The interaction of thrombomodulin with Ca2+
The interaction of thrombomodulin with Ca2+
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DOI：
10.1046/j.1432-1327.1999.00398.x
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发表时间：
1999-06-01
期刊：
影响因子：
--
通讯作者：
Morser, J
中科院分区：
文献类型：
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作者：
Light, DR;Glaser, CB;Morser, J
凝血调节蛋白（TM）是凝血酶激活蛋白C的辅助因子，第6表皮生长因子结构域（EGF6）一致Ca2+位点的每个残基对这种辅助因子的活性至关重要[Nagashima， M., Lundh， E., Leonard， J.C, Morser， J. & Parkinson， J.F.(1993)]。化学学报，26(2):588 - 592。通过对Ca2+-贫缓冲液的彻底透析，制备了TM胞外结构域的三种可溶性类似物，即solulin （Glu4-Pro490）、TM(E)1-6 （Cys227-Cys362）和TM(E)14-6 (Va134-Cys462)，用于平衡透析实验。然而，这三种类似物仍然含有一个紧密结合的Ca2+ (K-d接近2 μ M)，只能通过EDTA去除。使用Ca2+依赖的EGF6单克隆抗体进行表位定位，进一步定位了这个紧密的Ca2+位点。在10 ~ 200 μ M之间的[Ca-45(2+)]中对可溶性TM类似物进行平衡透析，发现在solulin和TM(E)1-6中都有第二个Ca2+位点（K-d = 30 +/- 10 μ M），但在TM(E) 4-6中没有。Ca2+结合到第二个位点不受结合凝血酶的影响，我们将其归因于EGF3中一致的Ca2+位点。通过在BIAcore（TM）仪器中测量k（assoc）和k（diss）率，观察到用EDTA处理的固定化solulin去除高亲和力Ca2+，凝血酶与TM的结合亲和力降低了75倍。通过远紫外CD光谱检测到的Ca2+依赖构象跃迁表明Ca2+结合后结构更有序。与EDTA的蛋白酶处理相比，结合Ca2+稳定的可溶性TM在EGF6的Arg456和His457之间的胰蛋白酶样蛋白酶敏感位点抗蛋白酶消化。最后，TM含有EGF结构域4-6，但缺乏EGF3和egf4之间的结构域间环（TM(E)4-6），对蛋白C的激活具有与TM(E)4-6相同的Ca2+依赖性，表明该结构域间环不参与Ca2+结合。
Thrombomodulin (TM) is a cofactor for protein C activation by thrombin and each residue of a consensus Ca2+ site in the sixth epidermal growth factor domain (EGF6) is essential for this cofactor activity [Nagashima, M., Lundh, E., Leonard, J.C., Morser, J. & Parkinson, J.F. (1993) J. Biol. Chem. 268, 2888-2892]. Three soluble analogs of the extracellular domain of TM, solulin (Glu4-Pro490), TM(E)1-6 (Cys227-Cys362) and TM(E)14-6 (Va134-Cys462) were prepared for equilibrium dialysis experiments by exhaustive dialysis against Ca2+-depleted buffer. However, all three analogs still contained one tightly bound Ca2+ (K-d approximate to 2 mu M), which could only be removed by EDTA. Epitope mapping with Ca2+-dependent monoclonal antibodies to EGF6 provided further localization of this tight Ca2+ site. Equilibrium dialysis of the soluble TM analogs in [Ca-45(2+)] between 10 and 200 mu M revealed a second Ca2+ site (K-d = 30 +/- 10 mu M) in both solulin and TM(E)1-6, but not in TM(E)i4-6. Ca2+ binding to this second site was unaffected by bound thrombin and we attribute it to the consensus Ca2+ site in EGF3. A 75-fold decrease in the binding affinity of thrombin to TM was observed with immobilized solulin treated with EDTA to remove the high affinity Ca2+ by measuring k(assoc) and k(diss) rates in a BIAcore(TM) instrument. Ca2+-dependent conformational transitions detected by CD spectroscopy in the far UV indicate a more ordered structure upon Ca2+ binding. Bound Ca2+ stabilized soluble TM against protease digestion at a trypsin-like protease-sensitive site between Arg456 and His457 in EGF6 compared with protease treatment in EDTA. Finally, TM containing EGF domains 4-6, but lacking the interdomain loop between EGF3 and 4 (TM(E)4-6), has an identical Ca2+ dependence for the activation of protein C as found for TM(E)i4-6, indicating this interdomain loop is not involved in Ca2+ binding.