Research Center for Low Temperature and Materials Sciences, Kyoto University, Kyoto, Kyoto, Japan.
Institute for Chemical Research, Kyoto University, Uji, Kyoto, Japan.
PLoS One. 2014 Jan 21;9(1):e85045. doi: 10.1371/journal.pone.0085045. eCollection 2014.
Annexin A2 (ANXA2), a member of the annexin family of cytosolic Ca(2+)-binding proteins, plays a pivotal role in vascular biology. Small amounts of this protein and S100A10 protein are exposed on the surface of endothelial cells (ECs). They control fibrinolysis by recruiting tissue-type and urokinase-type plasminogen activators from the plasma. Nutritional studies indicate that two major long-chain polyunsaturated fatty acids (PUFAs), i.e., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), provide benefits for EC functions. The effects of EPA and DHA on the plasminogen/plasmin system have not been characterized.
METHODOLOGY/PRINCIPAL FINDINGS: Proteomic analysis of a cultured human umbilical vein EC-derived cell line, HUV-EC-C, showed that cell-associated ANXA2 decreased with EPA treatment and increased with DHA. A small fraction of ANXA2 was bound to the cell surface, which was also affected by these PUFAs following the same trends. Cell surface expression was negatively regulated by protein kinase C (PKC) α-mediated Ser-phosphorylation, which was up- and down-regulated by EPA and DHA, respectively. These PUFAs differentially affected a small fraction of caveolae/rafts-associated ANXA2. In addition to chymotrypsin-like activity in the serum, newly activated plasmin cleaved the ANXA2 on the cell surface at distinct sites in the N-terminal sequence. ANXA2 also bound to membranes released in the medium, which was similarly processed by these proteases. Both the PUFAs did not directly affect the release.
CONCLUSION/SIGNIFICANCE: These results suggest that EPA and DHA reciprocally control cell surface location of ANXA2. Moreover, cleavage of this protein by plasmin likely resulted in autodigestion of the platform for formation of this protease. In conjunction with termination of the proteolysis by rapid inactivation of plasmin by α-2-antiplasmin and other polypeptide inhibitors, this feedback mechanism may emphasize the benefits of these PUFA in regulation of the initiation of fibrinolysis on the surface of ECs.
膜联蛋白 A2（ANXA2）是细胞溶质 Ca2+结合蛋白 annexin 家族的成员，在血管生物学中发挥着关键作用。少量的这种蛋白质和 S100A10 蛋白暴露在血管内皮细胞（EC）的表面。它们通过从血浆中募集组织型和尿激酶型纤溶酶原激活物来控制纤维蛋白溶解。营养研究表明，两种主要的长链多不饱和脂肪酸（PUFAs），即二十碳五烯酸（EPA）和二十二碳六烯酸（DHA），对 EC 功能有益。EPA 和 DHA 对纤溶酶原/纤溶系统的影响尚未得到描述。
方法/主要发现：对培养的人脐静脉内皮细胞衍生细胞系 HUV-EC-C 的蛋白质组学分析表明，细胞相关的 ANXA2 随着 EPA 处理而减少，随着 DHA 处理而增加。一小部分 ANXA2 与细胞表面结合，这些 PUFAs 也以相同的趋势影响其结合。细胞表面表达受蛋白激酶 C（PKC）α介导的 Ser 磷酸化负调控，EPA 和 DHA 分别上调和下调该磷酸化。这些 PUFAs 以不同的方式影响一小部分 caveolae/rafts 相关的 ANXA2。除了血清中的胰凝乳蛋白酶样活性外，新激活的纤溶酶在 N 端序列的特定位点切割细胞表面上的 ANXA2。ANXA2 还与释放到培养基中的膜结合，这些蛋白酶也同样对其进行加工。这两种 PUFAs 都不会直接影响释放。
结论/意义：这些结果表明，EPA 和 DHA 相互控制 ANXA2 在细胞表面的位置。此外，纤溶酶对这种蛋白质的切割可能导致该蛋白酶形成平台的自身消化。与纤溶酶通过 α-2-抗纤溶酶和其他多肽抑制剂的快速失活来终止蛋白水解的结合，这种反馈机制可能强调了这些 PUFAs 在调节 EC 表面纤维蛋白溶解的起始中的益处。
