嘌呤核苷酸结合结构域和羧基端的卷曲螺旋基序组成[4].GIMAPs蛋白家族通过控制淋巴细胞成熟,凋亡等生物学过程在淋巴细胞相关疾病中发挥关键作用[5].在小鼠淋巴组织内,条件性敲除GIMAP1基因导致成熟T细胞和B细胞几乎完全丧失[6].Carter等[7]研究发现,GIMAP4蛋白可与Bcl-2家族的促凋亡蛋白Bax结合促进T细胞凋亡.Patterson等[8]实验表明,GIMAP5基因缺陷可导致CD4+ T细胞发生DNA损伤,而转化生长因子-β可修复其DNA损伤并使其向Th17 谱系分化,进而分泌大量促炎性因子白细胞介素17A,导致过敏性气道疾病的发生.

免疫相关GTP结合蛋白2(GTPase of immunity-associated protein 2,GIMAP2)是GIMAPs蛋白家族的重要成员之一.目前发现GIMAP2蛋白仅存在于人细胞中,目前未发现其鼠源的同源物[3],可能是人机体所特有的蛋白.GIMAP2隶属于GTP结合蛋白范畴,是信号传导过程中的重要分子开关,但在细胞凋亡,细胞分裂,骨架动力学改变以及能量代谢等生命活动中功能鲜有报道,有待深入研究.

本研究中,我们利用生物信息学软件对GIMAP2蛋白进行分析,构建了GIMAP2与红色荧光蛋白mCherry融合表达载体,对于GIMAP2的亚细胞定位进行了初步研究,为进一步深入研究GIMAP2蛋白分子的功能奠定了基础.

1. 材料与方法

1.1. 材料

人乳腺癌细胞系MDA-MB-436,人胚肾细胞HEK293FT细胞,质粒pBABE-GIMAP2-EGFP,质粒pQCXIP-mCherry-N1由中国军事医学研究院生物工程研究所保存并提供,大肠杆菌感受态细胞Trans10,逆转录试剂盒,脱氧核苷酸dNTPs购自中国全式金公司,Q5超保真DNA聚合酶,XhoⅠ,BamHⅠ限制性内切酶,T4 DNA 连接酶均购自美国NEB公司,琼脂糖凝胶DNA回收试剂盒,无内毒素质粒小提中量试剂盒购自中国天根生化公司,Opti-MEM减血清培养基,嘌呤霉素,脂质体Lipofectamine 2000,ER-TrackerTM Green 染料,Mito Tracker®Green染料,BODIPY493/503染料和Hoechst33342染料购自美国Thermo Fisher公司,胎牛血清购自德国PAN-Biotech公司,高糖DMEM(Dulbecco's modified eagle medium)培养基,磷酸缓冲盐溶液(phosphate buffer saline,PBS)购自中国迈晨科技公司,兔抗人GIMAP2抗体购自美国Proteintech公司,Alexa Fluor®488标记的山羊抗鼠二抗购自美国Invitrogen公司,4',6-二脒基-2-苯基吲哚(4', 6-diamidino-2-phenylindole,DAPI)封片剂购自北京中杉金桥公司,激光扫描共聚焦显微镜(laser scanning confocal microscope,LSCM),超分辨率显微镜N-SIM购自日本Nikon公司.

1.2. 方法

1.2.1 GIMAP2蛋白的生物信息学分析 利用NCBI官方网站,查找GIMAP2的蛋白序列(编号:NP_056475.1)作为参考蛋白序列.运用以下几种生物软件对GIMAP2蛋白进行生物信息学预测分析.首先,使用TMHMM Server v.2.0网站(http://www.cbs.dtu.dk/services/TMHMM)预测GIMAP2蛋白跨膜结构;其次,分别使用cNLS Mapper网站(http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Map-per_form.cgi),NetNES 1.1 Server网站(http://www.cbs.dtu.dk/services/NetNES/)预测GIMAP2蛋白的核定位信号((nuclear localization signal,NLS),核输出信号(nuclear export signal,NES);最后,使用Cell-PLoc 2.0网站(http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/)预测GIMAP2蛋白的亚细胞定位[9,10,11].

