Angiogenin activates Erk1/2 in human umbilical vein endothelial cells.

Angiogenin is a potent angiogenic factor that binds to endothelial cells and is endocytosed and rapidly translocated to the nucleus where it is concentrated in the nucleolus and binds to DNA. Angiogenin also activates cell-associated proteases, induces cell invasion and migration, stimulates cell proliferation, and organizes cultured cells to form tubular structures. The intracellular signaling pathways that mediate these various cellular responses are not well understood. Here we report that angiogenin induces transient phosphorylation of extracellular signal-related kinase1/2 (Erk1/2) in cultured human umbilical vein endothelial cells. Angiogenin does not affect the phosphorylation status of stress-associated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein (MAP) kinases. PD98059--a specific inhibitor of MAP or Erk kinase 1 (MEK 1), the upstream kinase that phosphorylates Erk1/2--abolishes angiogenin-induced Erk phosphorylation and cell proliferation without affecting nuclear translocation of angiogenin. In contrast, neomycin, a known inhibitor of nuclear translocation and cell proliferation, does not interfere with angiogenin-induced Erk1/2 phosphorylation. These data indicate that both intracellular signaling pathways and direct nuclear functions of angiogenin are required for angiogenin-induced cell proliferation and angiogenesis.

Fibroblast growth factors are translocated to the nucleus of human endothelial cells in a microtubule- and lysosome-independent pathway.

Exogenous acidic and basic fibroblast growth factors undergo rapid nuclear translocation in human umbilical vein endothelial cells. When nuclear translocation reaches saturation, more than 70% of the internalized growth factors are in the nuclear fraction. Lysosomal inhibitors, such as leupeptin and chloroquine, and microtubule inhibitors including colchicine and 2-methoxyl-beta-estradiol neither increase nor decrease nuclear translocation. The results suggest that nuclear translocation of fibroblast growth factors does not require cytosolic accumulation or lysosomal processing and that the transportation of exogenous growth factors across the cytoplasm is independent of microtubules.

Human angiogenin is rapidly translocated to the nucleus of human umbilical vein endothelial cells and binds to DNA.

Human angiogenin is translocated to the nucleus of human umbilical vein endothelial cells in a time-dependent manner. Exogenous angiogenin appears in the nucleus in 2 min, reaches saturation in 15 min when 85% of the internalized angiogenin is in the nuclei, and remains associated with the nucleus for at least 4 h. Endothelial cells cultured at low density have a much higher capacity to translocate angiogenin to the nucleus than do those cultured at high density. This observation is consistent with previous findings that both the ability of endothelial cells to proliferate in response to angiogenin and the expression of an angiogenin receptor on the cell surface depend on cell density. Nuclear (125)I-angiogenin is not degraded and is neither spontaneously dissociated nor replaced by unlabeled angiogenin. It is, however, released by deoxyribonuclease I, but not by ribonuclease A, suggesting that angiogenin binds to DNA in the nucleus. These results suggest that in addition to acting as a ribonuclease, angiogenin may play a role in regulating gene expression by direct binding to DNA.

Modulation of juxtamembrane cleavage ("shedding") of angiotensin-converting enzyme by stalk glycosylation: evidence for an alternative shedding protease.

The role of juxtamembrane stalk glycosylation in modulating stalk cleavage and shedding of membrane proteins remains unresolved, despite reports that proteins expressed in glycosylation-deficient cells undergo accelerated proteolysis. We have constructed stalk glycosylation mutants of angiotensin-converting enzyme (ACE), a type I ectoprotein that is vigorously shed when expressed in Chinese hamster ovary cells. Surprisingly, stalk glycosylation did not significantly inhibit release. Introduction of an N-linked glycan directly adjacent to the native stalk cleavage site resulted in a 13-residue, proximal displacement of the cleavage site, from the Arg-626/Ser-627 to the Phe-640/Leu-641 bond. Substitution of the wild-type stalk with a Ser-/Thr-rich sequence known to be heavily O-glycosylated produced a mutant (ACE-JGL) in which this chimeric stalk was partially O-glycosylated; incomplete glycosylation may have been due to membrane proximity. Relative to levels of cell-associated ACE-JGL, rates of basal, unstimulated release of ACE-JGL were enhanced compared with wild-type ACE. ACE-JGL was cleaved at an Ala/Thr bond, 14 residues from the membrane. Notably, phorbol ester stimulation and TAPI (a peptide hydroxamate) inhibition of release-universal characteristics of regulated ectodomain shedding-were significantly blunted for ACE-JGL, as was a formerly undescribed transient stimulation of ACE release by 3, 4-dichloroisocoumarin. These data indicate that (1) stalk glycosylation modulates but does not inhibit ectodomain shedding; and (2) a Ser-/Thr-rich, O-glycosylated stalk directs cleavage, at least in part, by an alternative shedding protease, which may resemble an activity recently described in TNF-alpha convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765-27767].

