針對類風溼性關節炎病患滑膜液中巴拉松酶一號蛋白之活性與其轉譯後修飾之探討

Investigation of differential post-translational modification profile and biological activity of paraoxonase 1 in synovial fluid from rheumatoid arthritis patients

李芳瑜(Fang-Yu Li)

指導教授 ： 林景堉

共同指導教授 ： 林榮俊

臺北醫學大學/醫學科技學院/醫學檢驗暨生物技術學系所/碩士(2014年)

摘要

類風溼性關節炎為常見的自體免疫疾病，全球約0.5 %人口罹患。這種慢性炎症會造成關節變形，甚至影響全身各處組織，包括皮膚、心臟、血管等。根據先前研究顯示，類風濕性關節炎患者血清/血漿內之「巴拉松酶一號 (PON1)」活性低於正常健康受試者，然而，造成活性改變的因素目前仍有待研究。在本篇論文中，巴拉松酶一號在類風溼性關節炎患者滑膜液中的濃度低於退化性關節炎患者，且類風溼性關節炎患者滑膜液中巴拉松酶一號的活性較低。研究指出，轉譯後修飾可能是細胞調節酵素活性或蛋白間交互作用的機制之一，因此在接下來的實驗中，將利用奈米級液相層析串聯質譜 (Nano-LC-MS/MS) 和生物資訊系統 Peaks software針對類風溼性關節炎與退化性關節炎患者滑膜液中巴拉松酶一號蛋白的轉譯後修飾進行研究。結果顯示，類風溼性關節炎患者和退化性關節炎患者滑膜液巴拉松酶一號具有不同的甲基化和乙醯化等轉譯後修飾，而這些轉譯後修飾的差異可能會影響巴拉松酶一號和高密度脂蛋白 (HDL) 相關蛋白的交互作用、巴拉松酶一號穩定性和活性上的差異。本研究包含三個部分：第一部份為測定類風溼性關節炎和退化性關節炎患者巴拉松酶一號活性；第二部分為確定類風溼性關節炎患者滑膜液中巴拉松酶一號蛋白的表現量；第三部分則是分析類風溼性關節炎患者滑膜液的巴拉松酶一號具何種特異性的轉譯後修飾。目的是透過蛋白體技術，分析類風溼性關節炎患者滑膜液與血清中巴拉松酶一號表現量及轉譯後修飾的變化，探討蛋白轉譯後修飾在類風溼性關節炎扮演的致病角色，同時根據上述實驗結果評估巴拉松酶一號蛋白在類風溼性關節炎患者滑膜液中特有的轉譯後修飾種類發展為類風溼性關節炎生物標誌的可行性。

關鍵字

類風溼性關節炎 ； 巴拉松酶奈米級液相層析串聯質譜儀 ； 轉譯後修飾

並列摘要

Rheumatoid arthritis (RA) is a common autoimmune disease that affects about 0.5 % of the world’s population. The RA patient is characterized by chronic synovitis and vasculitis, which leads to destruction of articular cartilage, ankylosis of joints, and systemic organ damages. The reduced activity of paraoxonase 1 (PON1) was widely observed in the plasma/serum sample of RA patients. Nevertheless, the molecular mechanism involved in affecting its activity remains unclear. In the present study, the relatively low level of PON1 protein was examed in the synovial fluid (SF) from the RA as compared with the osteroarthritis (OA) patients; moreover, the reduced activity of PON1 was observed in the SF sample of RA patients recruited into this project. Due to the regulatory effect of differential post-translational modifications (PTMs) on the physiological features, including enzymatic activity of individual protein, the PTM profiles of PON1 from the SF of OA and RA patients was identified by using nano LC-MS/MS-coupled PEAKS software analysis. The mass spectrometry data showed the differential PTM profiles of PON1 from the SF of RA patients, including acetylation and methylation, which may interfere with its association with HDL-related proteins. Taken together, the results suggested that the insufficient activity and the RA-specific PTM residues of PON1 may be considered as a novel indicator for the diagnosis of RA. Furthermore, the differential PTMs may constitute a molecular mechanism in regulating the activity of PON1 in the SF of RA patients, which correlated with the severity of synovitis and joints inflammation.

並列關鍵字

rheumatoid arthritis ； paraoxonase 1 ； mass spectrometry ； post-translational modification

參考文獻

1. Y. Kochi, A. Suzuki, R. Yamada, K. Yamamoto, Ethnogenetic heterogeneity of rheumatoid arthritis-implications for pathogenesis. Nature reviews. Rheumatology 6, 290 (May, 2010).

2. D. L. Jacobson, S. J. Gange, N. R. Rose, N. M. Graham, Epidemiology and estimated population burden of selected autoimmune diseases in the United States. Clinical immunology and immunopathology 84, 223 (Sep, 1997).

3. C. F. Kuo et al., Rheumatoid arthritis prevalence, incidence, and mortality rates: a nationwide population study in Taiwan. Rheumatology international 33, 355 (Feb, 2013).

4. A. M. Abdel-Nasser, J. J. Rasker, H. A. Valkenburg, Epidemiological and clinical aspects relating to the variability of rheumatoid arthritis. Seminars in arthritis and rheumatism 27, 123 (Oct, 1997).

5. M. C. Symmons D, Pfleger B., The global burden of rheumatoid arthritis in the year 2000. (2003).