Bovine Angiogenin Research Articles

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.

A droplet-based fluorescence polarization immunoassay (dFPIA) platform for rapid and quantitative analysis of biomarkers.

Herein, we describe for the first time the integration of pneumatic micro-pumps with droplet-based microfluidic systems as basic platform for the rapid detection and quantitation of biomarkers. Specifically, we combine this microfluidic platform with fluorescence polarization detection to identify and quantify the potent blood vessel inducing protein bovine angiogenin within cow's milk in high-throughput. The droplet-based fluorescence polarization immunoassay is successful in accurately determining the concentration (4.84±1.21 μg/mL) of bovine angiogenin in cow's milk, affords a 10 fold reduction in dead volumes when compared to conventional droplet-based microfluidic experiments and requires an total sample volume of less than 1 nL.

Purification and identification of lactoperoxidase in milk basic proteins as an inhibitor of osteoclastogenesis

A milk protein fraction with alkaline isoelectric points (milk basic protein, MBP) inhibits both bone resorption and osteoclastogenesis for in vitro models. We previously identified bovine angiogenin as a component of MBP that inhibits bone resorption. However, purified angiogenin had no effect on osteoclastogenesis, suggesting that MBP contains unidentified component(s) that inhibit osteoclast formation. In this study, we purified lactoperoxidase (LPO) as the predominant inhibitor of osteoclastogenesis in MBP. The LPO treatment downregulated levels of reactive oxygen species in osteoclasts. Signaling by receptor activator of NF-kappa-B ligand/receptor activator of NF-kappa-B (RANKL/RANK) was downregulated in LPO-treated cells, and, in particular, the ubiquitination of tumor necrosis factor receptor associate factor 6 (TRAF6) and activation of downstream signaling cascades (JNK, p38, ERK, and NFκB) were suppressed. Ultimately, LPO treatment led to decreased expression of c-Fos and NFAT2. These results suggest that MBP contains at least 2 components that independently suppress bone resorption through a unique mechanism: angiogenin inhibits bone resorption and LPO inhibits RANKL-induced osteoclast differentiation. These data explain many of the positive aspects of milk consumption on bone health.

NMR Assignment of des [40-93] Mutant of Bovine Angiogenin

Angiogenin is a potent inducer of blood vessel formation and is overexpressed in many cancers. It is a member of the pancreatic RNase superfamily. Angiogenin has 33% sequence identity with RNase A. All RNases family proteins except angiogenin and turtle RNase have four disulfide bonds. Angiogenin has three disulfide bonds, [C27-C82], [C40-C93] and [C58-C108], respectively. To examine the role of disulfide bond [C40-C93] near the nuclear localization site, recombinant des [40-93] mutant by replacement of cysteines at positions 40 and 93 with serines was cloned, expressed, and purified. CD spectrum of des [40-93] was similar to that of wild type and implies that the overall folding of two forms is similar. 2D and 3D NMR experiments for 15 N and/or 13 C-isotope labeled des [40-93] were performed and the backbone resonance assignment was done. Based on the result of backbone assignment and chemical shift index (CSI), we confirmed that deletion of [C40-C93] disulfide bond affected the local structural stability of bovine angiogenin near the nuclear localization site and ribonucleolytic active site. Based on this assignment, studies on structure and dynamics of des [40-93] mutant will be performed.

Identification of angiogenin as the osteoclastic bone resorption-inhibitory factor in bovine milk

We identified, for the first time, the factor responsible for inhibiting osteoclast-mediated bone resorption in the basic protein fraction of bovine milk (milk basic protein, MBP). The protein was purified by a combination of ion and gel column chromatography from MBP, based on its activity to prevent unfractionated rabbit bone cells from forming pits on dentine slices. It was found to have a molecular weight of 15 kDa on SDS-PAGE, and the sequence of the N-terminal 25 amino acid residues was identical to that of bovine angiogenin. The purified bovine angiogenin inhibited the pit-forming activity of both unfractionated bone cells and purified osteoclasts in a dose-dependent manner, and the inhibitory activity was markedly suppressed by treatment with anti-bovine angiogenin antibody. The inhibitory activity was confirmed in mice both in vitro and in vivo. Treatment of osteoclasts with bovine angiogenin resulted in an impairment of the formation F-actin ring and a reduction in the mRNA levels of TRAP and cathepsin K, both known to be essential for the bone resorption activity of osteoclasts. These results suggest that bovine angiogenin is the substance mainly responsible for the inhibitory effect of bovine milk on osteoclast-mediated bone resorption, and that it exerts its activity by acting directly on the osteoclasts.

