Altered posttranslational modification on U1 small nuclear ribonucleoprotein 68k in systemic autoimmune diseases detected by 2D Western blot.
Altered posttranslational modification on U1 small nuclear ribonucleoprotein 68k in systemic autoimmune diseases detected by 2D Western blot.
复制标题
通过 2D Western blot 检测系统性自身免疫性疾病中 U1 小核核糖核蛋白 68k 翻译后修饰的改变。
DOI：
10.1002/elps.201200058
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发表时间：
2012
期刊：
影响因子：
2.9
通讯作者：
Tomohiro Kato
中科院分区：
文献类型：
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作者：
Kouhei Nagai; Mitsumi Arito;Yukiko Takakuwa;Seido Ooka;Toshiyuki Sato;Manae S Kurokawa;Kazuki Okamoto;Teisuke Uchida;Naoya Suematsu;Tomohiro Kato
抗核糖核蛋白（抗RNP）抗体是可在系统性红斑狼疮（SLE）和混合性结缔组织病（MCTD）患者中检测到的代表性自身抗体之一。通常，提出自身抗原上的翻译后修饰（PTM）参与自身抗体的产生。在这项研究中，我们试图检测U1小核RNP 68 k亚基（U1 - 68 k）（抗RNP抗体的主要抗原）上PTM的变化。外周血单核细胞（PBMC）从患有MCTD、SLE和类风湿性关节炎（RA）的患者以及从健康供体获得。通过2D Western blot（WB）检测PBMC中的U1 - 68 k，其中提取的核蛋白通过2DE分离，然后使用WB检测U1 - 68 k。检测到超过20种PTM亚型，分子量为65.0、66.5和68.0kDa，pI在6.0和8.5之间。重要的是，与RA和健康组相比，MCTD和SLE组中具有66.5 kDa和pI7.5的斑点的相对强度显著增加。此外，发现这种U1 - 68 k同种型，特别是在其RS结构域中，与其他同种型相比具有显著降低的磷酸化。PTM交替可能是产生抗RNP抗体的步骤之一。
Anti‐ribonucleoprotein (anti‐RNP) antibodies are one of the representative autoantibodies detectable in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Generally, posttranslational modifications (PTMs) on autoantigens are proposed to be involved in the production of autoantibodies. In this study, we tried to detect the alteration in PTMs on a U1 small nuclear RNP 68k subunit (U1‐68k), a major antigen of anti‐RNP antibodies. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with MCTD, SLE, and rheumatoid arthritis (RA), and from healthy donors. U1‐68ks in the PBMCs were detected by 2D Western blot (WB), where extracted nuclear proteins were separated by 2DE, followed by the detection of U1‐68k using WB. More than 20 PTM isoforms were detected with different molecular weights of 65.0 , 66.5, and 68.0kDa, and different pIs between 6.0 and 8.5. Importantly, the relative intensity of the spot with 66.5 kDa and pI7.5 was significantly increased in the MCTD and SLE groups compared to the RA and healthy groups. Further, this U1‐68k isoform, in particular, in its RS domain, was found to have significantly decreased phosphorylation compared to the other isoforms. The PTM alternation may be one of the steps to generate the anti‐RNP antibodies.