The angiogenins.

The angiogenic and other biological functions of the angiogenins, members of the pancreatic RNase superfamily of proteins, are reviewed in the context of their primary and tertiary structures. The ribonucleolytic activity and interactions with the placental ribonuclease inhibitor have seen much study in the last few years. The mechanism of the angiogenic activity of angiogenin has recently been postulated as involving multiple interactions with other proteins through specific regions on the molecular surface of angiogenin. These molecular partners include heparin, plasminogen, elastase, angiostatin, actin and most importantly a 170-kilodalton receptor on subconfluent endothelial cells. The existence of the latter receptor was established in conjunction with a mitogenic activity of angiogenin on subconfluent cells. The levels of angiogenin in various physiological and disease states are summarized, including various studies on pregnancy and angiogenin. Correlations are seen between states of enhanced angiogenesis and angiogenin levels. An overview of the relationship of angiogenin and the other RNases of the superfamily showed that their genes all are in relative close proximity on human chromosome 14. Examination of the many expressed sequence tags published in the public databanks, for angiogenin and the other RNases, revealed that angiogenin and RNase-4 (the most evolutionarily conserved RNase), share various identical 5'-untranslated regions on their sets of messenger RNAs, suggesting that their genes are in very close proximity on chromosome 14 and that they are products of differential splicing. This in turn suggests that, in both humans and mice, expression of these two proteins is under identical control, with obvious implications for their biological activities. The evolutionary history of the angiogenins is examined briefly on the basis of the protein sequences of the human, rabbit, pig, two bovine and four mouse angiogenins, and two mouse angiogenin pseudogene sequences. The discrepancy between the conventional requirement for conservatism in structure to allow multimolecule interactions, and the actual fast-changing sequence of the angiogenins, in concert with the wide-ranging activity even in birds, of human angiogenin, is discussed.

An angiogenic protein from bovine serum and milk--purification and primary structure of angiogenin-2.

Bovine serum and milk contain a basic angiogenic protein that binds tightly to placental ribonuclease inhibitor. It was purified from both sources by ion-exchange and reversed-phase chromatographies. Its amino acid sequence revealed that it is a member of the ribonuclease superfamily. It contains 123 amino acids in a single polypeptide chain, is cross-linked by three disulfide bonds, is glycosylated at Asn33, and is 57% identical to bovine angiogenin. The amino-terminal and carboxyl-terminal residues are pyroglutamic acid and proline, respectively. The protein has ribonucleolytic activity that is similar to, but somewhat lower than, that of bovine angiogenin, i.e. very low relative to RNase. It is angiogenically potent on chicken chorioallantoic membrane, but less so than angiogenin. The sequence and activities demonstrate that this protein is a second, distinct, member of the angiogenin sub-family of pancreatic ribonucleases, and is referred to as angiogenin-2.

Interaction of heparin with human angiogenin.

HT-29 human colon adenocarcinoma cells adhere rapidly to human angiogenin (Ang) via interactions with cell-surface heparan sulfate moieties (Soncin, F., Shapiro, R., and Fett, J. W. (1994) J. Biol. Chem. 269, 8999-9005). Soluble heparin inhibits adhesion, and Ang itself binds tightly to heparin-Sepharose. In the present study, the interaction of Ang with heparin has been further characterized. The basic cluster Arg-31/Arg-32/Arg-33 has been identified as an important component of the heparin binding site. Mutations of these residues, and of Arg-70 as well, decrease both the affinity of Ang for heparin-Sepharose and the capacity of Ang to support cell adhesion. Replacements of four other basic residues do not affect heparin binding. Heparin partially protects Ang from cleavage by trypsin at Lys-60, suggesting that heparin also binds to the region of Ang that contains this residue. The map here determined indicates that the heparin recognition site on Ang lies outside the catalytic center; indeed, heparin has no significant effect on the ribonucleolytic activity of Ang. It also does not influence the angiogenic activity of this protein. Light scattering measurements on Ang-heparin mixtures suggest that 1 heparin chain (mass of 16.5 kDa) can accommodate approximately 9 Ang molecules. The minimum size required for a heparin fragment to effectively inhibit HT-29 cell adhesion to Ang was determined to be 6 disaccharide units. The implications of these findings for inhibition of Ang-mediated tumor establishment in vivo are discussed.