RAAV-mediated angiogenin gene transfer induces angiogenesis and modifies left ventricular remodeling in rats with myocardial infarction

In vitro studies have demonstrated that bovine angiogenin (ANG) significantly stimulates both the migration of endothelial cells and the formation of tubelike structures. The aim of this study was to explore whether ANG gene transfer could enhance vascularization, modify left ventricular remodeling, and attenuate cardiac dysfunction in rats with myocardial infarction (MI). We constructed a recombinant adeno-associated virus vector encoding the ANG gene (rAAV-ANG) and evaluated its angiogenic potential after regional transfection by intramyocardial injection immediately after left anterior descending artery ligation in rats. Four weeks after coronary artery ligation, rAAV-ANG transfection upregulated the myocardium ANG protein expression level in both normal and MI rats, and immunohistochemistry showed that the overexpressed ANG was distributed in the cytoplasm of cardiomyocytes. In rats with MI, rAAV-ANG treatment altered left ventricular remodeling, as indicated by a decrease in left ventricular end diastolic diameter, left ventricular end systolic diameter, cardiomyocyte diameter, ventricular weight to body weight ratio and interstitial fibrosis infiltration. We also found an increase in capillary density and partly restored cardiac function in the group receiving rAAV-ANG treatment. These results confirmed that in rats with MI, ANG gene transfer could induce angiogenesis, alter left ventricular remodeling, and attenuate cardiac dysfunction. This study provides a new choice of treatment for ischemic heart disease.

High level production of bovine angiogenin in E. coli by an efficient refolding procedure

Recombinant bovine angiogenin (rbAng) was expressed in E. coli at up to 30% of total cell proteins but was produced as inclusion bodies. By investigating the effect of various factors on the refolding yield, we obtained about 60% refolding. After chromatographic purification, about 60 mg purified angiogenin was obtained from 1 l culture. The purified recombinant bovine angiogenin was identical to native bovine angiogenin (nbAng) obtained from cow's milk. Our approach is highly efficient and can be generally used for the production of various types of angiogenin for functional and structural studies as well as therapeutic purposes.

우유로부터 Angiogenin의 정제

This study was carried out to establish the purification protocol of angiogenin(ANG) from bovine milk. The purification of ANG from bovine milk was performed by using cation chromatography, high-performance liquid chromatography and gel-filtration. We obtained the ANG protein have the molecular weight of about 14 kD by SDS-PAGE analysis. This protein was confirmed as ANG by Nlb-terminal sequence analysis of the first 15 amino acids. Identified amino acids revealed the protein to be identical to that previously reported for bovine ANG.㜊谀Ѐ㌸㠻⤀䍯浭敲捥Ⱐ捯浭畮楣慴楯湳…⁴牡湳灯牴慴楯渀

Computer modeling and molecular dynamics simulations of ligand bound complexes of bovine angiogenin: dinucleotide topology at the active site of RNase a family proteins.

We have undertaken the modeling of substrate-bound structures of angiogenin. In our recent study, we modeled the dinucleotide ligand binding to human angiogenin. In the present study, the substrates CpG, UpG, and CpA were docked onto bovine angiogenin. This was achieved by overcoming the problem of an obstruction to the B1 site by the C-terminus and identifying residues that bind to the second base. The modeled complexes retain biochemically important interactions. The docked models were subjected to 1 ns of molecular dynamics, and structures from the simulation were refined by using simulated annealing. Our models explained the enzyme's specificity for both B1 and B2 bases as observed experimentally. The nature of binding of the dinucleotide substrate was compared with that of the mononucleotide product. The models of these complexes were also compared with those obtained earlier with human angiogenin. On the basis of the simulations and annealed structures, we came up with a consensus topology of dinucleotide ligands that binds to human and bovine angiogenins. This dinucleotide conformation can serve as a starting model for ligand-bound complex structures for RNase A family of proteins. We demonstrated this capability by generating the complex structure of CpA bound to eosinophil-derived neurotoxin (EDN) by fitting the consensus topology of CpA to the crystal structure of native EDN.

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.