Inhibition of human angiogenin by DNA aptamers: nuclear colocalization of an angiogenin-inhibitor complex.

Specific ligands (aptamers) for angiogenin were selected from a 72-mer oligodeoxynucleotide library consisting of 28 randomized positions flanked by two constant regions of 22 residues each. From a starting pool of approximately 10(14) molecules, 19 angiogenin-binding ligands were obtained. Among them, two oligonucleotides showed significant inhibition of the ribonucleolytic activity of angiogenin with apparent Kis of 0.65 and 0.60 micro M, respectively. One of them was shortened on the basis of its secondary structure to provide a 45-mer oligonucleotide that retained much of the inhibitory properties of the parent molecule. It inhibits both the angiogenic and cell proliferative activities of angiogenin but does not interfere with its nuclear translocation in human endothelial cells. Importantly, the inhibitor is cotranslocated to the nucleus with angiogenin in a approximately 1:1 stoichiometric ratio. These results demonstrate that the inhibition of angiogenin-induced cell proliferation and angiogenesis by the oligonucleotide is due to suppression of the ribonucleolytic activity of angiogenin, an event that occurs most likely within the cell nucleus.

Organogenesis and angiogenin.

Our search for an angiogenesis-inducing factor in culture medium conditioned by human colon adenocarcinoma cells (HT-29) was inspired by the 'organizer' hypothesis originally postulated by Spemann. It led us to the isolation of angiogenin, a 14 kD protein homologous to pancreatic ribonuclease and one of the most potent stimulators of blood vessel formation known. This review summarizes the properties of angiogenin, its enzymatic and three-dimensional relationship to ribonuclease A (RNase A), those aspects of its structure that are critical for its biological function, and the therapeutic potential of angiogenin inhibition. Despite having the same arrangement of catalytic residues as RNase A, angiogenin has very low enzymatic activity. It lacks one of the four disulphide loops of RNase A; instead, the corresponding residues form part of a cell binding region. Both the catalytic activity and cell binding site are essential for angiogenesis. Angiogenin binds to cell-surface actin in confluent endothelial cells and to an as yet uncharacterized receptor on proliferating cells. Internalization and translocation to the nucleolus are also required for activity. Inhibitors of angiogenin can block angiogenesis in vitro and prevent tumour growth in vivo. Thus, a noncytotoxic neutralizing monoclonal antibody prevents the establishment of HT-29 human tumour xenografts in up to 65% of treated athymic mice. In those tumours that develop, the number of vascular elements is reduced. Actin also prevents the establishment of tumours while exhibiting no toxic effects at daily doses > 50 times the molar amount of circulating mouse angiogenin. These antagonists also inhibit the appearance of tumours derived from two other human tumour cell lines. Inhibition of the action of angiogenin may prove to be an effective therapeutic approach for the treatment of malignant disease.

Nuclear translocation of human angiogenin in cultured human umbilical artery endothelial cells is microtubule and lysosome independent.

