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

177

Biochimica et BiophysicaActa, 1162 (1993) 177-186

© 1993 Elsevier Science Publishers B.V. All fights reserved 016%4838/93/$06.00

BBAPRO 34423

Characterization and sequencing of rabbit, pig and mouse angiogenins: discernment of functionally important residues and regions * Michael D. Bond a

,,1, Daniel

J. Strydom a,b and Bert L. Vallee a

Centerfor Biochemical and Biophysical Sciences and Medicine, Harvard Medical Schoo~ Boston, MA (USA) and b Department of Pathology, Harvard Medical School, Boston, MA (USA)

(Received 13 July 1992)

Key words: Angiogenesis; Ribonuclease superfamily; Ribonuclease inhibitor; Tryptophan fluorescence; Homology 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-ll4, 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 a n d / o r protein synthesis inhibition activities of human angiogenin.

Introduction

Angiogenin is a 14.1 kDa protein that was identified based on its capacity to stimulate blood vessel growth in vivo in the chick chorioallantoic membrane (CAM)

Correspondence to: B.L. Vallee, Center for Biochemical and Biophysical Sciences and Medicine, Seeley G. Mudd Building, 250 Long,wood Avenue, Harvard Medical School, Boston, MA 02115, USA. 1 Present address: Genetics Institute, One Burtt Road, "Andover, MA 01810, USA. * Data supplementary to this article are deposited with, and can be obtained from Elsevier Science Publishers B.V., BBA Data Deposition, P.O. Box 1345, 1000 BH Amsterdam, The Netherlands. Reference should be made to No. BBA/DD/530/34423/l162 (1993) 177. The supplementary information includes data on enzymatic digests used, and amino-acid composition and sequence tables. Abbreviations: CAM, chorioallantoic membrane; RNase, ribonuclease(s); RNase A, bovine pancreatic ribonuclease A; PRI, human placental ribonuclease inh~itor; HPLC, high-performance liquid chromatography; Mes, 4-morpholineethanesulfonic acid; Hepes, 4-(2-hydroxTethyl)-l-piperazineethanesulfonic acid; < Glu or < Q, pyroglntamic acid.

and rabbit cornea assays [1,2]. It was initially isolated from human tumor-cell-conditioned medium, but has also been found in normal body fluids, such as blood and milk [3,4]. Angiogenin is homologous to the ribonuclease (RNase) superfamily of proteins that includes the pancreatic RNases and numerous others [5-7]. It exhibits ribonudeolytic activity, albeit of markedly different magnitude from that of other RNases [8-10]: it cleaves most RNA substrates at rates 105-106-fold slower than does RNase A. Both the ribonucleolytic and blood-vessel-inducing activities of angiogenin are abolished upon binding to the 51 kDa protein, placental ribonuclease inhibitor (PRI) [11]. The homology between angiogenin and the RNases provides an exceptional opportunity for structure/ function analysis of these proteins, owing to the extensive sequence and structure information available for the RNases. Complete sequences of 42 mammalian pancreatic RNases have been determined [7,12] from which a 'consensus' sequence can be compiled. Not surprisingly, crystallographic and o t h e r studies have shown that m a n y o f the residues that are conserved across species have defined functional a n d / o r structural roles (reviewed in Refs. 13 and 14).

178 The sequences of human, bovine and mouse angiogenin were reported previously [4,5,15,16] and those of two additional angiogenins, from rabbit and pig sera, are presented here. We have derived a consensus-type sequence for these proteins and compared it with that of the pancreatic RNases. Key differences between these two classes of proteins have been identified which suggest specific residues of angiogenin to be involved in its biological activities. We have specifically examined the binding of angiogenin to PRI in detail. In human angiogenin, this interaction minimally encompasses the catalytic site and the region around Trp-89 [17-19]. Since some of the nonhuman angiogenins lack Trp-89, we have used them to monitor the effects of changes in this region on inhibitor binding. The Trp fluorescence spectra and the rate and equilibrium constants of binding for the interaction of PRI with rabbit, pig and bovine angiogenins are all consistent with the role of this region of angiogenin as a contact area for the inhibitor. Materials and Methods

