白细胞介素-4通过上调蛋白质精氨酸甲基转移酶1的表达水平引起对氧磷酶2的精氨酸甲基化修饰

IL-4 induces paraoxonase 2 methylation by upregulating PRMT1 expression

目的验证质谱筛选的结果,确定对氧磷酶2(PON2)是否为蛋白质精氨酸甲基转移酶1(PRMT1)的底物蛋白。方法用白细胞介素-4(IL-4)刺激A549细胞后,用免疫沉淀法捕获发生精氨酸非对称性二甲基化修饰的蛋白,用梯度聚丙烯酰胺凝胶电泳分析捕获的蛋白,通过银染显示差异性条带,质谱分析差异性条带中的蛋白种类。用免疫沉淀检验PON2的甲基化修饰状态。检测PON2和PRMT1对IL-4表达的浓度依赖性和时间依赖性。用免疫共沉淀检测PON2和PRMT1的相互作用,并在MS203抑制Ⅰ型PRMT后检测PON2的甲基化修饰水平的变化。诱导小鼠哮喘模型,检测PON2表达的组织细胞类型,以及肺组织中PON2和PRMT1的表达水平。结果(1)质谱分析显示在IL-4处理的A549细胞中检出311个差异显示的蛋白,PON2为其中之一。(2)免疫沉淀显示,IL-4可促进A549细胞中PON2发生精氨酸非对称性二甲基化修饰,但IL-4刺激对PON2的表达水平无影响。(3)PON2与PRMT1有相互作用,IL-4可促进PON2与PRMT1结合,抑制Ⅰ型PRMT活性可以削弱IL-4对PON2发生精氨酸非对称性二甲基化修饰的促进作用。(4)PON2主要在小鼠气道上皮表达,且PON2的表达水平在对照组与哮喘组组织间差异无统计学意义(P>0.05)。结论PON2为PRMT1的底物蛋白。IL-4可以促进PON2发生精氨酸非对称性二甲基化修饰。

Objective To verify the results of mass spectrometry screening,and to confirm that paraoxonase 2(PON2)was the substrate protein of protein arginine methyltransferase 1(PRMT1).Methods A549 cell line was stimulated with IL-4,the protein with asymmetric dimethylation at arginine residues was captured by immunoprecipitation,and the captured protein was analyzed by gradient polyacrylamide gel electrophoresis.The differential bands were displayed by silver staining,and the protein species in the differential bands were analyzed by mass spectrometry.The methylation status of PON2 was examined by immunoprecipitation.The concentration and time dependence of PON2 and PRMT1 expression on IL-4 were analyzed.Immunocoprecipitation was used to detect the interaction between PON2 and PRMT1,and the methylation modification of PON2 was detected after MS203 inhibited Type Ⅰ PRMT.The mouse asthma model was established,and the cell types in which PON2 expressed and the expression levels of PON2 and PRMT1 in lung tissue were detected.Results Mass spectrometry showed that 311 differentially displayed proteins were detected in A549 cells treated with IL-4,and PON2 was one of them.Immunoprecipitation showed that IL-4 could promote asymmetric dimethylation at arginine residues in PON2 in A549 cells.The expression level of PON2 was not affected by IL-4 stimulation.There was an interaction between PON2 and PRMT1.IL-4 could promote PON2 binding with PRMT1;and inhibit the activity of Type Ⅰ PRMT thus weaken the promotion of IL-4 on asymmetric dimethyl modification at arginine residues in PON2.PON2 was mainly expressed in mouse airway epithelium,and there is no significant difference between control group and asthma tissue in PON2 expression.Conclusion PON2 is the substrate protein of PRMT1.IL-4 can promote asymmetric dimethylation at arginine residues in PON2.