Exogenous angiogenin undergoes rapid nuclear translocation in cultured human umbilical artery endothelial cells at 37 degrees C but not at 4 degrees C. Treatment of cells with colchicine, nocodazole and taxol, which disrupt the microtubule system, does not affect the nuclear translocation process of angiogenin, suggesting that cells transport internalized angiogenin in a microtubule independent fashion. Lysosomal inhibitors, chloroquine and leupeptin, neither inhibit nor enhance the nuclear translocation of angiogenin, indicating that lysosomal targeting and processing are not required for, and do not compete with, the nuclear translocation. Moreover, treatment of cells with a tyrosine kinase antagonist, genistein, does not change the ability of the cells to translocate angiogenin into the nucleus. We suggest that exogenous angiogenin is translocated to the nucleus by a mechanism that does not require activation of tyrosine kinase, but includes receptor-mediated endocytosis, microtubule and lysosome independent transport across the cytoplasm, and nuclear localization sequence-assisted nuclear import.

Limited proteolysis of human kidney angiotensin-converting enzyme and generation of catalytically active N- and C-terminal domains.

The somatic form of angiotensin converting enzyme is a class I ectoenzyme that is bound to the surface of endothelial calls. It consists of two homologous, catalytic domains of approximately 600 residues each; a juxtamembrane "stalk" region; a transmembrane, hydrophobic sequence; and a 30 residue, C-terminal cytosolic domain. We have used limited proteolysis to probe the structural and functional properties of the enzyme. Endoproteinase Asp-N cleaves both the Thr615-Asp616 and the Leu1219-Asp1220 peptide bonds to generate the two catalytic domains which were isolated by a combination of immunoaffinity and lisinopril Sepharose affinity chromatography. The enzymatic characteristics of the N and C fragments were examined with angiotensin I, hippuryl-His-Leu, and luteinizing hormone-releasing hormone and indicate that both fragments contain catalytically active sites that retain their individual functional integrity.

Proteolytic release of membrane proteins: studies on a membrane-protein-solubilizing activity in CHO cells.

Diverse membrane proteins are solubilized by a specific proteolytic cleavage in the stalk sequence adjacent to the membrane anchor, with release of the extracellular domain. Examples are the amyloid precursor protein, membrane-bound growth factors and angiotensin-converting enzyme (ACE). The identities and characteristics of the responsible proteases remain elusive. We have studied this process in Chinese hamster ovary (CHO) cells stably expressing wild-type ACE (WT-ACE) or juxtamembrane (stalk) deletion or chimaera mutants. Determination of the C termini (i.e. the cleavage sites) of released, soluble wild-type and mutant ACE by MALDI-TOF mass spectrometry indicated that the membrane-protein-solubilizing protease (MPSP) in CHO cells is not constrained by a particular cleavage site motif or by a specific distance from the membrane, but instead may position itself with respect to the putative proximal, folded extracellular domain adjacent to the stalk. Nevertheless, kinetic analyses of release rates indicated that a minimum distance from the membrane must be preserved. Interestingly, soluble full-length (anchor-plus) WT-ACE incubated with fractions of, or intact, CHO cells was not cleaved. In all cases, release was stimulated by a media change or by the addition of phorbol ester, with rate enhancements of 5- and 50-fold, respectively, for WT-ACE. The phorbol ester effect was abolished by staurosporine, a protein kinase C (PKC) inhibitor. We propose that the CHO cell MPSP that solubilizes ACE: (1) only cleaves proteins embedded in a membrane; (2) requires an accessible stalk and cleaves at a minimum distance from both the membrane and proximal extracellular domain; (3) positions itself primarily with respect to the proximal extracellular domain and (4) is regulated in part by a PKC-dependent mechanism.

A putative angiogenin receptor in angiogenin-responsive human endothelial cells.

Angiogenin stimulates both [3H]thymidine incorporation and proliferation of human endothelial cells in sparse cultures. Under these conditions, a 170-kDa cell surface protein can be detected that binds angiogenin specifically. Angiogenin-stimulated cell growth is concentration-dependent and is completely inhibited by an anti-angiogenin monoclonal antibody, but not by a nonimmune control antibody. It is not affected by the nonangiogenic homolog, RNase A, nor by other angiogenic proteins, such as basic fibroblast growth factor and its antibody. Results suggest that under specific conditions, endothelial cells express an angiogenin receptor that may mediate angiogenin-stimulated DNA synthesis and proliferation and play an important role in angiogenin-induced angiogenesis.

