ABSTRACT
Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn2+-dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin.
Subject(s)
Angiogenesis Inhibitors/metabolism , Angiogenesis Inhibitors/pharmacology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Kininogen, High-Molecular-Weight/metabolism , Kininogen, High-Molecular-Weight/pharmacology , Tropomyosin/metabolism , Allantois/blood supply , Allantois/drug effects , Animals , Antibodies, Monoclonal/pharmacology , Apoptosis/drug effects , Base Sequence , Cells, Cultured , Chick Embryo , Chorion/blood supply , Chorion/drug effects , DNA, Complementary/genetics , Endothelium, Vascular/cytology , Humans , Kininogen, High-Molecular-Weight/genetics , Neovascularization, Physiologic/drug effects , Protein Binding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Tropomyosin/antagonists & inhibitorsABSTRACT
A partial clone of caldesmon, coding for the C-terminal 288 amino acids, was isolated from a human fetal liver cDNA library and sequenced. Expression of the clone in Escherichia coli produced a peptide called H1 (M(r) 32,549), which inhibited tropomyosin-enhanced actomyosin Mg(2+)-ATPase activity by 90% with half maximal inhibition at 0.03-0.04 mol H1 per mol actin. The inhibition could be reversed by Ca(2+)-calmodulin. H1 bound actin, Ca(2+)-calmodulin and tropomyosin and smooth muscle myosin with high affinities. This latter finding shows the presence of a second myosin-binding site in caldesmon. This was confirmed in thrombic digests of native sheep aorta and chicken gizzard caldesmon.