Morphological observation of nuclear sub-localization of human angiogenin in HUVECs

Angiogenin, a potent angiogenic factor, was cloned and expressed by Escherichia coli and then purified with gel filtration chromatography. Approximately 90% pure angiogenin was obtained to generate a monoclonal antibody. Using western immunoblotting and ELISA, we confirmed that monoclonal antibody C46 secreted from hybridoma cells stably and specifically binds to angiogenin. The fused protein angiogenin-EGF was then expressed in HUVECs, and the subcellular localization of the recombinant protein was determined by confocal microscopy and TEM assay. Recombinant angiogenin was found to mainly concentrate in the pars granulosa of the nucleus, where the protein accumulates to form ribonucleoprotein particles.     

High level expression of soluble angiogenin in Escherichia coli

Human angiogenin was genetically engineered and contained the E. coli Omp A signal sequence for secreting soluble angiogenin to the periplasm under tac promoter control. The angiogenin sequence was encoded in a single gene and expressed as a 14.4 kilodalton soluble protein in E. coli. It was purified by CM-Sepharose ion-exchange chromatography and by a heparin-Sepharose affinity chromatography procedure. The biological activity of angiogenin was established by its ability to inhibit mRNA-dependent rabbit reticulocyte cell-free translation.     

Expression of Met-(-1) angiogenin in Escherichia coli: conversion to the authentic less than Glu-1 protein

A method for obtaining authentic human angiogenin utilizing an Escherichia coli recombinant expression system is described. A synthetic gene encoding angiogenin was placed into a vector for direct expression under the control of a modified E. coli trp promoter. The protein was produced by the bacteria in an insoluble form and purified to homogeneity by cation-exchange and reversed-phase HPLC following reduction/solubilization and reoxidation. The protein isolated was identified as Met-(-1) angiogenin by amino acid analysis and tryptic peptide mapping; the latter demonstrated that all three disulfide bonds had formed correctly. Both the enzymatic and angiogenic activities of the Met-(-1) protein were equivalent to those of native angiogenin. A Met-(-1) Leu-30 derivative of angiogenin was also isolated and found to be fully active. Conversion of Met-(-1) angiogenin to the authentic less than Glu-1 protein was achieved by treatment with Aeromonas aminopeptidase under conditions in which the new N-terminal glutamine readily cyclizes nonenzymatically. This aminopeptidase treatment may have more general applicability for removal of undesirable N-terminal methionine residues from foreign proteins expressed in bacteria.     

血管生成素相互作用蛋白的发现及其在哺乳动物细胞中的验证

通过RT-PCR从人外周血白细胞中钓取血管生成素(angiogenin,Ang)cDNA,构建诱饵蛋白载体pAS-2-1-Ang,对其自身转录激活活性进行鉴定后,通过酵母双杂交系统筛选人肝细胞cDNA文库,获得两个双阳性克隆.序列分析和同源检索表明所获候选蛋白分别为人上皮细胞素和λ晶体蛋白.构建Ang及候选蛋白标签融合表达载体并共转染COS-7细胞,利用免疫共沉淀和蛋白质印迹方法在哺乳动物细胞中验证了Ang与候选蛋白间的相互作用.为阐明Ａng促血管新生的分子机制创造了条件.     

The complete amino acid sequence of bovine milk angiogenin

The amino acid sequence of angiogenin isolated from bovine milk was deduced by gas-phase sequencing of the protein and its fragments. The protein contains 125 residues and has a calculated molecular mass of 14,577 Da. The sequence is highly homologous (65% identity) to the sequence of human angiogenin, most of the differences being the result of conservative replacements. Like human angiogenin, the bovine protein is also homologous to bovine pancreatic RNase A (34% identity) and the three major active site residues known to be involved in the catalytic process, His-14, Lys-41 and His-115, are conserved. When tested against conventional substrates for RNase A activity, bovine angiogenin displays the same selective ribonucleolytic activity as human angiogenin. The sequence of bovine angiogenin contains the cell recognition tripeptide Arg-Gly-Asp which is not present in the human protein.     