Materials. Trace-hemolyzed sera from pig and rabbit and hemolyzed serum from mouse were obtained from Pel-Freez Biologicals; other materials were obtained as described [20,21]. Purification of angiogenins from serum. Angiogenin was purified from 0.5-10 liter quantities of pig, rabbit, and mouse sera as described for bovine angiogenin [21], except that 50 mM (mouse) or 70 mM (pig, rabbit) sodium phosphate (pH 6.6) was used as starting buffer in the CM-cellulose ion-exchange step. The sera were first diluted with water to reduce conductivities to below that of the starting buffer, adjusted to pH 6.6 with 3 M HCI and then loaded onto the resin. After washing with 2-4 volumes of starting buffer, the column was treated with 1 M NaC1 in starting buffer to elute bound angiogenin. Further purification was accomplished by Mono-S cation-exchange (Tris buffer (pH 8.0)) and ClS HPLC, as described [21]. Peak fractions from this column were pooled, lyophilized, redissolved in Milli-Q purified water and assayed for protein concentration by amino-acid analysis. Detection of angiogenin in chromatographic fractions. Angiogenin was detected by a combined PRI-binding/ RNase activity assay [20]. The first assay detects any protein that binds to PRI, whether angiogenin or an RNase. The results are expressed in RNase A equivalents, i.e., the amount of RNase A that would be required to bind the same amount of inhibitor. The second assay detects RNase activity toward yeast RNA; the results are also expressed in RNase A equivalents. Angiogenin-containing fractions exhibit a high PRIbinding activity and a very low RNase activity.

Enzymatic and biological activity measurements on the purified angiogenins. Ribonucleolytic activity toward yeast RNA was measured by a modification of the precipitation method of Blackburn et al. [22] as described [20]. Activity toward wheat germ RNA, poly(C) and poly(U) was determined according to Shapiro et al. [8]. All water and buffer solutions used in the assays were passed through Sep-Pak C18 cartridges (Millipore) to remove trace levels of ribonucleolytic activity. Angiogenic activity was assessed by the chicken embryo CAM assay of Knighton et al. [23] as described [1]. Cleavage of intact ribosomes and inhibition of cell-free protein translation by the angiogenins was examined by the procedure of St. Clair et al. [10] as modified by Harper and Vallee [24].

Interaction of rabbit, pig and bovine angiogenins with PR/. Fluorescence measurements were made as described [18]. The apparent second-order rate constant, ka, for association of PRI with bovine, pig and rabbit angiogenins was measured by a competition binding experiment with RNase A [17,18]; at least six determinations were employed in each case. The dissociation rate constant, k d, was determined by cation-exchange HPLC to monitor the release of angiogenin from the protein:PRI complex in the presence of excess scavenger for PRI, i.e., RNase A [17,19]. Ki values were calculated from the ratio of the association and dissociation constants. Simultaneous measurements on a sample of human angiogenin Yielded values that were in reasonable agreement with those obtained previously [191.

Amino-acid analysis and protein sequence determination. Amino-acid composition was determined by PicoTag analysis (Waters Associates) on samples that had been subjected to vapor phase hydrolysis, precolumn derivatization with phenylisothiocyanate, and separated on reverse-phase HPLC [5]. Samples for cysteine analysis were oxidized with performic acid prior to hydrolysis, and samples for tryptophan determination were hydrolyzed in 4 M methanesulfonic acid containing 0.2% (w/v) tryptamine and neutralized with NaOH before derivatization. The phenylthiocarbamyl derivatives were separated on 3.9 ×300 mm 'PicoTag' columns for enhanced resolution. Amino-acid sequences were determined with a Beckman 890C spinning-cup sequencer upgraded to microsequencing status [5]; reagents and solvents were used as supplied by the manufacturer. Addition of 0.1% (v/v) water to the heptafluorobutyric acid, and 0.1% (v/v) ethanethiol to the 25% (v/v) TFA converting reagent enhanced the sensitivity of the procedure. Identification and quantitation of the phenylthiohydantoin amino acids was performed with the aid of a Nelson Analytical data system. The amino-terminal sequences were obtained with 2 nmol pig angiogenin and 0.7 nmol pyroglutaminase-treated rabbit angio-

