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

Differential recognition of proteins due to methylation status by anti-Sm autoantibodies from SLE patients And Proteomic analysis of autoantigens of antineutrophil cytoplasmic autoantibodies (c-ANCA) from vasculitis patients

張宏豪(Hong-How Chang)

指導教授 ： 李娟

中山醫學大學/口腔醫學院/口腔醫學研究所/碩士(2005年)

https://doi.org/10.6834/CSMU.2005.00029

摘要

摘要1

在全身性紅斑性狼瘡自體免疫疾病的病人中，常會自發性的產生許多自體免疫抗體去對抗細胞內的成分。Anti-Sm自體免疫抗體是屬於紅斑性狼瘡疾病中特有的，它主要會辨識Sm蛋白，包括：Sm B/B’, D1, D3, E, F, G及辨識程度較微弱的D2。Sm D1、D3蛋白在近期的研究中被發現為對稱型精胺酸甲基化蛋白，且當它們的C端peptides上之精胺酸甲基化為對稱型時，就會被一些不同的anti-Sm自體免疫抗體所辨識；若為非對稱性或無甲基化時，則anti-Sm就無法辨識。由此可知精胺酸甲基化在自體免疫抗體的辨識上有其重要性。因此我們利用了本地紅斑性狼瘡病人的anti-Sm血清，來觀察是否會辨識帶有甲基化的Sm D蛋白，並且當我們去改變細胞內蛋白質甲基化程度時，是否有其他的蛋白也有類似辨識上不同的情形。利用西方點墨法比較三位不同病人的anti-Sm自體免疫血清，對有加（使細胞內蛋白為低度甲基化）或沒加AdOx（細胞內蛋白為正常甲基化的程度）處理的HeLa cell細胞萃取液之辨識程度。Sm D1蛋白沒有因著甲基化程度的不同，而造成辨識上信號差異。然而當比較有加和沒加AdOx處理的細胞萃取液時，無論是一維或二維膠電泳都有發現在40 kDa左右及21到18 kDa之間有一些辨識上減弱的信號。利用質譜儀分析這些可能的抗原蛋白。其中我們對CArG binding factor和ZNF9 protein這兩個可能的自體免疫抗原感到興趣，因為它們可能包含了GAR domain，並且它們也可能為精胺酸甲基化蛋白。所以，我們推論CArG binding factor和ZNF9 protein其上可能有精胺酸的甲基化，以致於因它們本身甲基化程度造成anti-Sm自體免疫抗體辨識上的差異。

摘要2

Antineutrophil cytoplasmic autoantibodies（c-ANCA）常顯著地出現在血管炎的病人中（如：韋格納肉芽腫）。Proteinase 3（PR3）是已被證實為c-ANCA特異性抗原。是否可發現其他一些有特異性的自體免疫抗原可以做為c-ANCA自體抗體的研判，為本篇研究的目的。我們以蛋白質體學的研究方法來鑑定c-ANCA的自體免疫抗原。利用一維和二維膠電泳展開並分離淋巴母細胞和HeLa細胞萃取液，再以本地病人的c-ANCA自體免疫血清做西方點墨法來鑑定其可能抗原。從膠上將這些經蛋白脢水解的可能抗原挖下並用質譜儀分析做蛋白質身份鑑定。在這些鑑定出的抗原中，我們對於Hsp70、Hsp60和vimentin這三個蛋白較感興趣，因為在已過的研究中，他們在一些自體免疫疾病中常做為自體免疫抗原。然而，利用7位病人中有6位的c-ANCA自體免疫血清與4位健康之人的血清會辨識純化的Hsp70、Hsp60和vimentin重組蛋白。因此，Hsp70、Hsp60和vimentin有可能是在人體中很普遍的抗原蛋白。我們也鑑定出另外兩個c-ANCA可能的自體免疫抗原：ATP synthase和eukaryotic translation elongation factor 1 beta 2。所以也可採用本研究的實驗方法來證實它們是否為c-ANCA的自體免疫抗原。

關鍵字

全身性紅斑性狼瘡 ； Anti-Sm自體免疫抗體 ； 精胺酸甲基化蛋白. c-ANCA ； 蛋白質體學 ； 自體免疫抗原

並列摘要

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.

並列關鍵字

systemic lupus erythematosus ； anti-Sm autoantibodies ； methylarginine containing protein. c-ANCA ； proteomic ； autoantigen

參考文獻

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