Alpha-actinin-2, a cytoskeletal protein, binds to angiogenin

Angiogenin is an angiogenic factor which is involved in tumorigenesis. However, no particular intracellular protein is known to interact directly with angiogenin. In the present study, we reported the identification of alpha-actinin-2, an actin-crosslinking protein, as a potential angiogenin-interacting partner by yeast two-hybrid screening. This interaction was confirmed by different approaches. First, angiogenin was pulled down together with His-tagged alpha-actinin-2 by Ni(2+)-agarose resins. Second, alpha-actinin-2 was coimmunoprecipitated with angiogenin by anti-angiogenin monoclonal antibody. Third, the in vivo interaction of these two proteins was revealed by fluorescence resonance energy transfer analysis. Since members of alpha-actinin family play pivotal roles in cell proliferation, migration, and invasion, the interaction between alpha-actinin-2 and angiogenin may underline one possible mechanism of angiogenin in angiogenesis. Our finding presents the first evidence of an interaction of a cytosolic protein with angiogenin, which might be a novel interference target for anti-angiogenesis and anti-tumor therapy.     

Angiogenin is a cytotoxic, tRNA-specific ribonuclease in the RNase A superfamily.

Angiogenin is a 14.4-kDa human plasma protein with 65% homology to RNase A that retains the key active site residues and three of the four RNase A disulfide bonds. We demonstrate that recombinant angiogenin functions as a cytotoxic tRNA-specific RNase in cell-free lysates and when injected into Xenopus oocytes. Inhibition of protein synthesis by angiogenin correlates with degradation of endogenous oocyte tRNA. Exogenous, radiolabeled tRNA is also hydrolyzed by angiogenin, whereas oocyte rRNA and mRNA are not detectably degraded by angiogenin. Protein synthesis was restored to angiogenin-injected oocytes by injecting the RNase inhibitor RNasin plus total Xenopus or calf liver tRNAs, thereby demonstrating that the tRNA degradation induced by angiogenin was the sole cause of cytotoxicity. A similar tRNA-reversible inhibition of protein synthesis was seen in rabbit reticulocyte lysates. Angiogenin therefore appears to be a specific cellular tRNase, whereas five homologues in the RNase A superfamily lack angiogenin's specificity for tRNA. One of these homologues purified from human eosinophils, eosinophil-derived neurotoxin, nonspecifically degrades oocyte RNA similar to RNase A and is also cytotoxic at very low concentrations.     

Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin

The binding of human placental ribonuclease inhibitor (PRI) to angiogenin, a human protein that induces neovascularization, occurs with a 1:1 stoichiometry and is accompanied by a 50% increase in tryptophan fluorescence. In contrast, the binding of PRI to bovine pancreatic RNase A or to angiogenin oxidized at its single tryptophan residue results in a quenching of fluorescence. These observations suggest that there is a change in the local environment of Trp-89 of angiogenin. Quenching experiments with acrylamide are consistent with the view that Trp-89 is exposed in the native protein and becomes less accessible upon formation of the complex with PRI. Stopped-flow kinetic measurements monitoring the fluorescence enhancement indicate a two-step mechanism for the binding of PRI to angiogenin. The first step involves rapid formation of an enzyme-inhibitor complex, EI, followed by a slower isomerization of EI to a tight enzyme-inhibitor complex, EI*: (Formula: see text). In 0.1 M NaCl at pH 6 and 25 degrees C, the values of K1 and K2 are 0.53 microM and 97 s-1, respectively. The apparent second-order rate constant of association at protein concentrations much less than K1 is approximated by K2/K1 and equals 1.8 X 10(8) M-1 s-1. The corresponding value for the association of PRI with RNase A is only slightly higher, 3.4 X 10(8) M-1 s-1. The effects of pH and sodium chloride concentration on the association rate of PRI with angiogenin suggest the importance of ionizable groups and ionic interactions, respectively, in the association process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutagenesis of residues flanking Lys-40 enhances the enzymatic activity and reduces the angiogenic potency of angiogenin.