179 genin. Hydroxylamine and CNBr cleavage were performed as described [5] and enzymatic digests data are deposited in the BBA Data bank (see footnote on first page of this article). Gel electrophoresis of proteins. SDS-PAGE was performed with 5% and 15% (w/v) acrylamide concentrations for the stacking and running gels, respectively, according to the method of Laemmli [25]; samples contained 5% (v/v) 2-mercaptoethanol. Proteins were visualized by silver staining with an ICN kit, and molecular weight standards from 3000-43000 were from Bethesda Research Laboratories. Results

Purification of serum angiogenins Angiogenin was isolated from pig, rabbit and mouse sera by the procedure described previously for bovine angiogenin [21], except that in the first step of purification the sample was applied to CM-cellulose in 50 or 70 rather than 100 mM sodium phosphate (pH 6.6). This increased the total amount of bound protein and led to significantly better yields of the angiogenins (up to 10-fold). At the next step of purification, Mono-S cation-exchange HPLC, fractions with activity indicative of angiogenin, i.e., high PRI binding with little or no detectable RNase activity, were readily identified in the rabbit (at 38-46 min) and mouse (at 63-68 min) sera profiles (Fig. la and c). Identification of pig angiogenin was more tentative, since the sole peak of PRI binding activity overlaps with that of RNase activity (Fig. lb). Fractions indicated by the bars in Fig. l a - c were pooled separately and subjected to further purification by C18 HPLC; in all cases a peak with activity characteristic of angiogenin was clearly identified. The pig and mouse proteins eluted as symmetrical peaks with no detectable RNase contamination (Fig. 2b and c), whereas the rabbit protein peak was irregular and overlapped a small amount of RNase activity at its leading edge. To remove the RNase, peak fractions were pooled, diluted with water and reinjected onto the Cls column (Fig. 2a). Sequencing of the three proteins later confirmed their identities as angiogenins. The asymmetric shape of the rabbit angiogenin Cla peak appears to result both from different equilibrium forms of the protein (also observed for human angiogenin, which elutes as a double "peak on the C18 column [1,3]) and perhaps from microheterogeneity. Samples of fractions collected at 0.5-rain intervals across the peak in Fig. 2a showed identical mobilities on SDS-PAGE, with no contaminating bands. Their amino-acid compositions were the same within experimental error except that the small trailing shoulder contained slightly less histidine.

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Fig. 1. Mono-S cation-exchange HPLC of angiogenin-containing fraction from CM-cellulose chromatography of (a), 6 liter rabbit; (b), 10 liter pig or (c), 0.6 liter mouse sera. Samples were applied to a Mono-S H R 5 / 5 column equilibrated in 10 mM Tris, 50 mM NaCI (pH 8.0) and eluted with a linear gradient to 0.4 M NaCI over 80 min; the flow rate was 0.8 m l / m i n . Fractions were assayed for PRI binding (e) and RNase (,x) activities, which are plotted as RNase A equivalents, and those fractions containing angiogenin (indicated by the horizontal bar) were pooled and subjected to further purification. RNase activity is shown amplified 20-fold. No absorbance tracing was obtained for mouse sera owing to a chart recorder malfunction.

Final yields were typically approx. 100 /~g/1 serum for porcine and rabbit angiogenins, similar to the amounts obtained from human and bovine plasma or sera. The recovery from two mouse angiogenin preparations was only 40/~g/1 serum. Whether this amount reflects lower angiogenin levels in mouse serum or simply the smaller scale at which the purifications were carried out (0.6 l) could not be determined. Most of the mouse angiogenin served for amino acid and sequence analysis, limiting the amount available for other studies.

