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

目的:对免疫相关GTP结合蛋白2(GTPase of immunity-associated protein 2,GIMAP2)进行亚细胞定位分析,为深入研究GIMAP2蛋白的功能奠定基础。方法:使用国家生物技术信息中心(National Center for Biotechnology Information,NCBI)数据库查询获取GIMAP2的蛋白序列,再利用生物信息学在线分析工具对GIMAP2蛋白的跨膜结构、核定位信号(nuclear localization signal,NLS)、核输出信号(nuclear export signal,NES)及亚细胞定位进行分析预测。采用PCR技术扩增GIMAP2基因片段,并插入至pQCXIP-mCherry-N1表达载体,用氨苄青霉素抗性筛选阳性克隆。测序正确的重组质粒pQCXIP-GIMAP2-mCherry经过提取、纯化步骤后,与逆转录病毒包装质粒VSVG、Gag/Pol在脂质体介导下共同转入HEK293FT细胞中进行病毒包装。转染48 h后收集病毒上清,直接感染人乳腺癌细胞系MDA-MB-436。使用免疫荧光染色方法检测内、外源性GIMAP2在MDA-MB-436胞内表达定位情况。使用绿色荧光化学染料分别标记稳定表达GIMAP2-mCherry融合蛋白的MDA-MB-436活细胞中的线粒体、内质网、脂滴,在超分辨率显微镜N-SIM下观察其与红色荧光的GIMAP2蛋白的定位情况。结果:生物信息学分析数据显示,由337个氨基酸组成的GIMAP2蛋白在羧基端可能有2个跨膜螺旋结构,其中跨膜螺旋含预期氨基酸数为40~41个,紧随跨膜螺旋结构之后的蛋白结构朝细胞质侧;羧基端第279~281位氨基酸有NES但无NLS;可能定位在内质网。测序结果表明,成功构建表达载体pQCXIP-GIMAP2-mCherry。荧光染色结果证实,GIMAP2-mCherry融合蛋白成功在MDA-MB-436细胞内表达,并与内源性GIMAP2定位一致,分布于内质网和脂滴。结论:免疫相关GTP结合蛋白2定位于内质网和脂滴,可能与脂代谢相关。

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.