Identification of actin isoforms after in situ hydroxylamine cleavage on sequencer membranes: serum actin is a cytoplasmic isoform.

Actin was isolated from fetal bovine serum by means of affinity chromatography on DNase I-Sepharose, reversed phase chromatography and SDS-gel electrophoresis. It was identified as a cytoplasmic isoform by direct micro sequence analysis of hydroxylamine cleavage products which had been generated in situ after covalent linkage of the protein to sequencer membrane disks through arylamine or isothiocyanato functionalities.

Comparison of amino acid analyses by phenylisothiocyanate and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate precolumn derivatization.

An extensive retrospective comparison was conducted of the long-term repeatability and consistency of amino acid analyses using phenylisothiocyanate and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) precolumn derivatization. Amino acid standards were analyzed more than 130 times on more than 60 independent occasions by each of these two precolumn derivatization methodologies, during routine amino acid analysis procedures. Similar coefficients of variation were seen only when very freshly prepared derivatives were analyzed. When realistic aging for < 20 h was taken into account, the extreme stability of the AQC derivatives stood out. Chromatography of AQC derivatives using HPLC solvents prepared on a large scale provided the lowest coefficients of variation. The superiority of AQC over PTC methodology was clearly apparent.

The amino acid sequence of human ribonuclease 4, a highly conserved ribonuclease that cleaves specifically on the 3' side of uridine.

A ribonuclease (RNase) that cleaves specifically on the 3' side of uridine [Shapiro, R., Fett, J. W., Strydom, D. J. & Vallee, B. L. (1986a) Biochemistry 25, 7255-7264] was purified from human plasma and its amino acid sequence was determined. This protein is a 119-residue single-chain polypeptide cross-linked by four disulfide bonds and has an amino-terminal pyroglutaminyl residue. No post-translational modifications were observed during extensive sequence studies on peptide fragments, except for the amino-terminal pyroglutamic acid and a possible deamidation of Asn66. The protein is homologous to the pancreatic ribonucleases and angiogenin, but differs substantially from both of these proteins; the protein sequence has 43% identity with human pancreatic ribonuclease and 39% identity with human angiogenin, as compared to 35% identity between human angiogenin and pancreatic ribonuclease. It is referred to as RNase 4, based on the nomenclature currently used for the genes of pancreatic RNase (RNase 1) and the eosinophil-derived RNases (RNase 2 and RNase 3). Virtually all of the RNase active-site components, including the catalytic residues His12, His119 and Lys41, are preserved. However, some invariant residues of RNase 1 are replaced, e.g. Lys7 by arginine, Asp14 by histidine, and Pro42 by arginine. RNase 4 contains a unique two-residue deletion at the position corresponding to amino acids 77 and 78 of pancreatic RNase, and its carboxyterminal sequence is truncated at position 122. The deletion in angiogenin at position 21 is also found in RNase 4. RNase 4 is very similar to two RNases isolated from bovine and porcine liver, and together they form a new family in the RNase superfamily. The degree of inter-species similarity (90%) is much greater than within the pancreatic RNase and angiogenin families, which suggests that this ribonuclease could possess a physiologically important function other than general RNA catabolism.

Characterization and sequencing of rabbit, pig and mouse angiogenins: discernment of functionally important residues and regions.

