Proteomic identification of heterogeneous nuclear ribonucleoprotein K as a novel cold-associated autoantigen in patients with secondary Raynaud's phenomenon.
Proteomic identification of heterogeneous nuclear ribonucleoprotein K as a novel cold-associated autoantigen in patients with secondary Raynaud's phenomenon.
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DOI：
10.1093/rheumatology/keu325
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发表时间：
2015-02
期刊：
影响因子：
5.5
通讯作者：
Lingli Yang;M. Fujimoto;H. Murota;S. Serada;M. Fujimoto;H. Honda;Kohji Yamada;Katsuya Suzuki;A. Nishikawa;Y. Hosono;Y. Yoneda;K. Takehara;Y. Imura;T. Mimori;T. Takeuchi;I. Katayama;T. Naka
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文献类型：
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作者：
Lingli Yang;M. Fujimoto;H. Murota;S. Serada;M. Fujimoto;H. Honda;Kohji Yamada;Katsuya Suzuki;A. Nishikawa;Y. Hosono;Y. Yoneda;K. Takehara;Y. Imura;T. Mimori;T. Takeuchi;I. Katayama;T. Naka
目的：本研究的目的是鉴定继发于CTD的RP患者中的冷相关自身抗体。方法用患者血清对非透性冷刺激的正常人真皮微血管内皮细胞（dHMVECs）进行间接免疫荧光染色。通过等压标签进行相对和绝对定量（iTRAQ）分析，分析冷诱导的细胞表面蛋白质组变化。应用血清蛋白质组分析（SERPA）筛选冷相关自身抗原。通过ELISA法测定了290名继发于CTD（SSc、SLE或MCTD）的RP患者、10名原发性RP患者和27名健康对照者中候选自身抗体的患病率。结果：在冷刺激的dHMVECs中，当与继发性RP患者血清孵育时，检测到细胞表面免疫反应性增强。通过iTRAQ分析，发现冷刺激后dHMVECs细胞表面的许多蛋白质，包括异质核核糖核蛋白K（hnRNP-K），增加。通过SERPA方法，hnRNP-K被鉴定为继发性RP患者的候选自身抗原。通过免疫印迹和流式细胞术证实冷诱导的hnRNP-K易位到细胞表面。通过ELISA分析，继发RP患者显示抗hnRNP-K自身抗体的患病率（30.0%，61/203）显著高于无RP患者（9.2%，8/87，P = 0.0001）、原发RP患者（0%，0/10，P = 0.0314）或健康对照（0%，0/27，P = 0.0001）。结论通过综合蛋白质组学研究，我们确定hnRNP-K为继发性RP患者的一种新的冷相关自身抗原。抗hnRNP-K自身抗体可能作为继发于各种CTD的RP的生物标志物。
OBJECTIVE The aim of this study was to identify cold-associated autoantibodies in patients with RP secondary to CTDs. METHODS Indirect immunofluorescence staining was performed on non-permeabilized cold-stimulated normal human dermal microvascular endothelial cells (dHMVECs), using patients' sera. Cold-induced alterations in cell surface proteomes were analysed by isobaric tag for relative and absolute quantitation (iTRAQ) analysis. Serological proteome analysis (SERPA) was applied to screen cold-associated autoantigens. The prevalence of the candidate autoantibody was determined by ELISA in 290 patients with RP secondary to CTDs (SSc, SLE or MCTD), 10 patients with primary RP and 27 healthy controls. RESULTS Enhanced cell surface immunoreactivity was detected in cold-stimulated dHMVECs when incubated with sera from patients with secondary RP. By iTRAQ analysis, many proteins, including heterogeneous nuclear ribonucleoprotein K (hnRNP-K), were found to be increased on the cell surface of dHMVECs after cold stimulation. By the SERPA approach, hnRNP-K was identified as a candidate autoantigen in patients with secondary RP. Cold-induced translocation of hnRNP-K to the cell surface was confirmed by immunoblotting and flow cytometry. By ELISA analysis, patients with secondary RP show a significantly higher prevalence of anti-hnRNP-K autoantibody (30.0%, 61/203) than patients without RP (9.2%, 8/87, P = 0.0001), patients with primary RP (0%, 0/10, P = 0.0314) or healthy controls (0%, 0/27, P = 0.0001). CONCLUSION By comprehensive proteomics, we identified hnRNP-K as a novel cold-associated autoantigen in patients with secondary RP. Anti-hnRNP-K autoantibody may potentially serve as a biomarker for RP secondary to various CTDs.