ZNF9精胺酸甲基化分析及其對anti-Sm抗體抗原性影響

Anti-Sm是一種在紅斑性狼瘡﹙SLE﹚病人中發生的自體免疫抗體。過去的研究指出，Sm D1蛋白上不同程度的精胺酸甲基修飾會造成anti-Sm對其產生辨識性差異。之前以三位本地SLE病人的Anti-Sm血清對經AdOx處理過的HeLa細胞蛋白﹙細胞內蛋白呈現低度甲基化﹚及未經處理的細胞蛋白﹙細胞內蛋白呈正常甲基化程度﹚進行西方點墨法實驗，結果並沒有觀察到細胞蛋白中的Sm D1蛋白因甲基化程度不同而使anti-Sm產生辨識性差異。然而不論是在一維或二維電泳膠上，都可以看到和未經AdOx處理的細胞蛋白相比，anti-Sm對經AdOx處理的樣本之中數個蛋白的辨識性明顯降低。這些蛋白經質譜儀鑑定後，發現其中之ㄧ為ZNF9蛋白。ZNF9蛋白具有精胺酸甲基轉移酶受質特徵的GAR domain，且放射性in vitro蛋白甲基化實驗中，RMT1及PRMT1分別能對GST-ZNF9甲基化。雖然GST-ZNF9重組蛋白進行非放射性in vitro蛋白甲基化實驗後，西方點墨法實驗分別以anti-Sm、7E6及ASYM24分析，都沒有觀察到和未經甲基化反應的GST-ZNF9相比有辨識性差異。但是轉殖入FLAG-ZNF9質體的HeLa細胞所表現之重組蛋白，卻會因轉殖細胞經AdOx處理與否，而使7E6及anti-Sm對其產生辨識性差異。後續的細胞轉殖實驗中，野生性的FLAG-ZNF9蛋白分別能被SYM10及ASYM24抗體所辨識，而刪除GAR domain的兩種FLAG-ZNF9則不被辨識；且在共免疫沉澱實驗中，也觀察到FLAG-ZNF9與PRMT1及PRMT5可能有交互作用，因此我們推測，ZNF9可能是精胺酸甲基轉移酶的受質，並具有非對稱型及對稱型雙甲基精胺酸修飾。

關鍵字

精胺酸甲基化

並列摘要

Anti-Sm is one type of autoantibodies from systemic lupus erythematosus (SLE) patients. Methylation status of specific arginine residues on SmD-1 has been reported to cause differential recognition by anti-Sm. Previous studies in our laboratory used Anti-Sm autosera from three different local SLE patients to analyze AdOx-treated (proteins presumably at hypomethylation state) and untreated (proteins at normal methylation state) HeLa cell extracts by western blots. There were no significant differences between the signals corresponding to SmD1 in samples of different methylation status. However, a few weaker signals were consistently detected from cell extracts treated with AdOx than the ones without. One interesting protein identified by mass spectrometry is ZNF9, which has the GAR domain, a common feature in substrates of protein arginine methyltransferases. We observed that recombinant GST-ZNF9 expressed in Escherichia coli could be methylated by recombinant RMT1 and PRMT1 in vitro. However, no significant differences could be detected between the signals corresponding to GST-ZNF9 of different methylation status after a non-radiation in vitro methylation experiment by western blot with a methylarginine specific antibody 7E6. Therefore, the E. coli expressed recombinant ZNF9 protein could not be used to investigate whether the arginine methylation status affect the recognition of anti-Sm. Nevertheless, FLAG-ZNF9 expressed in HeLa cells could be detected by 7E6 and anti-Sm, while the ones expressed in AdOx-treated HeLa cells has much weaker signals. Furthermore, wild-type FLAG-ZNF9 could be recognized by SYM10 (symmetric dimethylarginine specific antibody) and ASYM24 (asymmetric dimethylarginine specific antibody) but GAR-domain deleted mutant ZNF9 could not. We also observed that FLAG-ZNF9 may interact with two different protein arginine methyltransferase PRMT1 and PRMT5 by an co-immunoprecipitation experiment. In summery, in this study we demonstrate that ZNF9 has both symmetric and asymmetric dimethylarginine modifications and can be differentially recognized by anti-Sm due to the methylation status.

並列關鍵字

Arginine methylation

參考文獻

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2. Abramovich, C., Yakobson, B., Chebath, J., and Revel, M. (1997). A protein-arginine methyltransferase binds to the intracytoplasmic domain of the IFNAR1 chain in the type I interferon receptor.

3. Tang, J., Gary, J. D., Clarke, S., and Herschman, H. R. (1998). PRMT 3, a type I protein arginine N-methyltransferase that differs from PRMT1 in its oligomerization, subcellular localization, substrate specificity, and regulation. The Journal of biological chemistry 273(27), 16935-16945

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6. Lee, J., Sayegh, J., Daniel, J., Clarke, S., and Bedford, M. T. (2005). PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family. The Journal of biological chemistry 280(38), 32890-32896

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ZNF9精胺酸甲基化分析及其對anti-Sm抗體抗原性影響

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