Rabbit, pig and mouse angiogenins have been purified from blood serum and characterized, and the rabbit and pig proteins have been sequenced fully. A partial sequence of the mouse protein is consistent with the sequence deduced from the genomic DNA (Bond, M.D. and Vallee, B.L. (1990) Biochem. Biophys. Res. Commun. 171, 988-995). All three angiogenins are homologous to the pancreatic RNases and contain the essential catalytic residues His-13, Lys-40 and His-114, and the 6 half-cystines of the human protein. Like human angiogenin they display extremely low ribonucleolytic activities toward wheat-germ RNA, yeast RNA, poly(C) and poly(U). The rabbit and pig proteins induce neovascularization in vivo and also inhibit protein synthesis in vitro. The interaction of rabbit, pig and bovine angiogenins with placental ribonuclease inhibitor, a potent inhibitor of angiogenin, was examined by fluorescence spectroscopy. Rate and equilibrium binding constants indicate that rabbit angiogenin binds to the inhibitor much like human angiogenin, whereas the pig and bovine proteins show significant differences. A comparison of the five angiogenin sequences now available points to specific residues that are highly conserved among them but differ from the corresponding residues in the RNases. These residues are clustered in particular regions of the three-dimensional structure, two of which contribute to the angiogenic, second-messenger and/or protein synthesis inhibition activities of human angiogenin.

Actin is a binding protein for angiogenin.

The 42-kDa angiogenin binding protein isolated previously has been purified to electrophoretic homogeneity. It has been identified as a member of the actin family by peptide mapping and partial amino acid sequencing. The interaction of bovine muscle actin with angiogenin is similar to that of the angiogenin binding protein. Angiogenin induces the polymerization of actin below the critical concentration for spontaneous polymerization. The interaction occurs both in solution and on a poly(vinylidene difluoride) membrane. It is inhibited by excess unlabeled angiogenin and also by platelet factor 4 and protamine, which are known inhibitors of angiogenesis. Two other angiogenic molecules, basic fibroblast growth factor and tumor necrosis factor alpha, bind to 125I-labeled actin and can be crosslinked by a water-soluble carbodiimide. Both actin and an anti-actin antibody inhibit the angiogenic activity of angiogenin in the chicken embryo chorioallantoic membrane assay. The results indicate that the angiogenin binding protein is a cell surface actin and suggest that the reaction between angiogenin and this actin is an essential step in the angiogenesis process induced by angiogenin.

Importance of asparagine-61 and asparagine-109 to the angiogenic activity of human angiogenin.

Two distinct regions of angiogenin are critical for angiogenic activity: a catalytic site capable of cleaving RNA and a noncatalytic site, encompassing residues 60-68, which may bind to an endothelial cell-surface receptor [Hallahan, T. W., Shapiro, R., & Vallee, B. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2222-2226]. We have now shown that Asn-61 is an essential residue within the cell-binding site and that in addition a segment containing Asn-109 is part of this site. Both asparagines undergo nonenzymatic deamidation during long-term storage or treatment at alkaline pH. While the isolated desamido-61 and desamido-109 derivatives retain nearly full enzymatic activity, their angiogenic activity on the chicken embryo chorioallantoic membrane is markedly attenuated and they do not inhibit angiogenin-induced neovascularization. Tryptic peptide mapping and Edman degradation demonstrate that the isolated deamidated derivatives primarily contain isoaspartic rather than aspartic acid at the positions in question (83% for desamido-61, greater than 99% for desamido-109). Aspartic acid replacement of Asn-61 and Asn-109 by site-directed mutagenesis results in the same ribonucleolytic and angiogenic activities as those of the spontaneous deamidation products. However, the aspartyl derivatives differ strikingly from their isoaspartyl counterparts in that they do inhibit angiogenin-induced angiogenesis. These results indicate that the combination of ribonucleolytic activity and receptor-binding capacity is not sufficient for angiogenic activity and that Asn-61 and Asn-109 within the noncatalytic site are required for some additional function, as yet undefined.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of human testicular angiotensin-converting enzyme: the testis isozyme is identical to the C-terminal half of endothelial angiotensin-converting enzyme.