1.2.2 pQCXIP-GIMAP2-mCherry表达载体的构建 以pBABE-GIMAP2-EGFP质粒为模板,利用SnapGene软件设计PCR引物,其中GIMAP2的5'端引入限制性核酸内切酶切位点 XhoⅠ,3'端引入限制性核酸内切酶切位点 BamHⅠ,通过PCR扩增技术获得GIMAP2基因片段.引物序列为GIMAP2-F: 5'-CCGCTCGAGCGGATGGACCAAAATGAACACAGTC-ACTGG-3',GIMAP2-R: 5'-CGGGATCCCGTAACCTA-GGAGTCTTGCGTTCTAGTCT-3'.PCR扩增条件: 98 ℃预变性30 s,98 ℃变性10 s,61 ℃退火30 s,72 ℃延伸1 min 30 s,32个循环,72 ℃延伸2 min.GIMAP2基因片段和载体pQCXIP-mCherry-N1分别加入XhoⅠ/BamHⅠ双酶切,回收酶切产物,用T4 DNA连接酶22 ℃连接1 h,然后转化到大肠杆菌感受态细胞Trans10中;取连接产物涂布在含100 mg/L氨苄青霉素的固体培养基,37 ℃培养16 h;挑取单克隆菌落进行菌液PCR,并将阳性菌落送北京天一辉远生物公司测序鉴定.测序正确后进行质粒提取,获得纯化的重组表达载体pQCXIP-GIMAP2-mCherry.

1.2.3 病毒包装,感染 取HEK 293FT细胞按1.0×106/孔的密度接种于6孔板内,加入含10%(体积分数)胎牛血清的高糖DMEM培养基,在37 ℃,5% CO2条件下培养过夜.当细胞汇合度为80%~90%时,取400 ng pQCXIP-mCherry-N1质粒或pQCXIP-GIMAP2-mCherry质粒分别与200 ng VSVG和250 ng Gag/Pol包装质粒共同稀释在100 μL Opti-DMEM培养基中,使用Lipofectamine 2000作为转染介质,共转入HEK293FT细胞中,孵育6 h后换成DMEM培养基继续培养 48 h;收集含病毒上清液,使用0.45 μm孔径的滤器过滤,收集滤液.将人乳腺癌细胞系MDA-MB-436按3.0×105/孔的密度铺于6孔板中,每孔加入0.5 mL DMEM培养基,1 mL 病毒上清液,1.5 μL 聚凝胺,在48 h后使用嘌呤霉素(1 g/L)药筛3~4 d,获得稳定表达mCherry红色蛋白或GIMAP-mCherry融合蛋白的MDA-MB-436细胞株.

1.2.4 免疫荧光染色检测GIMAP2蛋白在MDA-MB-436细胞内定位 分别选取表达pQCXIP-mCherry-N1,pQCXIP-GIMAP2-mCherry的MDA-MB-436细胞各1.0×105/孔接种在含盖玻片的12孔板内,37 ℃,5% CO2培养12 h.加入1 mL 4%(质量分数)多聚甲醛,室温固定10 min;弃除培养基,用1 mL PBS清洗3次;用1 mL 0.15% (体积分数)Triton X-100室温透膜3 min,加入PBS清洗3次;加入5% (质量分数)BSA室温封闭1 h;封闭结束后加入兔抗人GIMAP2抗体(稀释比为1 ∶200), 4 ℃孵育过夜;次日使用PBS洗涤3次;加入Hoechst33342染料(稀释比为1 ∶1 000), Alexa Fluor®488标记的山羊抗鼠二抗(稀释比为1 ∶200),室温染色1 h,加入PBS清洗3次,每次10 min;加入封片剂,避光30 min.使用LSCM进行图像采集.

1.2.5 GIMAP2-mCherry融合蛋白亚细胞定位 分别选取表达pQCXIP-mCherry,pQCXIP-GIMAP2-mCherry的MDA-MB-436细胞各1.5×105个/孔接种在玻璃底小皿内, 37 ℃, 5%CO2培养12 h.弃培养基,用1 mL PBS清洗1次;分别加入Mito Tracker®Green染料(50 nmol/L),ER-TrackerTM Green 染料(1 μmol/L),BODIPY493/503染料(1 g/L), 37 ℃染色30 min;加入PBS清洗1次;加入新鲜DMEM培养基1 mL.使用超分辨率显微镜N-SIM进行图像采集.

