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。此蛋白甲基化與否和自體免疫抗體辨識是否有關正在研究中。
In this project we are going to find out the relationships between protein methylation, mostly protein N-arginine methylation to form symmetric or asymmetric NP G P, NP G P: dimethylarginines (sDMA or aDMA), and autoimmune disease. Many methylarginine containing proteins such as fibrillarin, several hnRNP, myelin basic protein and SmD1 and D3 are known to be autoantigens of different autoimmune diseases. Sm protein D1 and D3 were reported to 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 by the anti-Sm sera due to their methylation states. We treated HeLa cells with adenosine dialdehyde (AdOx), an inhibitor of protein methylation. Anti-Sm autosera from three different SLE patients were used in western blot analyses of AdOx-treated (proteins presumably at hypomethylation state) and untreated (proteins at normal methylation states) HeLa cell extracts. Reduced signals between molecular mass of 18 to 21 kDa and about 31 kDa were consistently detected from cell extracts treated with AdOx compared to the ones without AdOx treatment by one-dimensional SDS-PAGE analyses. However, there were no significant differences between the signals corresponding to SmD1 in samples of different methylation status. By two-dimensional electrophoresis, the differentially detected signals were further pinpointed and putative spots were picked and digested by trypsin. The peptide fragments were analyzed by MS and MS/MS analyses. Interestingly, two proteins (ZNF9 and Carg binding protein 1) contain typical arginine and glycine (RGG) sequences of the arginine methyltransferase substrates were identified. We prepared recombinant GST-ZNF-9 and GST-Carg-bp1 fusion proteins expressed in E. coli. The recombinant proteins can be in vitro methylated by the arginine methyltransferase PRMT1 or RMT1. The confirmation of the proteins differentially detected by anti-Sm due to their methylation status are under investigation.