Angiotensin-converting enzyme (ACE; EC 3.4.15.1) is a zinc-containing dipeptidyl carboxypeptidase widely distributed in mammalian tissues and is thought to play a critical role in blood pressure regulation. Testis contains a unique, androgen-dependent ACE isozyme of unknown function. We have determined the cDNA sequence for human testicular ACE; it encodes a protein that is identical, from residue 37 to its C terminus, to the second half or C-terminal domain of the endothelial ACE sequence [Soubrier, F., Alhenc-Gelas, F., Hubert, C., Allegrini, J., John, M., Tregear, G. & Corvol, P. (1988) Proc. Natl. Acad. Sci. USA 85, 9386-9390]. The full-length human testis ACE cDNA was constructed from a composite of cloned cDNAs, obtained by a combination of (i) immunoscreening and hybridization screening of a human testicular cDNA library in lambda gt11 and (ii) hybridization screening of human testis cDNAs constructed with ACE-specific primers and amplified by the polymerase chain reaction. The protein sequence inferred consists of a 732-residue preprotein including a 31-residue signal peptide. The mature polypeptide has a molecular weight of 80,073. The testis enzyme contains the second of the two putative metal-binding sites (His-Glu-Met-Gly-His) identified in endothelial ACE. This indicates that the functionally active catalytic site is within the C-terminal domain of the endothelial enzyme, accounting for the previous finding that these two structurally dissimilar isozymes are virtually identical catalytically. Of 22 testis ACE cDNAs cloned and sequenced, 3 have unique 5' regions, consisting of inserted, deleted, or substituted sequences up to 328 base pairs long, which have apparently arisen by alternative pre-mRNA splicing.

Amino acid sequence of bovine angiogenin.

The amino acid sequence and disulfide bridges of bovine plasma derived angiogenin were determined by sequencer analysis of the intact protein and fragments derived by enzymatic and chemical digestion. Bovine angiogenin is a single-chain protein of 125 amino acids; it contains six cysteines and has a calculated molecular weight of 14,595. In contrast to the human protein its amino terminus is unblocked. It has the following sequence: H2N-Ala1-Gln-Asp-Asp-Tyr-Arg-Tyr-Ile-His-Phe10-Leu-Thr-Gln-His-Tyr -Asp-Ala-Lys- Pro-Lys20-Gly-Arg-Asn-Asp-Glu-Tyr-Cys-Phe-Asn-Met30-Met-Lys- Asn-Arg-Arg-Leu-Thr - Arg-Pro-Cys40-Lys-Asp-Arg-Asn-Thr-Phe-Ile-His-Gly-Asn50-Lys- Asn-Asp-Ile-Lys-Ala - Ile-Cys-Glu-Asp60-Arg-Asn-Gly-Gln-Pro-Tyr-Arg-Gly-Asp-Leu70- Arg-Ile-Ser-Lys-Ser - Glu-Phe-Gln-Ile-Thr80-Ile-Cys-Lys-His-Lys-Gly-Ser-Ser-Arg90- Pro-Pro-Cys-Arg-Tyr - Gly-Ala-Thr-Glu-Asp100-Ser-Arg-Val-Ile-Val-Val-Gly-Cys-Glu-Asn1 10-Gly-Leu-Pro- Val-His-Phe-Asp-Glu-Ser-Phe120-Ile-Thr-Pro-Arg-His-OH. Disulfide bonds link Cys(27)-Cys(82), Cys(40)-Cys(93), and Cys(58)-Cys(108). Bovine angiogenin is 64% identical with human angiogenin; like the human protein, it is homologous to the pancreatic ribonucleases, with conservation of active site residues. Two regions, 6-22 and 65-75, are highly conserved between the angiogenins but are significantly different from those of the ribonucleases, suggesting a possible role in the molecules' biological activity.

A micromethod for the determination of carboxyl-terminal amino acids of peptides and proteins: phenylisothiocyanate reaction with hydrazinolysates.

Proteins and peptides are digested with anhydrous hydrazine and the free amino acids, released from their carboxyl termini, are analyzed by reverse-phase chromatography as phenylthiocarbamyl (PTC) derivatives. Most of the potentially interfering hydrazides are removed due to the insolubility of their derivatives in the solution used for dissolving the phenylthiocarbamyl-amino acids. This methodology can be used to analyze peptides and proteins in quantities smaller than 100 pmol. The method is here employed for the screening of carboxyl termini in peptide fractions originating from an HPLC chromatogram.