The primary structure of the blood vessel inducing protein angiogenin is 35% identical with that of pancreatic ribonuclease (RNase) and contains counterparts for the critical RNase active-site residues His-12, Lys-41, and His-119. Although angiogenin is a ribonucleolytic enzyme, its activity toward conventional substrates is lower than that of pancreatic RNase by several orders of magnitude. Comparison of the amino acid sequences of RNase and angiogenin reveals several striking differences in the region flanking the active-site lysine, including a deletion and a transposition of aspartic acid and proline residues. In order to examine how these sequence changes alter the functional properties of angiogenin, an angiogenin/RNase hybrid protein (ARH-II), in which residues 38-41 of angiogenin (Pro-Cys-Lys-Asp) have been replaced by the corresponding segment of bovine pancreatic RNase (Asp-Arg-Cys-Lys-Pro), was prepared by regional mutagenesis. Compared to angiogenin, ARH-II has markedly diminished angiogenic activity on the chick embryo chorioallantoic membrane but 5-75-fold greater enzymatic activity toward a variety of polynucleotide and dinucleotide substrates. In addition, the specificity of ARH-II toward dinucleotide substrates differs from that of angiogenin and is qualitatively similar to that of pancreatic RNase. Thus, non-active-site residues near Lys-40 in angiogenin appear to play a significant role in determining enzymatic specificity and reactivity as well as angiogenic potency. An additional angiogenin/RNase hybrid protein (ARH-IV), in which residues 59-71 of ARH-II have been replaced by the corresponding segment of pancreatic RNase, was also prepared.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiogenin and its role in angiogenesis]

The review is devoted to angiogenin, one of the factors that induce formation of blood vessels, which is unique among them in that it is a ribonuclease. Consideration is given to the tertiary structure of human angiogenin; the catalytic and cell-receptor binding sites, their significance for angiogenic activity; the human angiogenin gene structure, chromosomal localization, and expression; the specificity of angiogenin as a ribonuclease and abolishment of protein synthesis; the nuclear localization of angiogenin in proliferating endothelial cells and its significance for angiogenic activity; angiogenin binding to a cell-surface actin as a plausible mechanism of inducing neovascularization (enhancement of plasminogen activation by actin with angiogenin, stimulation of the cell-associated proteolytic activity by angiogenin; promotion of the cultured cells invasiveness); modulation of mitogenic stimuli in endothelial, smooth muscle, and fibroblast cells by angiogenin. The importance of angiogenin as an adhesive molecule for endothelial and tumor cells is discussed too, as well as the modulation of tubular morphogenesis by bovine angiogenin, prevention of tumor growth in vivo by angiogenin antagonists, prospects of the use of angiogenin and angiogenin-encoding recombinant plasmids and vaccinia virus in therapeutic practice.     

Angiogenin and Its Functions in Angiogenesis

The review is devoted to angiogenin, one of the factors that induce formation of blood vessels, which is unique in that it is a ribonuclease. Consideration is given to the tertiary structure of human angiogenin; the catalytic and cell receptor binding sites, their significance for angiogenic activity; the human angiogenin gene structure, chromosomal localization, and expression; the specificity of angiogenin as a ribonuclease and abolishment of protein synthesis; the nuclear localization of angiogenin in proliferating endothelial cells and its significance for angiogenic activity; angiogenin binding to cell surface actin as a plausible mechanism of inducing neovascularization (enhancement of plasminogen activation by actin, stimulation of the cell-associated proteolytic activity; promotion of the cultured cell invasiveness); modulation of mitogenic stimuli in endothelial, smooth muscle, and fibroblast cells by angiogenin. The importance of angiogenin as an adhesive molecule for endothelial and tumor cells is discussed too, as well as the modulation of tubular morphogenesis by bovine angiogenin, prevention of tumor growth in vivoby angiogenin antagonists, prospects of the use of angiogenin and angiogenin-encoding recombinant plasmids and vaccinia virus in therapeutic practice.     