Properties of the angiogenins Rabbit, pig and mouse angiogenins each migrate as a single, discrete band on SDS-PAGE, with apparent molecular weights of about 16 000. Amino-acid compositions are similar to those of human and bovine angio-

180 120

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TABLE I

A

Amino-acid composition of mammalian angiogenins 0

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18 10 6 9 6 13 6 4 7 6 4 2 9 4 6 9 6 0

14.9 (15) 10.0(10) 5.6 (6) 10.5 (10) 4.9 (5) 10.5 (11) 9.1 (10) 5.8 (6) 7.2 (7) 4.6 (5) 3.1 (3) 1.3 (1) 7.1 (8) 3.3 (3) 7.0 (7) 9.9 (10) 6.3 (6) 0.1 (0)

15.4 (16) 9.2 (9) 9.1 (10) 9.8 (9) 5.8 (6) 9.0 (9) 7.5 (8) 4.1 (3) 7.3 (7) 4.6 (5) 6.4 (8) 2.4 (2) 3.6 (4) 3.4 (3) 6.8 (7) 11.3 (12) 6.1 (6) 0.9 (1)

13.3 (13) 7.9 (8) 9.7 (11) 8.9 (8) 5.9 (6) 11.2 (11) 7.1 (7) 5.9 (6) 8.3 (8) 3.7 (4) 4.6 (5) 2.4 (3) 3.9 (4) 5.4 (5) 7.1 (7) 8.8 (9) 6.0 (6) 0.4 (0)

a Average of multiple analyses from at least two separate purifications; hydrolysis was for 18-20 h at 110°C; Cys was determined as cysteic acid; Trp was determined after hydrolysis in methanesulfonic acid in the presence of tryptamine. Values for human and bovine are from the sequence, as those shown in parentheses for pig, rabbit and mouse.

60

ELUTION T I M E ( r a i n )

Fig. 2. Reverse-phase HPLC of (a), rabbit; (b), pig and (c), mouse angiogenins. Fractions from Fig. la or c, or 100 /~1 aliquots from fractions in Fig. lb, were pooled and injected directly onto a SynChropak RP-P Cls column (0.46 x 25 cm, SynChrom) equilibrated in 70% solvent A (0.1% TFA) and 30% solvent B (2-propanol/acetonitrile/water, 0.08% TFA, 3:2:2). Elution was accomplished with a linear gradient to 42% solvent B over 54 min; the flow rate was 0.8 ml/min. Fractions were then assayed for PRI binding (e) and RNase ( z~) activities; RNase activity is shown amplified 100-fold. The RNase from Fig. l b does not elute under these gradient conditions.

genins (Table I), with the 6 Cys/mol clearly distinguishing them from other RNases. Rabbit angiogenin, like human, contains 1 Trp/mol. All of them bind PRI with 1:1 stoichiometry, based on comparison of concentration determinations from PRI-binding assays and amino-acid analyses. All display very low levels of activity toward conventional substrates for RNase A (Table II).

Angiogenesis The rabbit and pig angiogenins were assayed on the chick embryo CAM (Table III) and both gave responses comparable to those obtained for human angiogenin. They show a dose response over the range of 0.1 to 10 ng and reach maximal values of 52 and 57% positive reaction, respectively.

Inhibition of protein synthesis Human angiogenin is an effective inhibitor of cellfree protein synthesis: It selectively cleaves the 18 S rRNA component of the 40 S ribosomal subunit when present in the intact ribosome or the isolated 40 S subunit [10]. At concentrations of 40-70 nM, both

TABLE II

Relative ribonucleolytic activities of serum-derived angiogenins compared to RNase A Values should be considered as upper limits of activity for the nonhuman angiogenins; n.d. not determined. Assay conditions are: 5 m g / m l yeast RNA, 1 mM EDTA, 0.1 M Tris (pH 7.5), 25°C; 2.7 m g / m l wheat germ RNA, 50 mM sucrose, 150 mM NaC1, 30 mM Tris (pH 8.5), 37"C; 0.4 m g / m l poly(C) or poly(U), 40 mM Tris (pH 8), 37°C. Although the mouse and rabbit proteins show somewhat higher activities, these two also were at greatest risk of contamination by RNases during purification owing to the small amount ot protein recovered or to the necessity of pooling large numbers ot fractions in the final chromatography step. Hence, these values can only be taken as upper limits of RNase activity.

