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

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.