人類凝血酶調節素不同區段之表現、純化以及分析

Expression, Purification and Structural Analysis of Various Domains of Human Thrombomodulin

吳東祈(Dung-Chi Wu)

指導教授 ： 樓國隆

共同指導教授 ： 李玉梅 ； 沈三泰(San-Tai Shen)

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01713

摘要

人類凝血酶調節素（Thrombomodulin; TM）是一大小約為70kDa的I型跨膜糖蛋白，主要表現在於上皮細胞表面。人類TM基因無內含子，其轉譯出的蛋白質含多個結構域，從N-端開始，為一類C型凝集素結構區段（Lectin-C like domain; TMD1），主要和發炎反應相關，其後則是接了6個類EGF重複序列（6x EGF-like repeats; TMD2），TM即是透過這6個類EGF重複與凝血酶（thrombin）或是和其他蛋白質如蛋白質C （protein C）或凝血酶啟動纖溶抑制物（thrombin-activatable fibrinolysis inhibitor）相互作用，再來則是一段絲氨酸/蘇氨酸豐富的區域（Serine/Threonine-rich domain; TMD3），其上有四個潛在的O-連接糖基化位點，最後則是一個跨膜段（TMD4）和位於細胞質中的短尾巴（TMD5）。
TM在生理上表現了多種功能：包含血液凝結（coagulation），血纖維蛋白溶解（fibrinolysis），發炎反應（inflammation），以及細胞粘附與增生（cell adhesion and proliferation）。但是，TM的最主要功能則是作為輔助因子與凝血酶 （thrombin）形成複合物，與TM的結合會幫助凝血酶誘導抗凝途徑中蛋白質C （protein C) 活化，而這可提高thrombin活化protein C的速度將近千倍。與TM在功能上的發現的重要性相比，目前對於TM的結構的研究，無論是結構域或是整體，都是相對有限的，目前唯一解出的晶體結構是TM 的一個片段，EGF4-6，與thrombin形成的複合物（Thrombin-Thrombomodulin EGF4-6 complex）。而為了了解TM是藉由何種機制來增強thrombin-induced的 protein C activation達到千倍，這可能需要對TM的結構有更進一步的了解才有辦法解釋，因此我們便想試著表現不同結構區段的TM來對其進行結構的研究。
從先前的數據，大腸桿菌與酵母菌似乎都不是一個適合表現TM的系統。 先前TMD1（只有Lectin-C like domain）在大腸桿菌中的表達，結果會形成包涵體。然而在酵母菌表現系統中的結果，再次發現有蛋白質聚集的情形。所以，這一次我們決定使用不同的系統來表現TM的各個區段。最後我們選擇的是果蠅細胞表現系統，（Drosophila Schneider 2 cell; S2 cells）。 S2 cells表現系統的優點是，昆蟲細胞的醣基化修飾較酵母菌單純，並且也容易培養。同時我們為了探討TM是藉由何種機制來調節凝血酶活化蛋白質C，我們也試著進一步利用S2 cells來表現蛋白質C以及凝血酶。
在本篇論文中，我們成功地克隆到TM的不同結構域區段，包括了Lectin-C domain（LC; TMD1），EG-like domain（EGF; TMD2），以及soluble TM（sTM; TMD12）。同時我們也克隆了wild-type/mutant thrombin。將這些成功構築的質體轉染進S2 cells中，並且利用hygromycin B來做篩選，直到stable clone篩選完成後才開始進行重組蛋白的表現。我們利用ELISA和LC-MS/MS來確認表現的重組蛋白，同時也利用酵素活性分析來驗證表現的重組蛋白確實具有生理活性，而這些結果表明，利用S2細胞作為蛋白質表現系統是適合於我們的研究標的。而在本篇論文中，我們嘗試利用目前已經日趨成熟的小角度X光散射（SAXS）實驗技術來研究不同區段的TM結構。在小角度X光散射實驗中，我們利用 ATSAS 程式分析 TMD12的蛋白分子數據以及TMD2在添加鈣離子以及不添加鈣離子的情形下的蛋白分子數據，並利用 DAMMIF 及 Situs 重建蛋白表面構造（ab-initio envelope）。同源模型(homology model)的構築則是利用Swiss-model database進行同源模擬來模擬TMD1的結構，而TMD2的EGF1-3亦同樣利用Swissmodel database，利用TMD2已解出的EGF4-6晶體結構來模擬，並將這些模擬與小角度X光散射實驗所得之表面構造進行比較，並且反計算出同源模擬之小角度X光散射曲線後與實際實驗數值比較，結果顯示同源模型與重建之表面構造符合。