Angiogenin Is Involved in Morphological Changes and Angiogenesis in the Ovary

Angiogenin is a potent angiogenic factor secreted by cultured tumor cells and is found in various normal organs and tissues. The ovary is one of the adult organs in which angiogenesis normally occurs during the female reproductive cycle. In this study, we examined whether angiogenin protein is localized and if angiogenin mRNA is expressed in the normal bovine ovary by immunohistochemistry using polyclonal rabbit anti-bovine angiogenin IgG and byin situhybridization using bovine angiogenin probe, respectively. The localization and mRNA expression of angiogenin in the ovarian follicle and in the corpus luteum were different in their developmental stages. The intensities of immunoreactivities and angiogenin transcripts in the follicle increased from the primordial to the tertiary (or Graafian) follicle. The early corpus luteum contained strong immunoreactivities and mRNA expression of angiogenin but these intensities weakened during regression. The results suggest that angiogenin is involved in morphological changes and angiogenesis in the ovary.

Modeling of Angiogenin - 3′-NMP Complex

Angiogenin belongs to the Ribonuclease superfamily and has a weak enzymatic activity that is crucial for its biological function of stimulating blood vessel growth. Structural studies on ligand bound Angiogenin will go a long way in understanding the mechanism of the protein as well as help in designing drugs against it. In this study we present the first available structure of nucleotide ligand bound Angiogenin obtained by computer modeling. The importance of this study in itself notwithstanding, is a precursor to modeling a full dinucleotide substrate onto Angiogenin. Bovine Angiogenin, the structure of which has been solved at a high resolution, was earlier subjected to Molecular Dynamics simulations for a nanosecond. The MD structures offer better starting points for docking as they offer lesser obstruction than the crystal structure to ligand binding. The MD structure with the least serious short contacts was modeled to obtain a steric free Angiogenin 3′ mononucleotide complex structure. The structures were energetically minimized and subjected to a brief spell of Molecular Dynamics. The results of the simulation show that all the ligand-Angiogenin interactions and hydrogen bonds are retained, redeeming the structure and docking procedure. Further, following ligand protein interactions in the case of the ligands 3′-CMP and 3′-UMP we were able to speculate on how Angiogenin, a predominantly prymidine specific ribonuclease prefers Cytosine to Uracil in the first base position.

Detection, Quantitation, and Localization of Bovine Angiogenin by Immunological Assays

Bovine angiogenin (bAng) is a potent blood vessel inducing protein found in bovine serum and milk. Antisera have been raised against bAng. Western blot analysis for bAng indicated that the polyclonal antibody recognized bAng specifically, and no cross-reactivity with bovine RNase A, a protein homologous to bAng, was observed. The sandwich enzyme-linked immunosorbent assay for bAng was sensitive to 10 pg of bAng, and this assay was able to quantitate bAng in bovine serum (100–180 ng/mL) and milk (4–8 μg/mL). Strong positive immunohistochemical reactions were detected in alveolar cells, the secretion of alveolar cells and excretory ducts in sections of cow mammary gland, epithelial cells of visceral peritoneum and bile-duct in sections of cow liver, and epithelial cells and mucous glands in sections of cow gallbladder. These results suggest that epithelial cells and secretory cells are major sites of angiogenin synthesis.

Modulation by Bovine Angiogenin of Tubular Morphogenesis and Expression of Plasminogen Activator in Bovine Endothelial Cells

Angiogenin is a potent angiogenic molecule in the chick chorioallantoic membrane assay and rabbit corneal assay. However, no angiogenic activity has been reported in in vitro system. In this study,we isolated and purified angiogenin from bovine milk, and the biological effect of the bovine angiogenin on bovine endothelial cells was examined in in vitro angiogenesis models. Bovine angiogenin significantly stimulated both cell migration and formation of tube-like structures in the collagen gel by bovine aortic endothelial cells. Angiogenin at 10-100 ng/ml stimulated about 2-fold higher the tube formation when basic fibroblast growth factor (bFGF) at 10 ng/ml stimulated about 3-fold over the control. Tubular morphogenesis stimulated by bFGF or angiogenin was almost completely blocked in the presence of aprotinin, an inhibitor of serine proteases. Angiogenin upregulated both mRNA level and activity of urokinase type plasminogen activator, a key mediator of angiogenesis. Moreover, both bFGF and bovine angiogenin induced a marked increase of c-fos mRNA level at 30 min after stimulation. These novel effects of bovine angiogenin are to be discussed in relation to its structure specificity.

Angiogenin promotes invasiveness of cultured endothelial cells by stimulation of cell-associated proteolytic activities.

