紅斑性狼瘡病人anti-Sm自體免疫抗體因蛋白質甲基化程度造成的辨識差異與血管炎病人之c-ANCA所辨識自體免疫抗原的蛋白質體研究

Abstract

Abstract1 Patients suffering from the autoimmune disease systemic lupus erythematosus (SLE) spontaneously produce autoantibodies against a multitude of cellular components. Antibodies against Sm proteins (anti-Sm autoantibodies) are SLE-specific and react predominantly with the Sm proteins B/B’, D1, D3, E, F, G and, to a lesser extent, D2. Sm protein D1 and D3 were reported to contain symmetric di-methylarginines（sDMA）and a few different anti-Sm autoantisera recognized only the sDMA peptide of SmD1 and D3 but not unmethylated or asymmetric dimethylarginine peptides. The results indicate the importance of methylarginine modification for autoantibody recognition. We thus would like to know 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. 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. There were no significant differences between the signals corresponding to SmD1 in samples of different methylation status. However, in one-dimensional and 2D-dimensional gel electrophoresis, weaker signals between molecular mass of 18 to 21 kDa and about 40 kDa were both consistently detected from cell extracts treated with AdOx compared to the ones without AdOx treatment. However, We identified these proteins by mass spectrometry. We were interested in two putative autoantigens CArG binding factor and ZNF9 proteins, they contain GAR domain and are likely to be methylarginine containing proteins. We inferred that CArG binding factor and ZNF9 protein might contain methyl-arginines thus lead to the differential recognition by anti-Sm autoantibodies due to their mehtylation status. Abstract2 Antineutrophil cytoplasmic autoantibodies（c-ANCA）are predominantly found in patients with small-vessel vasculitis such as Wegener’s granulomatosis. Proteinase 3（PR3） was identified as the principle target antigen of c-ANCA. The purpose of our studies is to determine whether other autoantigens can be identified for c-ANCA. We used proteomic methods to identify the autoantigens of c-ANCA. c-ANCA autosera from local patients were used in western blot analyses of LCL and HeLa cell extracts separated by one- or two-dimensional gel electrophoresis. The protease digested putative antigens were taken out the gels and identified by mass spectrometry. Among these antigens, we were interested in Hsp70, Hsp60 and vimentin since they have been reported as autoantigens in some autoimmune diseases. However, recombinant Hsp70, Hsp60 and vimentin proteins could be recognized by six of seven c-ANCA and four normal sera tested. Thus Hsp70, Hsp60 and vimentin proteins are possibly common antigens in human. We have also identified ATP synthase and eukaryotic translation elongation factor 1 beta 2 as putative c-ANCA autoantigens. The same method can be used to determine if they are c-ANCA autoantigens.