Identification of N-linked glycosylation sites in human testis angiotensin-converting enzyme and expression of an active deglycosylated form.

The sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential N-linked glycosylation sites, Asn90 and Asn109, were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn155, Asn337, and Asn586, are partially glycosylated, whereas Asn72 appears to be fully glycosylated. The only potential site that was not modified is Asn620. Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser625. When expressed in the presence of the alpha-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies.

Expandable metal stents for non-malignant bronchial obstruction.

An expandable metal stent was inserted to relieve bronchial obstruction following lobectomy for localised squamous carcinoma which had not been relieved by bronchoplasty with a Goretex flap. This resulted in substantial improvement in lung function and exercise tolerance for nine months, following which severe inflammation around the stents required residual pneumonectomy.

Proteolytic release of membrane-bound angiotensin-converting enzyme: role of the juxtamembrane stalk sequence.

Many structurally and functionally diverse membrane proteins are solubilized by a specific proteolytic cleavage in the stalk sequence adjacent to the membrane anchor, with release of the extracellular domain. Examples are the amyloid precursor protein, membrane-bound growth factors, and angiotensin-converting enzyme (ACE). The identities and characteristics of the responsible proteases remain elusive. We have studied this process in Chinese hamster ovary (CHO) cells stably expressing wild-type ACE (WT-ACE; human testis isozyme) or one of four juxtamembrane (stalk) mutants containing either deletions of 17, 24, and 47 residues (ACE-JM delta 17, -JM delta 24, and -JM delta 47, respectively) or a substitution of 26 stalk residues with a 20-residue sequence from the stalk of the low-density lipoprotein receptor (ACE-JMLDL). The C termini of released, soluble WT-ACE and ACE-JM delta 17 and -JMLDL were determined by MALDI-TOF mass spectrometry analyses of C-terminal peptides generated by CNBr cleavage. Observed masses of 4264 (WT-ACE) and 4269 (ACE-JM delta 17) are in good agreement with an expected mass of 4262 for the C-terminal CNBr peptide ending at Arg-627, indicating cleavage at the Arg-627/Ser-628 bond in both WT-ACE and ACE-JM delta 17, at distances of 24 and 10 residues from the membrane, respectively. Data for ACE-JM delta 24 are also consistent with cleavage at or near Arg-627. For ACE-JMLDL, in which the native cleavage site is absent, observed masses of 4372 and 4542 are in close agreement with expected masses of 4371 and 4542 for peptides ending at Ala-628 and Gly-630, respectively, indicating cleavages at 17 or 15 residues from the membrane. These data indicate that the membrane-protein-solubilizing protease (MPSP) in CHO cells is not constrained by a particular cleavage site motif or by a specific distance from the membrane but instead may position itself with respect to the putative proximal, folded extracellular domain adjacent to the stalk. Nevertheless, cleavage at a distance of 10 residues from the membrane is more favorable, as ACE-JM delta 17 is cleaved 12-fold faster than WT-ACE. In contrast, ACE-JM delta 24 is released 17-fold slower, suggesting that a minimum distance from the membrane must be preserved. This is supported by results with the ACE-JM delta 47 mutant, which is membrane-bound but not cleaved, likely because the entire stalk has been deleted. Finally, soluble full-length (anchor-plus) WT-ACE is not cleaved when incubated with various CHO cell fractions or intact CHO cells. On the basis of these and other data, we propose that the CHO cell MPSP that solubilizes ACE (1) only cleaves proteins embedded in a membrane; (2) requires an accessible stalk and cleaves at a minimum distance from both the membrane and proximal extracellular domain; (3) positions itself primarily with respect to the proximal extracellular domain; and (4) may have a weak preference for cleavage at Arg/Lys-X bonds.

Assignment of free and disulfide-bonded cysteine residues in testis angiotensin-converting enzyme: functional implications.

