凝血酶調節素(Thrombomodulin ,TM )是一種表現於血管內皮細胞表面的醣蛋白，主要的功能是藉著結合凝血酶而活化蛋白質C進而達到抑制凝血反應。TM基因轉譯出557 個胺基酸，其蛋白質結構從氮端依序被分為lectin-like結構區, EGF-like 結構區, serine/ threonine-rich結構區, transmembrane結構區及碳端的cytoplasmic結構區。在先前的研究中，將突變的TM基因轉染至黑色素瘤細胞中顯示了TM基因調節細胞的生長速率是同時需要完整的lectin-like結構區及cytoplasmic結構區。因此我們認為TM的cytoplasmic結構區可能參與一些生理的功能。本篇研究中，我們觀察到在穩定表現TM基因的人類黑色素瘤A2058 細胞中，細胞是以很緊密相鄰的群落型態聚集生長，而且TM會明顯地聚集在細胞間的邊界上。相反的，在穩定表現缺失TM cytoplasmic結構區基因的人類黑色素瘤A2058 細胞中，細胞是獨立分散生長且失去了細胞與細胞間的黏著，這暗示著TM cytoplasmic結構區有參與調節細胞與細胞間黏著的角色。再更進一步，TM cytoplasmic結構區顯示有四個可能發生磷酸化的胺基酸位置包括 534 Tyr , 540 Ser , 550 Thr ,及 553 Thr，還有經由電腦軟體預測出含有PDZ蛋白結合的位置、WW蛋白結合的位置以及PKC磷酸化的位置；我們已藉由定點突變的方式將上述的胺基酸突變來分析它們對細胞形態的影響；我們注意到在PDZ 蛋白結合位置上542 Glu 以及WW 蛋白結合位置上兩個threonine(550,553)對於維持細胞聚集的形態是很重要的。除此之外，藉由MTT assay分析HEK293 細胞的生長速率，發現表現野生型TM 1.7 的細胞其生長速率比起表現TM △lec , TM △Cyt及空白載體的HEK293 細胞慢了許多，顯示TM lectin-like結構區及cytoplasmic結構區對於調節細胞生長扮演很重要的角色。最後，pERK以及PKC訊息傳遞路徑在表現TM的細胞中似乎有參於調節的角色。

　　Thrombomodulin (TM ) expresses on endothelial cell surface, and functions as a thrombin receptor to inhibit procoagulant reaction of thrombin by activation of protein C. TM gene encodes 557 amino acids, consisting of five distinct domains including N-terminal lectin-like domain, six epidermal growth factor-like (EGF-like) domain, serine/threonine -rich domain, transmembrane domain, and an intraceullar cytoplasmic tail. In previous study, transfection of melanoma cells with mutated TM constructs indicated that the effect of TM on proliferation required intact cytoplasmic and extracellular N-terminal lectin-like domain. It is possible that the cytoplasmic domain participates in some physiological functions. In this report, we observe that TM-expressed A2058 cells grow as closely clustered colonies, with TM localized prominently in the intercellular boundaries. Conversely, TM detected cytoplasmic domain-expressed A2058 cells grow singly with loose cell-cell adhesion. It implies that TM cytoplasmic domain plays a role in regulating cell-cell adhesion. Furthermore, TM shows 4 potential phosphorylated sites including one tyrosine534, one serine540, and two threonine(550,553), a computer predicted PDZ protein binding site, WW protein binding site and a protein kinase C phosphorylation site on its cytoplasmic domain. We analyze the role of these residues on cell morphology by site-directed mutagenesis. We notice that glutamine acid542 in the PDZ binding region and threonine(550,553) in WW binding site are important in affecting cell-cell adhesion morphology. Furthermore, the proliferation rates of HEK293 cell lines are performed by MTT assay that result shows that wild type TM1.7-expressed HEK293 cells with a decreased growth rate than HEK293-△lec , △cyt , and vector control. It suggests that TM’s lectin-like domain and cytoplasmic domain play an important role in regulating cell proliferation. Finally, pERK and PKC signaling pathway seem to play a role in modulating TM-expressed cell’s function.

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