| Summary: | 碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 101 === 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.
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