Primary structure of human placental ribonuclease inhibitor.

The primary structure of the human placental ribonuclease inhibitor (PRI), a tight-binding inhibitor of angiogenin and pancreatic ribonucleases, has been determined from the cDNA. The sequence of the mature protein is composed of 460 amino acids, yielding a molecular mass of 49,847 g/mol. Peptides comprising 92% of the predicted sequence were isolated from a tryptic digest of PRI, and direct sequence information obtained for 65% of the molecule agreed at all positions with the sequence predicted from the cDNA. The amino acid sequence of PRI contains seven direct internal repeat units, each 57 amino acids in length. These repeat units comprise 87% of the molecule. The average degree of identity between any two is 39%. A region within each repeat unit displays similarity to tandem, leucine-rich repeats found in six other proteins. Modification of PRI with iodoacetic acid, p-(hydroxymercuri)benzoate, and 5,5'-dithiobis(2-nitrobenzoic acid) reveals that at least 30 of the 32 cysteine residues of PRI are in the reduced form.

Amino acid analysis utilizing phenylisothiocyanate derivatives.

Advances in liquid chromatography have brought about the development of new techniques in amino acid analysis which take full advantage of precolumn derivatization procedures. Using phenylisothiocyanate as the reagent, detection limits under 1 pmol can be routinely achieved, allowing the analysis of submicrogram protein samples. Analysis times as short as 10 min for samples after hydrolysis and 1 h for physiologic samples are possible. Accurate, reproducible quantitation of amino acids can be obtained from complex matrices such as plasma, urine, feed, and food samples. This level of performance and flexibility gives the analyst the first realistic alternative to ion-exchange analysis without compromising desirable features of the traditional methodology.

Sites of modification of human angiogenin by bromoacetate at pH 5.5.

Human angiogenin is inactivated by treatment with bromoacetate at pH 5.5. Use of [14C]bromoacetate and tryptic peptide mapping have identified the sites of carboxymethylation as His-13 and His-114, with His-114 reacting approximately 1.5-fold more rapidly than His-13. At later stages in the reaction, both His-13 and -114 become modified with His-114 in part forming a bis derivative. Comparison with carboxymethylhistidine derivatives of known structure obtained from bovine pancreatic ribonuclease A indicates that the reaction order is N-1 of His-114 greater than N-3 of His-13 greater than N-3 of His-114.

Expression of human angiogenin in cultured baby hamster kidney cells.

Baby hamster kidney cells were transformed with DNA sequences derived from the gene for human angiogenin. Expression was under the transcriptional control of the inducible mouse metallothionein 1 promoter. Recombinant angiogenin was purified and shown to be chemically, biologically, and enzymatically indistinguishable from the natural product. The large-scale production of recombinant angiogenin achieved should facilitate detailed studies into the structure-function relationships of this potent angiogenic molecule.

Isolation of angiogenin from normal human plasma.

Angiogenin, a potent blood vessel inducing protein, was previously isolated from medium conditioned by a human adenocarcinoma cell line [Fett, J. W., Strydom, D.J., Lobb, R.R., Alderman, E.M., Bethune, J.L., Riordan, J.F., & Vallee, B.L. (1985) Biochemistry 24, 5480-5486]. We now report that a protein which is physically and functionally identical with angiogenin is present in normal human plasma and can be purified to homogeneity by CM 52 and Mono S cation-exchange chromatography. The plasma-derived angiogenin exhibits the same angiogenic and ribonucleolytic activities, amino acid composition, molecular weight, immunoreactivity, and chromatographic behavior as the tumor cell derived protein. Peptide mapping and sequencing studies indicate chemical identity of the two proteins. The present yield of angiogenin from either plasma or serum is 60-150 micrograms/L. These findings demonstrate that angiogenin is not a tumor-specific product and provide further opportunities for the investigation of the role and mechanism of action of angiogenin and its potential diagnostic or prognostic utility.

