Angiostatin binds ATP synthase on the surface of human endothelial cells
Angiostatin binds ATP synthase on the surface of human endothelial cells
复制标题
DOI：
10.1073/pnas.96.6.2811
复制
发表时间：
1999-03-16
影响因子：
11.1
通讯作者：
Pizzo, SV
中科院分区：
文献类型：
--
作者：
Moser, TL;Stack, MS;Pizzo, SV
血管抑素是纤溶酶原的蛋白水解片段，是一种有效的血管生成拮抗剂和内皮细胞迁移和增殖的抑制剂。为了确定血管抑素抑制内皮细胞迁移和/或增殖的机制是否涉及与细胞表面纤溶酶原受体的结合，我们从人脐静脉内皮细胞中分离出纤溶酶原和血管抑素的结合蛋白。结合研究表明，纤溶酶原和血管抑素结合的浓度依赖性，饱和的方式。纤溶酶原结合不受100倍摩尔过量的血管抑素的影响，表明存在一个独特的血管抑素结合位点。这一发现证实了分离的人脐静脉内皮细胞质膜组分的配体印迹分析，这表明纤溶酶原绑定到一个44 kDa的蛋白质，而血管抑素绑定到一个55 kDa的物种。氨基末端测序结合肽质量指纹分析和免疫分析鉴定纤溶酶原结合蛋白为膜联蛋白II，血管抑素结合蛋白为ATP合酶的α/β亚基。通过流式细胞术和免疫荧光分析证实该蛋白在细胞表面的存在，血管抑素也与重组人ATP合酶的α亚基结合，并且这种结合不受2，500倍摩尔过量的纤溶酶原的抑制。在抗α-亚单位ATP合成酶抗体存在下，血管抑素对内皮细胞的抗增殖作用被抑制高达90%。血管抑素与细胞表面ATP合酶α/β亚基的结合可能介导其抗血管生成作用和下调内皮细胞增殖和迁移。
Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studies demonstrated that plasminogen and angiostatin bound in a concentration-dependent, saturable manner. Plasminogen binding was unaffected by a 100-fold molar excess of angiostatin, indicating the presence of a distinct angiostatin binding site. This finding was confirmed by ligand blot analysis of isolated human umbilical vein endothelial cell plasma membrane fractions, which demonstrated that plasminogen bound to a 44-kDa protein, whereas angiostatin bound to a 55-kDa species. Amino terminal sequencing coupled with peptide mass fingerprinting and immunologic analyses identified the plasminogen binding protein as annexin II and the angiostatin binding protein as the alpha/beta-subunits of ATP synthase, The presence of this protein on the cell surface was confirmed by flow cytometry and immunofluorescence analysis, Angiostatin also bound to the recombinant alpha-subunit of human ATP synthase, and this binding was not inhibited by a 2,500-fold molar excess of plasminogen. Angiostatin's antiproliferative effect on endothelial cells was inhibited by as much as 90% in the presence of anti-alpha-subunit ATP synthase antibody. Binding of angiostatin to the alpha/beta-subunits of ATP synthase on the cell surface may mediate its antiangiogenic effects and the down-regulation of endothelial cell proliferation and migration.