Moser T L, Stack M S, Asplin I, Enghild J J, Højrup P, Everitt L, Hubchak S, Schnaper H W, Pizzo S V
Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA.
Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2811-6. doi: 10.1073/pnas.96.6.2811.
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.
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以及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了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以浓度依赖性、可饱和的方式结合。纤溶酶原的结合不受100倍摩尔过量血管抑素的影响，这表明存在一个独特的血管抑素结合位点。对分离出的人脐静脉内皮细胞质膜组分进行的配体印迹分析证实了这一发现，该分析表明纤溶酶原与一种44 kDa的蛋白质结合，而血管抑素与一种55 kDa的蛋白质结合。氨基末端测序结合肽质量指纹图谱和免疫学分析确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了该蛋白在细胞表面的存在。血管抑素也与人ATP合酶的重组α亚基结合，并且这种结合不受2500倍摩尔过量纤溶酶原的抑制。在存在抗ATP合酶α亚基抗体的情况下，血管抑素对内皮细胞的抗增殖作用被抑制了多达90%。血管抑素与细胞表面ATP合酶的α/β亚基的结合可能介导其抗血管生成作用以及内皮细胞增殖和迁移的下调。