2. 结果

2.1. GIMAP2蛋白生物信息学分析

GIMAP2基因编码的蛋白(编号:NP_056475.1)含有337个氨基酸(图1A).TMHMM Server v.2.0软件预测结果显示,GIMAP2蛋白羧基端有2个跨膜结构,其跨膜螺旋中的预期氨基酸数为40~41个,分别为第271~290位氨基酸,第294~316位氨基酸,而氨基端前60个氨基酸中预期跨膜螺旋氨基酸数为0个;羧基端在膜的细胞质侧的总概率在0.6~0.8(图1B).cNLS Mapper,NetNES 1.1 Server软件分析得出,GIMAP2蛋白无NLS,但具有NES(图1C),在第279~281位氨基酸.最后,利用Cell-PLoc 2.0软件预测,GIMAP2可能定位在内质网.根据以上所述,我们推测GIMAP2蛋白通过羧基端的跨膜螺旋结构锚定在内质网上,且其紧随跨膜螺旋结构之后的羧基端朝细胞质侧,为其他重要分子结合提供了空间位置.

1.

GIMAP2蛋白生物信息学分析

The bioinformatics analysis of GIMAP2 protein

A, protein sequence of GIMAP2, yellow parts represent its transmembrane regions and red part represents the amino acids of nuclear export signal(NES); B, prediction of GIMAP2 transmembrane regions; C, prediction of GIMAP2 NES, transmembrane possibility(B) or NES score(C) on the vertical Y-axis against sequence position on the horizontal X-axis.

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2.2. pQCXIP-GIMAP2-mCherry表达载体的构建与鉴定

根据pQCXIP-GIMAP2-mCherry表达载体的构建示意图(图2A),以pBABE-GIMAP2-EGFP质粒为模板,GIMAP2-F,GIMAP2-R为引物扩增GIMAP2基因片段,其PCR产物大小为1 033 bp.DNA凝胶电泳结果显示,与预期大小相符(图2B).将扩增片段酶切,连接插入pQCXIP-mCherry-N1表达载体,获得重组表达载体pQCXIP-GIMAP2-mCherry.随机挑取6个单菌落进行菌液PCR验证,结果显示编号1,5,6均有明显条带,表明pQCXIP-GIMAP2-mCherry表达载体构建成功(图2C).培养编号1阳性菌落,送测序公司测序,GIMAP2基因正确插入pQCXIP-mCherry-N1(图2D),表明pQCXIP-GIMAP2-mCherry表达载体构建成功,无碱基突变或缺失.

2.

pQCXIP-GIMAP2-mCherry重组载体构建过程

The construction process of pQCXIP-GIMAP2-mCherry recombinant vector

A, schema graph of pQCXIP-GIMAP2-mCherry recombinant vector; B, electrophoresis results of GIMAP2 gene fragment amplified by PCR; C, electrophoresis results of bacterial colony PCR; M, DNA marker Ⅲ; F, 1-6, PCR amplification products; D, sequencing result of pQCXIP-GIMAP2-mCherry recombinant vector.

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2.3. 内源性,外源性GIMAP2蛋白在MDA-MB-436细胞内共定位

将稳定表达GIMAP2-mCherry融合蛋白的MDA-MB-436细胞以1.0×105个/孔接种在含盖玻片的12孔板内,37 ℃培养12 h.经固定,透膜,封闭,兔抗人GIMAP2抗体免疫染色等步骤后,激光共聚焦显微镜下观察GIMAP2蛋白的定位情况,结果显示抗体标记的GIMAP2蛋白(绿色)大部分围绕蓝色的细胞核向外周铺展延伸分布,部分呈点状分布于细胞伪足(图3A).GIMAP2-mCherry红色融合蛋白已表达,与绿色区域几乎完全重叠,这证明了内源性,外源性GIMAP2在胞内定位一致,也表明了GIMAP2-mCherry融合蛋白可用于后续GIMAP2的定位,功能研究(图3B).

3.

内源性,外源性GIMAP2蛋白在MDA-MB-436细胞内共定位

Endogenous and exogenous GIMAP2 protein are co-localized in MDA-MB-436 cell

A, C-terminally mCherry-tagged GIMAP2 (red) and endogenous GIMAP2 (green) are co-localized in MDA-MB-436 cells, nuclei was stained with DAPI, white arrow indicates gray scan position; B, green and red curves are the gray scan results of the corresponding fluorescence channel, vertical Y-axis represents fluorescence intensity while the horizontal X-axis represents white arrow's distance.