Human testicular angiotensin-converting enzyme (tACE) is an extracellular protein that contains seven cysteine residues. The cysteines occur in a sequential distribution that is precisely mimicked in the tACE from rabbit and mouse, and in both domains of all known species of somatic ACE. One of the cysteines in human tACE, Cys496, is present in the reduced form as shown by labeling it with 5-[[2-(iodoacetyl)amino]ethylamino]naphthalene-1-sulfonic acid, isolating the fluorescent peptide from enzymatic digests by HPLC, and analyzing its sequence by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Thiol reagents have no significant effect on the activity of tACE, indicating that this Cys is not involved in catalysis. The other six cysteines exist as three disulfides. Mass spectral analysis of cyanogen bromide peptides has established that the cystine connectivities follow a nearest-neighbor, aabbcc, pattern i.e., Cys152-Cys158, Cys352-Cys370, and Cys538-Cys550, in which the disulfides form three small loops of five, 17, and 11 residues, respectively. Although these disulfide loops constitute less than 5% of the total sequence of the protein, they contribute to the overall structural stabilization of tACE.

The C-terminal region of human angiogenin has a dual role in enzymatic activity.

The ribonucleolytic activity of angiogenin (Ang) is essential to Ang's capacity to induce blood vessel formation. Previous x-ray diffraction and mutagenesis results have shown that the active site of the human protein is obstructed by Gln-117 and imply that the C-terminal region of Ang must undergo a conformational rearrangement to allow substrate binding and catalysis. As a first step toward structural characterization of this conformational change, additional site-directed mutagenesis and kinetic analysis have been used to examine the intramolecular interactions that stabilize the inactive conformation of the protein. Two residues of this region, Ile-119 and Phe-120, are found to make hydrophobic interactions with the remainder of the protein and thereby help to keep Gln-117 in its obstructive position. Furthermore, the suppression of activity by the intramolecular interactions of Ile-119 and Phe-120 is counterbalanced by an effect of the adjacent residues, Arg-121, Arg-122, and Pro-123 which do not appear to form contacts with the rest of the protein structure. They contribute to enzymatic activity, probably by constituting a peripheral subsite for binding polymeric substrates. The results reveal the nature of the conformational change in human Ang and assign a key role to the C-terminal region both in this process and, presumably, in the regulation of human Ang function.

Angiotensin II: biosynthesis, molecular recognition, and signal transduction.

1. Angiotensin II is a well-known vasopressive octapeptide that is the principal end-product of the renin-angiotensin system. In addition to its tonic effect on vascular smooth muscle cells, it also stimulates aldosterone secretion from the adrenals and promotes sodium reabsorption through renal tubular cells. 2. These physiological functions have been appreciated for some time, but as details of the molecular and cell biology of the angiotensin response mechanism become understood, it is increasingly apparent that the hormone has a much broader repertoire. Its functional variability is made possible by (i) different enzymatic routes for its generation, (ii) different receptors distributed in different tissues, (iii) different mechanisms for receptor regulation, and (iv) different signal transduction pathways. 3. This insight is the direct consequence of advances in pharmacology that led first to inhibitors of angiotensin converting enzyme and later to angiotensin II receptor antagonists. This review looks at the current status of angiotensin biochemistry and physiology and provides a basis for anticipation of future developments.

LRRning the RIte of springs.

The determination of the crystal structure of the ribonuclease inhibitor-ribonuclease A complex provides exciting new insight on how the leucine-rich repeat allows a single molecule to get around the problem of inhibiting an entire family of enzymes.

Crystal structure of bovine angiogenin at 1.5-A resolution.

The capacity of angiogenin (Ang) to induce blood vessel growth is critically dependent on its ribonucleolytic activity. Crystallography and mutagenesis of human Ang have previously shown that its pyrimidine binding site is obstructed by Gln-117, implying that a conformational change is a key part of the mechanism of Ang action. The 1.5-A-resolution crystal structure of bovine Ang, in which glutamic acid is substituted for Gln-117, now confirms that a blocked active site is characteristic of these proteins. Indeed, the inactive conformation of bovine Ang is stabilized by a more extensive set of interactions than is that of human Ang. The three-dimensional structure of the putative receptor binding site is also well conserved in the two proteins. The Arg-Gly-Asp segment of this site in bovine Ang, which is replaced by Arg-Glu-Asn in human Ang, does not have a conformation typical of an integrin recognition site.