Angiogenin, a potent inducer of neovascularization in the chicken chorioallantoic membrane and rabbit cornea, promotes endothelial cell invasion of Matrigel basement membrane. A transformed bovine aortic endothelial cell line, GM 7373, is 5 times more invasive when cultured in the presence of 1 microgram of bovine angiogenin per ml than in its absence. A polyclonal anti-angiogenin antibody and alpha 2-antiplasmin neutralize the effect of angiogenin, but an angiogenin-binding protein (actin) does not. Further, this concentration of angiogenin induces a 14-fold increase in the cell-associated proteolytic activity of cultured endothelial cells, determined with a tissue-type plasminogen activator-specific peptide as the substrate. In addition, cells cultured on a three-dimensional fibrin gel in the presence of angiogenin are 3 times more capable of dissolving the gel and forming focal defects in the underlying matrix. The results indicate that angiogenin can enhance the ability of endothelial cells to digest extracellular matrix components and degrade basement membrane, thereby facilitating cell invasion and migration. Binding of angiogenin to its cell-surface binding protein (actin) followed by dissociation of the angiogenin-actin complex from the cell surface and subsequent activation of tissue-type plasminogen activator/plasmin are likely steps involved in the processes of endothelial cell invasion and angiogenesis.

Proton resonance assignments and secondary structure of bovine angiogenin.

Angiogenins are 14-kDa proteins able to induce blood vessel growth in various preparations and are thus thought to be involved in the development of solid tumors. Angiogenins have significant similarities with extracellular ribonuclease and possess a characteristic nuclease activity against large RNA molecules. These proteins are also able to induce second-messenger pathways. We have undertaken the determination of the three-dimensional structure of bovine angiogenin by using nuclear magnetic resonance (NMR) spectroscopy. Since this protein was directly purified from cow milk, it was not possible to enrich angiogenin with 13C or 15N isotopes. However, extensive use of two-dimensional and three-dimensional proton NMR experiments enabled us to identify all but four spin systems and to assign all corresponding proton resonances. Identification of most backbone-backbone nuclear Overhauser enhancements led to the characterization of the secondary structure elements of the protein. Comparison with the structure of ribonuclease A and analysis of the location of conserved residues confirmed that the two molecules have very similar structures.

Angiogenin supports endothelial and fibroblast cell adhesion.

When coated on bacteriological plastic at doses greater than or equal to 0.1 microgram/cm2, human and bovine angiogenin support calf pulmonary artery endothelial and Chinese hamster fibroblast cell adhesion and spreading, but do not affect cell adhesion when in solution. The kinetics of endothelial cell attachment to angiogenin are indistinguishable from those in the presence of gelatin. Calcium and/or magnesium ions are critical for cell adhesion or spreading onto angiogenin but protein synthesis and glycoprotein secretion are not necessary. Adhesion to angiogenin is not altered by the addition to the incubation solution of fibronectin, fibrinogen, laminin, collagen I and IV, or vitronectin. The peptide Arg-Gly-Asp-Ser inhibits endothelial cell response to angiogenin whereas the reverse peptide Ser-Asp-Gly-Arg-Gly has no effect. These findings show that angiogenin can serve as an effective substratum for cell adhesion by inducing an interaction similar to but independent from that of other extracellular matrix molecules. Induction of cell adhesion and subsequent migration may be critical steps in the process of angiogenesis.

An angiogenin-binding protein from endothelial cells.

A 42-kDa bovine protein that binds bovine angiogenin [angiogenin binding protein (AngBP)] has been identified as a dissociable cell-surface component of calf pulmonary artery endothelial cells and a transformed bovine endothelial cell line, GM7373. Binding of 125I-labeled bovine angiogenin (125I-Ang) to AngBP occurs with an apparent Kd approximately 5 x 10(-10) M and is specific, saturable, and inhibited by excess unlabeled angiogenin. 125I-Ang can be crosslinked efficiently to AngBP by a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide. Bovine ribonuclease A competes with the binding of 125I-Ang to AngBP, but lysozyme does not. Direct binding to AngBP of 125I-labeled bovine ribonuclease A is, however, much weaker than that of 125I-Ang. Two enzymatically active derivatives of angiogenin cleaved at residues 60-61 and 67-68, respectively, fail to induce angiogenesis and also bind to AngBP only weakly. AngBP has been isolated by treatment of cells with heparan sulfate, affinity chromatography on angiogenin-Sepharose of the material dissociated from the cell surface, and gel filtration HPLC. The results suggest that AngBP has the characteristics of a receptor that may likely function in angiogenesis.

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.