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2.4. GIMAP2-mCherry融合蛋白定位于内质网和脂滴

根据GIMAP2定位于生物膜的生物信息学分析,我们分别使用Mito Tracker®Green染料,ER-TrackerTM Green染料,BODIPY493/503染料标记稳定表达GIMAP2-mCherry融合蛋白的MDA-MB-436细胞中的线粒体,内质网和脂滴,在超分辨率显微镜N-SIM下观察.实验结果表明,GIMAP2-mCherry融合蛋白在众多分支小管相互连接吻合的网状结构中分布,与线粒体的绿色荧光信号不重叠,而与ER-TrackerTM Green染料标记的内质网绿色荧光信号重叠共同合并成黄色信号,证明了GIMAP2-mCherry融合蛋白定位于内质网.另外,我们意外发现,GIMAP2-mCherry融合蛋白围绕在BODIPY493/503染料标记的绿色脂滴表面,呈指环状分布,证明了GIMAP2-mCherry融合蛋白也定位于脂滴(图4).脂滴是由内质网合成,由此推测,GIMAP2蛋白很有可能由内质网合成,然后转移到脂滴表面分布.

4.

GIMAP2-mCherry融合蛋白定位于细胞中内质网和脂滴

GIMAP2-mCherry fusion protein localizes to endoplasmic reticulum and lipid droplets

Mito, specific marker dyes for mitochondria; ER, specific marker dyes for endoplasmic reticulum; BODIPY, specific marker dyes for lipid droplets.

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3. 讨论

GIMAPs作为GTP结合蛋白的重要分支,参与T淋巴细胞分化,发育,成熟的过程[12].以往研究表明该蛋白家族与免疫介导的疾病相关,如白血病,哮喘,过敏,白塞病等[13,14,15].GIMAP2是GIMAP蛋白家族独特的成员且功能未明确,其国内外文献资料相对匮乏.Schwefel等[16,17]研究发现,GIMAP2蛋白锚定于Jurkat细胞中脂滴表面.脂滴起源于内质网,生长成熟后以萌芽形式脱离内质网.本文建立了稳定表达GIMAP2-mCherry的MDA-MB-436细胞系,并对GIMAP2蛋白的亚细胞定位进行研究,结果表明GIMAP2蛋白分布于胞质中网状结构的内质网以及脂滴的表面,与生物信息学预测结果一致,并提示GIMAP2可能参与从内质网合成到脂滴成熟的形成过程,可能与脂代谢相关.有研究表明,二磷酸腺苷-核糖基化因子1(ADP-ribosylation factor 1,ARF1)属于GTP结合蛋白家族的成员,其能直接活化磷脂酶D1(phospholipase D1,PLD1),二者共定位于脂滴表面[18].有趣的是,脂滴是丙型肝炎病毒(hepatitis C virus,HCV)组装的场所,而脂肪分化蛋白(adipose differentiation-related protein,ADRP)能够抑制HCV病毒的装配.活化的ARF1从脂滴解离ADRP,促进HCV病毒组装[19].与ARF1相似,GIMAP2作为GTP结合蛋白家族的成员,其GTP结合结构域可能具备GIMAP家族免疫功能,而且我们的研究提示了GIMAP2可能作为脂滴相关蛋白参与脂代谢通路,因此我们推测,GIMAP2可能是免疫异常相关疾病和脂代谢异常的"关键结合点分子",值得深入探究.

综上所述,本研究对GIMAP2蛋白的亚细胞定位分析为我们后续探索GIMAP2蛋白功能及相应机制提供了实验基础.

Funding Statement

国家自然科学基金（81872314）; 国家自然科学基金（31671432）; 国家重点研发计划（2016YFC1303303）; 国家重点研发计划（2018YFA0900804）

Supported by the National Natural Science Foundation of China(81872314); Supported by the National Natural Science Foundation of China(31671432); the National Key Research & Development Program of China(2016YFC1303303); the National Key Research & Development Program of China(2018YFA0900804)

Contributor Information

黄 红艳 (Hong-yan Huang), Email: hhongy1999@126.com.

王 小宁 (Xiao-ning WANG), Email: xnwang88@163.com.

References

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