马疱疹病毒1型gD囊膜蛋白磷酸化位点的鉴定及其对亚细胞定位的影响  

Identification of Phosphorylation Sites in EHV-1 gD Envelope Protein and Their Effect on the Subcellular Localization

作　　者：段琦颖 贾万欣 肖楚宁 屈春惠 李斌[1] 王世民[1] DUAN Qi-ying;JIA Wan-xin;XIAO Chu-ning;QU Chun-hui;LI Bin;WANG Shi-min(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi,Xinjiang,830052,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《动物医学进展》2023年第6期70-76,共7页Progress In Veterinary Medicine

基　　金：新疆维吾尔自治区自然科学基金项目(2018D01B17);国家自然科学基金项目(31860710)。

摘　　要：为了研究蛋白质磷酸化修饰对马疱疹病毒1型(EHV-1)gD囊膜蛋白高尔基体驻留的影响,通过蛋白质修饰质谱技术鉴定gD囊膜蛋白磷酸化位点,利用Overlap PCR技术构建磷酸化位点突变型真核表达质粒pCAGGS-gD-S391A-myc及pCAGGS-gD-S391D-myc,并转染至Hela细胞,经Western blot检测野生型及突变型gD囊膜蛋白表达情况,利用间接免疫荧光技术鉴定gD囊膜蛋白及其突变体的亚细胞定位。质谱结果显示,EHV-1 gD囊膜蛋白的S391位点发生磷酸化修饰;测序结果表明,成功构建模拟磷酸化突变型质粒pCAGGS-gD-S391D-myc及模拟去磷酸化突变型质粒pCAGGS-gD-S391A-myc;间接免疫荧光结果显示,模拟磷酸化突变体gD-S391D-myc与野生型gD-myc定位情况类似,都驻留于高尔基体;而模拟去磷酸化突变体gD-S391A-myc可转运至细胞膜。S391位点的磷酸化修饰对gD囊膜蛋白驻留于高尔基体起到重要作用,阻断该位点磷酸化修饰可促进gD囊膜蛋白转运至细胞膜,为进一步研究gD高尔基体驻留机制提供参考。To investigate the effect of phosphorylation on the Golgi-retention of glycoprotein D(gD)encoded by equine herpesvirus type 1,the phosphorylated sites of gD were identified using LC-MS in this study.The mutant plasmids pCAGGS-gD-S391A-myc and pCAGGS-gD-S391D-myc were constructed by Overlap PCR and then transfected into Hela cells.The expression and subcellular localization of gD and mutants were detected by Western blot and indirect immunofluorescence assay,respectively.The S391 site of EHV-1 gD was phosphorylated according to the LC-MS results.The phospho-mimic mutant plasmid pCAGGS-gD-S391D-myc and phosphorylation-deficient mutant plasmid pCAGGS-gD-S391A-myc were constructed and expressed successfully.Compared with the localization of gD wildtype,the phospho-mimic mutant protein gD-S391D-myc reproduced the Golgi-retention phenotype,while the phosphorylation-deficient mutant protein gD-S391A-myc was transported to cell membrane.The phosphorylation at S391 played an important role in the Golgi-retention of EHV-1 gD,and if the phosphorylation at this site was blocked,the gD could transport to the cell membrane.These results provide a reference for further research on the mechanism of gD Golgi-retention.

关 键 词：马疱疹病毒1型 gD囊膜蛋白 磷酸化 亚细胞定位 

分 类 号：S852.21[农业科学—基础兽医学] S852.4[农业科学—兽医学]