關鍵字

凝血酶調節素 ； 類EGF重複序列 ； 鈣離子 ； 小角度X光散射

並列摘要

Thrombomodulin (TM) is a 70 kDa type I transmembrane glycoprotein mainly expressed on the epithelial cell surface. The intron-less gene of human TM encoded a multiple domains protein including an N-terminal C-type lectin-like domain (TMD1) which may associate in inflammation, six EGF-like repeats (TMD2) which would interact with thrombin and the other proteins such as protein C or thrombin-activatable fibrinolysis inhibitor, a serine/threonine-rich region (TMD3) which have four potential O-linked glycosylation sites, a single transmembrane segment (TMD4) and a short cytoplasmic tail (TMD5).
TM exhibits a range of physiologically multiple functions: coagulation, fibrinolysis, inflammation, cell adhesion, and cell proliferation. But the major function of TM is as a cofactor in the thrombin-induced activation of protein C in the anticoagulant pathway by forming a complex with thrombin, and this would raises the speed of protein C activation thousand-fold. In spite of its importance, structural studies of TM and its domains are limited; only crystal structures of a fragment of TM EGF4-6 complex with thrombin are available. In order to understand the mechanism of how TM enhances thrombin-induced protein C activation by thousand-fold, besides, there are several counteracting molecules complexed with thrombomodulin and fine-tuning the haemostatsis, such as thrombin-thrombomodulin-protein C may turn the coagulation cascade off, thrombin-thrombomodulin-TAFI which would shut-down fibrinolysis cascade. These dynamic mechanisms of how thrombomodulin modulate thrombin are unclear and remained to be resolved. To date, the only structure been resolved for thrombomodulin is EGF domain 4-6. In this study, we aim for expressing TM for functional and structural studies.
From our previous data, yeast or E. coli seemed not be suitable for expressing TM. The expression of TMD1, consisting of only the C-type lectin-like domain, in E. coli results in the formation of inclusion bodies. And TMD1 expressed in yeast show again the protein aggregation. So, this time we decide to use the different system and plasmid to express the various domains of TM. We want to use the Drosophila Schneider 2 cell (S2 cell) line expression system. The advantage of S2 cell system is that the insect cell has relatively simple glycosylation to yeast, and it is also easily cultivated. And to explore how the EGF-like domains of TM modulate thrombin activity on protein C activation, we also aimed to express protein C and thrombin in S2 cell for further study.
In this study, we successfully cloned the lectin-C like domain (LC; TMD1), EGF-like domain (EGF; TMD2), and soluble form of TM (sTM; TMD12) for S2 cell expression. We also cloned the wild-type/mutant thrombin. The stable transfected clones have been selected by using hygromycin B. In the study, we successfully expressed pre-thrombin-2, Protein C and TM recombinant proteins in S2 cells analyzed by LC-MS/MS. Further, we also demonstrated that activated thrombin by ecarin indeed activates Protein C in the presence of TM. The activation activity is about thousand fold by ELISA. Then, we take advantage of the currently matured small-angle X-ray scattering (SAXS) technique to investigate the TM structure. In the SAXS experiment, we collected the SAXS data of TMD12, TMD2 with calcium and TMD2 without calcium, which have been characterized by programs implant in ATSAS, ab-initio envelope of each protein was reconstructed by DAMMIF and Situs. And the homology models of TMD12 and TMD2 have been built, TMD1 were built from swissmodel database according to sequence alignment by Swissmodel database and TMD2 were built by the crystal structure of resolved TM EGF4-6. And these models have been docked into average envelops and assessed. The results showed that the homology model can dock into SAXS envelope pretty well. And we found that EGF domain of TM may induce some conformational change by calcium. This is the first time, even though it is just an envelope, to view the conformation and calcium effect of EGF like domain 1~6 of TM structurally.

並列關鍵字

Thrombomodulin ； EGF-like repeats ； Calcium ； Small-angle X-ray Scattering(SAXS)

參考文獻

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