An angiogenin-binding protein from endothelial cells.

Abstract

Angiogenin, a 14-kDa protein first isolated from HT-29 human colon adenocarcinoma cells (1), is a potent blood vessel inducer present in mammalian plasma and milk (2)(3)(4). It has a unique ribonucleolytic activity (5,6) that is essential for neovascularization. It also stimulates endothelial cells to form diacylglycerol (7) and to secrete prostacyclin (8) by activating phospholipase C and phospholipase A2, respectively. It binds to calf pulmonary artery endothelial (CPAE) cells (9) and has been found to modulate a mitogenic effect in certain other cells (10). When angiogenin is modified by mutagenesis (11) or by limited proteolysis (12) in the region of residues 61-67, it loses angiogenin activity and will not compete with the native protein in the chicken embryo chorioallantoic membrane (CAM) assay. In contrast, angiogenin modified at either of the active-site histidine residues involved in ribonucleolytic activity, although inactive itself on the CAM, will compete with the native protein in this angiogenesis assay (13). These second-messenger binding and modulatory effects have led to the postulation of a dual-site model for the organogenic activity of angiogenin (12), which assumes that angiogenin interacts directly with cells, presumably through a membrane receptor, to exert this biological function.We report here the identification, isolation, and initial characterization of a bovine endothelial cell angiogeninbinding protein (AngBP) whose properties are consistent with its being a component of a cellular receptor of angiogenin.MATERIALS AND METHODS Materials. Bovine angiogenin, isolated from bovine milk as described (4), migrated as a single band in SDS/PAGE, induced vascularization in the chicken embryo CAM assay, and stimulated a second-messenger response in endothelial cells. Single-site mutants H13A and H114A in which the ribonucleolytic active-site histidine residues are changed to alanine,t the regional mutant ARH-Ij native human angiogenin, placental ribonuclease inhibitor (PRI), and bovine angiogenin were provided by R. Shapiro. The two proteolytic derivatives of human angiogenin, angiogenin K (cleaved at residues 60-61) and angiogenin E (cleaved at residues 67-68) were from T. Hallahan (12 2227The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Angiogenin antagonists prevent tumor growth in vivo.

“…Actin, an additional Ang antagonist, was tested for its capacity to delay or prevent tumor growth. It resembles the above anti-Ang mAbs in binding to and neutralizing the angiogenic activity of human Ang (16); indeed, its binding epitope overlaps that of mAb 36u (15). However, unlike the above mAbs, it also binds to mouse Ang.…”

“…It activates intracellular second-messenger pathways (12), supports tumor and endothelial cell adhesion (6,13), and undergoes nuclear translocation (14). A 42-kDa Ang-binding protein isolated from the surface of cultured bovine endothelial cells has been identified as a smooth muscle type of a-actin (15)(16)(17). It, like commercial actin preparations, binds tightly not only to bovine but also to mouse and human Ang (G.-F. Hu, personal communication).…”

Cell Surface-Dependent Generation of Angiostatin4.5

“…There have been a series of studies demonstrating the presence of one or more actin isoforms on the external cell surface. One or more actin isoform was found by several independent groups of researchers on the surface of lymphocytes (44,45), brain capillary endothelial cells (46), and bovine pulmonary endothelial cells (47)(48)(49)(50). Actin was also discovered as a cell surface binding site for plasminogen on endothelial cells (39), monocytoid cells, blood monocytes (34), and breast cancer cells (40).…”

Antiplasmin Activity of a Peptide That Binds to the Receptor-binding Site of Angiogenin

“…The fact that angiogenin activates phospholipases (7-9) and undergoes nuclear translocation (10) suggests that the biological action of angiogenin is mediated through a cell surface receptor. Human angiogenin can specifically bind to endothelial cells with high affinity (11), and a cell surface actin-like protein was identified as an angiogenin-binding protein (12)(13)(14). Several lines of evidence suggest that the interaction of angiogenin with an angiogenin-binding protein or actin occurs through the receptor-binding site of angiogenin.…”

“…Several lines of evidence suggest that the interaction of angiogenin with an angiogenin-binding protein or actin occurs through the receptor-binding site of angiogenin. Actin and anti-actin antibody inhibit the internalization of angiogenin into endothelial cells, and block the biological activity of angiogenin in the chick chorioallantoic membrane (CAM) 1 assay (10,12,13). Therefore, the interaction of angiogenin with endothelial cell surface actin is an essential step in angiogenin-induced neovascularization.…”