Ile73Asn mutation in protein C introduces a new N-linked glycosylation site on the first EGF-domain of protein C and causes thrombosis
Ile73Asn mutation in protein C introduces a new N-linked glycosylation site on the first EGF-domain of protein C and causes thrombosis
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蛋白 C 中的 Ile73Asn 突变在蛋白 C 的第一个 EGF 结构域上引入了新的 N 连接糖基化位点并导致血栓形成
DOI：
10.3324/haematol.2019.227033
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发表时间：
2020-06-01
期刊：
影响因子：
10.1
通讯作者：
Rezaie, Alireza R.
中科院分区：
文献类型：
--
作者：
Lu, Yeling;Mehta-D'souza, Padmaja;Rezaie, Alireza R.
活化的蛋白 C 通过有限的蛋白水解作用，通过蛋白 S 依赖性灭活因子 Va 和 VIIIa 来发挥其抗凝血活性。我们发现一名静脉血栓患者的血浆蛋白 C 抗原水平为 63%，活动水平为 44% 和 23%（通过显色和凝血检测监测）。遗传分析显示先证者在PROC中携带复合杂合突变（c.344T>A、p.I73N和c.1181G>A、p.R352Q）。我们单独表达了蛋白 C 突变，发现凝血酶-血栓调节蛋白正常激活这两种变体，并且所得的激活蛋白 C 突变体表现出正常的酰胺分解和蛋白水解活性。然而，虽然活化蛋白 C-R352Q 对因子 Va 的蛋白 S 依赖性催化活性正常，但活化蛋白 C-I73N 的催化活性显着受损。这些结果表明，Ile 至 Asn 的取代损害了活化蛋白 C-I73N 与蛋白 S 的相互作用。该结论得到了活化蛋白 C-I73N 在蛋白 S 缺陷中但在正常血浆中没有的正常抗凝活性的支持。进一步分析显示，Ile 替换为 Asn 在蛋白 C 的第一个 EGF 样结构域上引入了新的糖基化位点，从而对活化蛋白 C 与蛋白 S 的相互作用产生不利影响。活化蛋白 C-R352Q 仅在亚生理浓度的 Na+ 和 Ca2+ 下表现出活性降低，表明该残基有助于蛋白酶的金属离子结合亲和力，而没有明显的不利影响 其在金属离子生理水平存在下的功能。这些结果深入了解了活化蛋白 C 中的 I73N/R352Q 突变导致携带这种复合杂合突变的先证者血栓形成的机制。
Activated protein C exerts its anticoagulant activity by protein S-dependent inactivation of factors Va and VIIIa by limited proteolysis. We identified a venous thrombosis patient who has plasma protein C antigen level of 63% and activity levels of 44% and 23%, as monitored by chromogenic and clotting assays. Genetic analysis revealed the proband carries compound heterozygous mutations (c.344T>A, p.I73N and c.1181G>A, p.R352Q) in PROC. We individually expressed protein C mutations and discovered that thrombin-thrombomodulin activates both variants normally and the resulting activated protein C mutants exhibit normal amidolytic and proteolytic activities. However, while protein S-dependent catalytic activity of activated protein C-R352Q toward factor Va was normal, it was significantly impaired for activated protein C-I73N. These results suggest that the Ile to Asn substitution impairs interaction of activated protein C-I73N with protein S. This conclusion was supported by a normal anticoagulant activity for activated protein C-I73N in protein S-deficient but not in normal plasma. Further analysis revealed Ile to Asn substitution introduces a new glycosylation site on first EGF-like domain of protein C, thereby adversely affecting interaction of activated protein C with protein S. Activated protein C-R352Q only exhibited reduced activity in sub-physiological concentrations of Na+ and Ca2+, suggesting that this residue contributes to metal ion-binding affinity of the protease, with no apparent adverse effect on its function in the presence of physiological levels of metal ions. These results provide insight into the mechanism by which I73N/R352Q mutations in activated protein C cause thrombosis in proband carrying this compound heterozygous mutation.