在此研究計劃中，我們想要探討蛋白質精胺酸甲基化（分為對稱型或不對稱型雙甲基精胺酸）和自體免疫疾病的關係。許多蛋白質包含了甲基精胺酸，如：fibrillarin、部分的hnRNP蛋白、myelin basic protein以及SmD1、D3蛋白，而這些蛋白也已被發現是許多自體免疫疾病的自體免疫抗原。在這個計劃中，我們首先從紅斑性狼瘡病人中取得anti-Sm和anti-RNP自體免疫抗體血清，尋找抗血清中是否有因精胺酸甲基化差異而對蛋白質辨識差異之抗體，再利用蛋白質體學方法找出有甲基化的自體免疫抗原。我們用一位病人血清作西方點墨法時，發現低甲基化之細胞萃取蛋白和正常甲基化組比較，有數調訊號減低情況。將相關蛋白點挖出，以蛋白脢水解後作質譜分析，有二蛋白質ZNF9以及CARG binding protein含有典型精胺酸甲基化蛋白質中出現之RGG序列，我們建構此二蛋白GST-tag之重組蛋白，並證明此二蛋白在試管中可被精胺酸甲基轉移脢甲基化。將FLAG-tag之重組蛋白表現於HeLa細胞，FLAG-ZNF9蛋白可被對稱或非對稱雙甲基精胺酸的抗體辨識，同時於低甲基化條件下所得到的FLAG-ZNF9蛋白，其被anti-Sm自體免疫抗體辨識減低。將ZNF9蛋白中的RGG序列移除，則不會被甲基精胺酸的抗體及anti-Sm 自體免疫抗體辨識。 In this project we would like to find out the relationships between protein methylation, mostly protein N-arginine methylation (symmetric or asymmetric NG,NG-dimethylarginines; sDMA or aDMA) and autoimmune disease. Methylarginine containing proteins such as fibrillarin, several hnRNP, myelin basic protein and SmD1 and D3 are autoantigens of different autoimmune diseases. Sm protein D1 and D3 contain symmetric di-methylarginines and a few different anti-Sm autoantisera recognized only the sDMA peptide of SmD1 and D3 but not unmethylated or asymmetric dimethylarginine peptides. We thus examined if the anti-Sm sera from local SLE patients also preferentially recognize the methyl-modified Sm D proteins and if there are other proteins that can be differentially recognized due to their methylation states. Anti-Sm autosera from three different SLE patients were used for western blot analyses of adenosine dialdehyde (AdOx, an inhibitor of protein methylation) treated (presumably hypomethylated) and untreated (normal methylated) HeLa cell extracts. A few reduced signals but not SmD proteins were consistently detected from cell extracts treated with AdOx compared to the ones without AdOx treatment by SDS-PAGE analyses. By two-dimensional electrophoresis, the differentially detected signals were further pinpointed and putative spots were picked and analyzed by MS and MS╱MS analyses. Two proteins (ZNF9 and CArG binding protein 1) contain typical arginine and glycine (RGG) sequences of the arginine methyltransferase substrates were identified. Recombinant GST-ZNF-9 and GST-CArG-BP1 fusion proteins expressed in E. coli can be in vitro methylated by arginine methyltransferase PRMT1 or RMT1. The signals detected by 7E6 and anti-Sm for the FLAG-ZNF9 expressed in HeLa cells were much stronger than ones expressed in AdOx-treated cells. Furthermore, wild-type FLAG-ZNF9 could be recognized by SYM10 (sDMA specific antibody) and ASYM24 (aDMA specific antibody) but GAR-domain deleted mutant ZNF9 could not. Putative interaction of FLAG-ZNF9 with PRMT1 and PRMT5 is suggested by a co-immunoprecipitation experiment. In summery, we demonstrate that ZNF9 has both symmetric and asymmetric dimethylarginine modifications and can be differentially recognized by anti-Sm according to its methylation status.