Isolation and characterization of a human colon carcinoma-secreted enzyme with pancreatic ribonuclease-like activity.

A ribonuclease was isolated from serum-free supernatants of the human colon adenocarcinoma cell line HT-29. It was purified by cation-exchange and C18 reversed-phase high-performance liquid chromatography. The protein is basic, has a molecular weight of approximately 16,000, and has an amino acid composition that is significantly different from that of human pancreatic ribonuclease. The amino terminus is blocked, and the carboxyl-terminal residue is glycine. The catalytic properties of this ribonuclease resemble those of the pancreatic ribonucleases in numerous respects. Thus, it exhibits a pH optimum of approximately 6 for dinucleotide cleavage and employs a two-step mechanism in which transphosphorylation to a cyclic 2',3'-phosphate is followed by slower hydrolysis to produce a 3'-phosphate. It does not cleave NpN' substrates in which adenosine or guanosine is at the N position and prefers purines at the N' position. Like bovine ribonuclease A, the HT-29-derived ribonuclease is inactivated by reductive methylation or by treatment with iodoacetate at pH 5.5 and is strongly inhibited by the human placental ribonuclease inhibitor. However, in contrast, the tumor enzyme does not cleave CpN bonds at an appreciable rate and prefers poly(uridylic acid) as substrate 1000-fold over poly(cytidylic acid). It also hydrolyzes cytidine cyclic 2',3'-phosphate at least 100 times more slowly than uridine cyclic 2',3'-phosphate and is inhibited much less strongly by cytidine 2'-monophosphate than by uridine 2'-monophosphate. Other ribonucleases known to prefer poly(uridylic acid) were isolated both from human serum and from liver and were compared with the tumor enzyme. The physical, functional, and chromatographic properties of the serum ribonuclease are essentially identical with those of the tumor enzyme. The liver enzymes, however, differ markedly from the HT-29 ribonuclease. The potential utility of the tumor ribonuclease in the diagnosis of cancer is considered.

Human class 1 heparin-binding growth factor: structure and homology to bovine acidic brain fibroblast growth factor.

The structure of the major class 1 heparin-binding growth factor from human brain has been analyzed. Edman degradation performed on the native mitogen and on fragments generated by chemical and enzymatic cleavage allows the sequence to be described by four nonoverlapping segments. The sum of the amino acids of the four segments is in excellent agreement with the experimentally determined amino acid composition of the mitogen itself, suggesting that, jointly, they account for the entire molecule. The four segments can be aligned into a presumptive complete sequence that shows 92% identity with that of bovine acidic brain fibroblast growth factor. The data indicate that the human mitogen has the following sequence: Phe1-Asn-Leu-Pro-Pro-Gly-Asn-Tyr-Lys-Lys-Pro-Lys-Leu-Leu-Try15+ ++-Cys- Ser-Asn-Gly-Gly-His-Phe-Leu-Arg-Ile-Leu-Pro-Asp-Gly-Thr30-Val-Asp- Gly-Thr-Arg- Asp-Arg-Ser-Asp-Gln-His-Ile-Gln-Leu-Gln45-Leu-Ser-Ala-Glu-Ser-Val- Gly-Glu-Val- Tyr-Ile-Lys-Ser-Thr-Glu60-Thr-Gly-Gln-Tyr-Leu-Ala- Met-Asp-Thr-Asp-Gly-Leu-Leu-Tyr-Gly75-Ser-Gin-Thr-Pro-Asn-Glu-Glu- Cys-Leu-Phe- Leu-Glu-Arg- Leu-Glu90-Glu-Asn-His-Tyr-Asn-Thr-Tyr-Ile-Ser-Lys-Lys-His-Ala-Glu- Lys105-Asn- Trp-Phe-Val-Gly- Leu-Lys-Lys-Asn-Gly-Ser-Cys-Lys-Arg-Gly120-Pro-Arg-Thr-His-Tyr-Gly -Gln-Lys-Ala- Ile-Leu-Phe-Leu- Pro-Leu135-Pro-Val-Ser-Ser-Asp140.