人類凝血酶調節素(Thrombomodulin；簡稱TM)是血管內皮細胞表面的一種醣蛋白，它是身體中重要的抗凝血分子，主要是藉著結合凝血酶而限制凝血酶的促凝血活性及生理活性，科學家們利用包括生化分析及基因改造老鼠等方法探討凝血酶調節素的生理功能，發現凝血酶調節素也扮演抑制血塊溶解，抗發炎反應，抑制腫瘤細胞增生，維持正常的懷孕過程及胚胎發育等重要角色。在本實驗室最近的研究發現TM可以媒介細胞與細胞間的聯結，而且lectin-like結構區在此過程中是非常重要的，細胞間的連結作用對於細胞的爬行、增生及分化扮演重要的角色。而細胞連結的作用，主要是藉由細胞表面的蛋白與鄰近細胞表面(細胞對細胞)或細胞基質(細胞對細胞基質)上的蛋白結合，以及調節細胞內的訊息傳遞路徑來調控細胞連結所造成的細胞生理機能的改變。
ERM蛋白質已知是作為細胞骨架與膜蛋白之間的連結者，在ERM蛋白中，ezrin是被研究最多的蛋白。在實驗室初步的結果發現，確實ezrin可作為TM與細胞骨架的連結者。在本論文中，利用重組蛋白在細胞外做結合試驗(in vitro binding assay)的方式，來分析TM的胞內區域與ezrin的作用是直接結合或經由其他蛋白協助，此外，並更進一步探討在TM胞內區域上與ezrin結合有關的位置。
在本中研究證實，ezrin N端而非C端區域會直接結合於TM的胞內區域，不需其他分子輔助，這兩個分子結合呈現濃度依存性(dose-dependent)的現象，且其分離常數KD為0.98μM。另外利用截短TM胞內區域的方式，發現TM胞內區域中ezrin結合的區域是位於前九個胺基酸序列(RKKQGAARA)及最後九個胺基酸序列(RTERTRQRL)上。很多會與ERM蛋白質結合的膜蛋白，例如CD43, CD44 及 ICAM-2，在胞內區域靠近細胞膜附近的帶正電胺基酸區域被認為與ezrin的結合有關。利用在TM胞內區域靠近細胞膜附近的帶正電胺基酸區域胺基酸RKK以AAA取代的方式證明RKK確實為與ezrin結合有關的胺基酸。同時利用電腦分析預測TM胞內區域的特殊結構，發現在這個區域中有一個Class IV WW domain interaction motif (TP)，主要涉及與磷酸化有關的蛋白與蛋白結合作用。在這個motif上將threonine 571以glutamic acid取代後，會將其原本與ezrin結合的能力降低。TM與ezrin的結合方式可能涉及透過胞內區域靠近細胞膜附近的帶正電胺基酸區域以及與Class IV WW domain interaction motif有關。

Thrombomodulin (TM) is a glycoprotein that was originally found on vascular endothelium and is well characterized as a natural endothelial anticoagulant factor. Studies by several investigators have elucidated multiple aspects of TM functions in fibrinolysis, inflammation, proliferation, and in embryogenesis. Our recent study has demonstrated that TM may have a novel function mediating cell-cell adhesion through its lectin-like domain. Adhesive interactions play critical roles in directing the migration, proliferation, and differentiation of cells. These adhesive interactions, mediated by cell surface proteins that bind to others on adjacent cells (cell-to-cell) or in the extracellular matrix (cell-to-extracellular matrix), also regulate intracellular signal transduction pathways that control adhesion-induced changes in cell physiology.
The ERM(ezrin/radixin/moesin) family of proteins, of which ezrin is the best studied member, act as a linker between plasma membrane and the actin cytoskeleton. Previous work in our laboratory implicated that ezrin links TM to the actin cytoskeleton. In this study, we have examined whether the interaction of ezrin and TM is interact directly by in vitro binding assay. Furthermore, we identified the regions cytoplasmic domain of TM responsible for binding to ezrin.
Here we provide evidence that the ezrin N-terminal domain bound to the TM cytoplasmic domain with an estimated KD of 0.98μM and in absence of other cofactors . By truncating the TM cytoplasmic domain we showed that the ezrin-binding sequence was located in the juxtamembrane sequence nine amino acids residues (RKKQGAARA) and at C-terminal tail nine amino acids (RTERTRQRL). Most of ERM-binding proteins such as CD43, CD44 and ICAM-2 have a cluster of positively charged amino acids in the juxtamembrane region of the cytoplasmic tail that was found to be responsible for the binding. Alanine mutations of membrane-proximal basic amino acid residues in the cytoplasmic domain of TM identified RKK as a critical residue for ezrin interaction. Meanwhile, a Class IV WW domain interaction motif (TP) that involved phosphorylation-dependent protein-protein interaction in TM cytoplasmic domain was predicted. Glutamic acid mutations of threonine 571 in this region impaired its binding ability to ezrin. We therefore conclude both that positively charged amino acid cluster RKK and WW domain interaction motif in TM cytoplasmic domain were involved in ezrin binding.

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