Introduction of the human recombinant angiogenin at the early stages of postnatal development inhibits the growth of mice

We studied the influence of recombinant DNA containing the cloned angiogenin gene, plasmid DNA without angiogenin gene, and purified recombinant angiogenin injected to Tg8 mice at the age of two days on the body mass of 28- and 40-day old mice. The body mass of mice that were injected with the cloned angiogenin gene or purified angiogenin was less than in the control mice. The body mice of Tg8 mice injected with recombinant DNA containing the cloned angiogenin gene did not increase from day 2 to day 40, while in the mice with purified recombinant angiogenin and control mice it increased by 24 and 57%, respectively. These data suggest that the elevated level of angiogenin at the early developmental stages inhibits the increase of body mass. The effect we described as related, in al likelihood, to the known inhibitory effect of angiogenin on protein synthesis.     

血管生成素:RNA代谢过程中重要的核糖核酸酶

血管生成素是核糖核酸酶A超家族成员之一,具有较弱的核糖核酸酶活性.最新研究发现,血管生成素参与细胞内多种RNA的代谢过程.在生长条件下,血管生成素可以发生核转位聚集于细胞核中,促进rRNA转录,并可参与其剪切加工,同时它也调控一系列mRNA基因的转录,最终促进细胞的生长和增殖;在应激条件下,血管生成素能降解tRNA形成tiRNA,抑制细胞内整体蛋白质的翻译水平,并促进应激小体的形成,激活细胞内应激保护机制,从而促进细胞存活.此外,血管生成素还可参与非编码小RNA等RNA代谢过程.本文概述了血管生成素在RNA代谢中的作用与分子机制等方面的进展,并探讨了其在疾病发生和发展中的作用,以期开拓血管生成素的研究新思路.     

Introduction of Human Recombinant Angiogenin at the Early Stages of Postnatal Development Inhibits the Growth of Mice

We studied the influence of recombinant DNA containing the cloned angiogenin gene, plasmid DNA without angiogenin gene, and purified recombinant angiogenin injected to Tg8 mice at the age of two days on the body mass of 28- and 40-day old mice. The body mass of mice that were injected with the cloned angiogenin gene or purified angiogenin was less than in the control mice. The body mice of Tg8 mice injected with recombinant DNA containing the cloned angiogenin gene did not increase from day 28 to day 40, while in the mice with purified recombinant angiogenin and control mice it increased by 24 and 57%, respectively. These data suggest that the elevated level of angiogenin at the early developmental stages inhibits the increase of body mass. The effect we described is related, in al likelihood, to the known inhibitory effect of angiogenin on protein synthesis.     

Absorption of bovine angiogenin into peripheral blood of rats orally administered milk basic protein

Bovine angiogenin is a major component of the bone resorption inhibitory activity of milk basic protein (MBP). The intestinal absorption of bovine angiogenin was investigated in a rat model, where it was detected in an intact form in the peripheral blood after the oral administration of MBP. This finding demonstrates that orally administered bovine angiogenin is absorbed without being degraded.     

Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells

Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells.     

Internalization and processing of human angiogenin by cultured aortic smooth muscle cells

Human angiogenin is a 14-kDa plasma protein with angiogenic and ribonucleolytic activities. Angiogenin binds specifically to aortic smooth muscle cells, activates second messenger pathways, and inhibits their proliferation. Human and bovine aortic smooth muscle cells were used to study the internalization and intracellular fate of human angiogenin at 37 degrees C. Using a specific antibody against angiogenin, we found that the internalized native protein was localized in the perinuclear region at 30 min and then dispersed throughout the cytoplasm. In conditions favoring receptor-mediated endocytosis, internalization of iodinated angiogenin showed a first peak at 5 min and then further increased for up to 24 h. The half-life of the molecule, calculated as 12 h in chase experiments, could contribute to its intracellular accumulation. In cell extracts, in addition to the 14-kDa protein, a 8.7-kDa fragment was observed at 24 h, and three fragments with molecular mass of 10.5, 8.7, and 6. 1 kDa were detected at 48 h. Our data point to a specific internalization and processing of human angiogenin by aortic smooth muscle cells.