跨膜衔接蛋白PAG1棕榈酰化位点突变对CD59介导的Jurkat细胞信号转导的影响  

Effect of palmitoylation site mutation of PAG1 on CD95-mediated Jurkat cell signal transduction

作　　者：丛蓓蓓 王丽娜 王冰[3] 高美华 孙钰 杨心婷 CONG Bei-bei;WANG Li-na;WANG Bing;GAO Mei-hua;SUN Yu;YANG Xin-ting(Central Laboratory,Qingdao Stomatological Hospital,Qingdao 266001,China;Department of Blood Transfusion,Qilu Hospital,Shandong University,Qingdao 266035,China;Medical College,Qingdao University,Qingdao 266001,China;College of Stomatology,Binzhou Medical College,Yantai 264100,China)

机构地区：[1]青岛市口腔医院中心实验室,青岛266001 [2]山东大学齐鲁医院(青岛)输血科,青岛266035 [3]青岛大学医学院,青岛266001 [4]滨州医学院口腔学院,烟台264100

出　　处：《现代免疫学》2023年第1期16-21,共6页Current Immunology

基　　金：国家自然科学基金(81273206);青岛市卫生健康委员会项目(2018-wjzd108);青岛市医疗卫生重点学科建设项目(2020-2022)。

摘　　要：为探讨跨膜衔接蛋白——富含鞘磷脂的脂膜微区结合酪氨酸磷酸化蛋白(phosphoprotein associated with glycosphing-olipid-enriched microdomains 1,PAG1)棕榈酰化位点突变对CD59介导的Jurkat细胞活化、增殖等生物学效应的影响,通过慢病毒转染技术建立PAG1棕榈酰化位点突变的Jurkat细胞株,用CD59单克隆抗体刺激试验组和阴性对照组。用免疫荧光检测PAG1、CD59在细胞上的表达及定位关系;CCK-8法及FACS检测细胞的增殖、凋亡情况;Western blotting检测Jurkat细胞信号转导通路中相关信号蛋白的变化。结果显示,PAG1、CD59分子均定位于细胞膜上且表达位置重叠。棕榈酰化位点突变后,PAG1与CD59分子虽出现点簇状聚集现象,但二者并不重叠。位点突变不影响细胞增殖和凋亡(P>0.05),但CD59单克隆抗体刺激后,细胞凋亡水平显著下降(P<0.05),且TCR活化通路下游分子非受体酪氨酸激酶Fyn、淋巴细胞特异性蛋白酪氨酸激酶(lymphocyte-specific protein tyrosine kinase,Lck)、磷脂酶Cγ1(phospholipase Cγ1,PLC-γ1)表达均下降。该研究提示,PAG1棕榈酰化位点突变后不能抑制CD59介导的Jurkat细胞增殖。This study proposes to investigate the effect of palmitoylation site mutation of phosphoprotein associated with glycosphingolipid-enriched microdomains 1(PAG1)on CD59-mediated activation and proliferation of Jurkat cells.To this end,PAG1 with palmitoylation site mutations were transfected into Jurkat cell lines using lentivirus and the cells were stimulated by CD59 monoclonal antibody.The expressions and localizations of PAG1 and CD59 were measured by immunofluorescence cytochemistry.The proliferation and apoptosis of Jurkat cells were measured using CCK-8 assay and FACS.Western blotting was used to monitor the changes in protein expression levels.The results showed that PAG1 and CD59 molecules were both localized on the cell membrane and they displayed overlapped expression patterns.The mutation at the palmitoylation site abolished this overlap and caused the clusterization of PAG1 and CD59.Mutation at the palmitoylation site did not affect Jurkat cell proliferation and apoptosis(P>0.05).However,upon stimulation by CD59 monoclonal antibody,the level of cell apoptosis decreased significantly(P<0.05).In addition,upon antibody stimulation,the expressions of TCR pathway downstream genes including non-receptor tyrosine kinase Fyn,lymphocyte-specific protein tyrosine kinase(Lck),and phospholipase Cγ1(PLC-γ1)were all decreased.In conclusion,PAG1 palmitoylation site mutation does not inhibit the CD59-mediated proliferation of Jurkat cells.

关 键 词：棕榈酰化位点突变 富含鞘磷脂的脂膜微区结合酪氨酸磷酸化蛋白 CD59 信号转导 

分 类 号：R392.11[医药卫生—免疫学]