人類凝血酶調節素結構分析：I.人類凝血酶調節素類外源凝集素區域的表現、純化與結晶II.人類凝血酶調節素上皮生長因子區域小角度X 光散射及螢光共振能量轉移分析

Abstract

人類凝血酶調節素（Thrombomodulin， TM）為存在於血管內皮細胞表面之醣蛋白，其大小約為70kDa。人類凝血酶調節素參與了多種生化反應，如血液凝結、血纖維蛋白溶解、免疫反應、細胞附著及增生等。此蛋白質依其結構與功能由Ｎ端至C端可分成五個不同的區域：c-type lectin-like domain、連續六個EGF-like domains、serine/threonine-rich domain、transmembrane domain以及cytoplasmic domain。人類凝血酶調節素參與的各種生化功能和其結構有關，因此我們使用幾種不同的結構功能分析方法來探討人類凝血酶調節素的結構區域如何參與各種生化反應。藉由蛋白結晶繞射的技術，我們期待在原子層級觀察到人類凝血酶調節素如何利用不同的區域之結構，和其他分子(例如：thrombin、protein C等) 反應形成複合體來調控上述生化反應。此外，我們也透過小角度X光散射實驗得到蛋白質於溶液中的表面構形；這部份配合了螢光共振能量轉移分析 (FRET)，用來探討TM之dimerization。 我們使用酵母菌表現系統來表現TM之片段 (TMD-23) ，這包含了EGF-like domain 以及 serine/threonine-rich domain。這類 TMD-23，純化後透過 SDS-PAGE發現有醣化現象，而在non-reducing gel上則可觀察到 TMD23 存在monomer 及 dimer。所以在結構分析前必須以分子篩分開不同型態的TMD23。接著將酵母菌表現的TMD-23純化濃縮後進行結晶條件的篩選，另外將TMD23濃縮到不同濃度後，進行小角度X光散射實驗 （small-angle X-ray scattering） ，並配合電腦軟體分別模擬TMD-23 monomer， dimer 及 non-glycosylated TMD23在水溶液中和鈣離子結合前後的結構變化。結果發現鈣離子會使TMD23的結構改變。由於先前分子篩數據顯示 TMD-23 於溶液中可能以dimer的狀態存在，所以我們就將 TMD-23 構築於帶有CFP及YFP的質體上，轉殖入細胞後去觀察是否有 FRET 訊號產生。初步結果顯示FRET效率不高，但無法就此斷定 TMD23 於細胞內沒有形成 dimer，可能是螢光物質的位置影響結果。 另外TMD-1，我們則採用大腸桿菌系統來表現。由於先前利用大腸桿菌表現之TMD-1容易凝集沉澱，所以我就將TMD-1構築於帶有glutathione-S-transferase的質體上，觀察接上較大的蛋白是否會使TMD1不易凝集沉澱，但結果發現只要濃度高於 5 mg 仍會有凝集沉澱的現象產生。

Thrombomodulin (TM) is a 70 kDa multifunctional glycoprotein expressed on the epithelial cell surface. TM has different biological functions, impacting on coagulation, fibrinolysis, inflammation, cell adhesion, and cell proliferation. This glycoprotein is structurally organized into 5 distinct domains. From the N-terminus to the C-terminus, TM has an N-terminal C-type lectin-like domain, six EGF-like repeats, and a serine/threonine-rich region, a single transmembrane segment and a short cytoplasmic tail inside the membrane. For the works described in this thesis, including protein crystallization, SAXS (small-angle X-ray scattering) and FRET analyses, TMD-23 containing the EGF-like domains and Ser/Thr-rich domain was expressed with Pichia pastoris expression system. To understand how the distinct functions of this molecule can be achieved through each individual domains, the crystal structures of various domains in atomic resolutions should be anticipated. Meanwhile, before the successful crystallization trials are carried out, we have started to perform the SAXS experiments to deduce the structure envelope of TM in solution. From the non-reducing SDS-PAGE gel, there were two forms of TM (here, the TMD-23) -- monomer and dimer -- existed in our protein solution. Upon performance of the molecular sieving, monodispersed protein solutions were applied for SAXS analysis. According to our results, conformational changes are supposed to be induced by different concentrations of calcium in both TMD-23 monomer and dimer solutions. When compared with the unglycosylated monomer, glycosylated TMD-23 shows an additional structural region which may be comprehended as the mannose oligosaccharides on the EGF-like domain. As described above, the TMD-23 dimer has been observed on the SDS-PAGE. This may be brought from the nature of TM molecule in cells or from only the aggregation phenomenon in solution. Therefore it should be necessary to investigate the dimerization of TM in vivo. Experiments using FRET analysis through the transfection of plasmids constructed with TMD-23 containing CFP/YFP into HEK293 cells have been performed regarding this purpose. From our preliminary data, no apparent dimerization could be observed. However, we should not exclude that such results are probably due to the opposite orientation of TMD-23 dimer, with which the locations of CFP and YFP are far from each other, so that the distance required for the detection of FRET signal upon fluorescence emission might not be appropriate. The expression of TMD-1, consisting of only the C-type lectin-like domain, in E. coli results in the formation of inclusion bodies. We have constructed the plasmid containing TMD-1 and glutathione-S-transferase to improve the protein solubility through the attachment of a larger soluble part. However, the results show again the protein aggregation.