RNase A Angiogenin Human Bovine Pig Rabbit Mouse

yeast RNA

wheat germ RNA

poly(C)

poly(U)

100

100

100

100

n.d. 0.0002 0.002 0.01 0.04

0.0003 0.0006 0.001 0.03 0.02

0.0005 0.0007 0.0003 0.00003 0.001

0.0006 0.0007 0.001 0.006 0.008

181 TABLE III

100

Angiogenesis activity of rabbit, pig and human angiogenins: CAM-assay Individual assays employed sets of 10-20 eggs. Human angiogenin was obtained either from plasma or from BHK cell or E. coli expression systems [1,3,26]. The total number of eggs employed for each sample is given in parentheses. rabbit

pig

human

H20

10 1 0.1

52 (82) 40 (58) 31 (55)

10 1 0.1

57 (42) 47 (47) 39 (54)

10 1 0.1

52 (67) 47 (68) 28 (39)

-

22 (50)

human and bovine angiogenins completely abolish the uptake of [35S]Met by a rabbit reticulocyte lysate into newly synthesized proteins. Electrophoretic analysis of the reticulocyte rRNA reveals a characteristic pattern of limited cleavage products. Pig and rabbit angiogenins were also tested in this system for their effects on in vitro protein synthesis. Both proteins completely inhibit [35S]Met incorporation at concentrations of 70 nM. Moreover, they yield nearly identical rRNA cleavage products compared to those generated by human angiogenin (data not shown). As demonstrated by urea-polyacrylamide gel electrophoresis, all three proteins generate a doublet followed by a group of 7 to 10 faster migrating bands. Thus, all angiogenins tested to date have displayed very similar activities in this system.

Fluorescence changes on binding of angiogenins to PRI Characteristic changes in tryptophan fluorescence have been observed upon interaction of PRI with either human angiogenin or RNase A [18]. Rabbit angiogenin, like the human protein, contains a single tryptophan and exhibits a weak fluorescence spectrum when excited at 285 nm (Fig. 3). PRI contains 6 Trp/mol and displays a more intense fluorescence spectrum: the ~'max is about 340 nm. Complexes of PRI with either human or rabbit angiogenin yield identical fluorescence spectra: Their intensities are 55-60% greater than the sum of the individual spectra, and the ~'max shifts to approx. 345 nm. This change appears to result entirely from alterations in the environment of the angiogenin Trp residue, since oxidation of the indole ring of Trp-89 abolishes the fluorescence change [18], as does its mutagenesis to Met (Fox, E.A., personal communication). PRI complexes with the bovine and pig angiogenins, which do not contain tryptophan, show less extensive increases in fluorescence intensity, ap-

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340 360 380 400 WAVELENGTH (nm) Fig. 3. Fluorescence emission spectra of angiogenins and angiogenin:PRI complexes (Acx = 285 nm). Samples contain 0.4/~M angiogenin, 0 or 0.4 /zM PRI and 0.1 M Mes/0.1 M NaCI/1 mM EDTA (pH 6). Spectra are: (a), human angiogenin-PRI complex, and also rabbit angiogenin-PRI complex; (b), bovine angiogenin-PRI complex; (c), porcine angiogenin-PRI complex; (d), PRI alone; (e), rabbit angiogenin alone and (f), human angiogenin alone. Spectra for bovine and porcine angiogenins alone are essentially flat on this scale.

prox. 30%, and the )Lmax values shift toward shorter wavelengths. It should be noted that complexation with RNase A, which also lacks tryptophan, has virtually no effect on the fluorescence spectrum of PRI [18].

Rate and binding constants for the interaction of angiogenins with PRI Association rate constants, ka, were measured by competition experiments with RNase A, for which the k a is lolown. Rabbit, pig, and bovine angiogenins display faster association rates with PRI than the human protein (Table IV). Dissociation rate constants, kd, for the angiogenin: PRI complexes were determined by measuring the release of angiogenin from the complex over several days (Fig. 4), Release of bovine and pig angiogenins was substantiafly faster than that observed previously for the human; they both reached 50% TABLE IV

Rate and binding constants for the interaction of serum-derioed angiogenins with human PRI In 0.1 M NaC1, 1 mM EDTA, 50-120/.~M dithiothreitol, 0.1 M Mes (pH 6), 25"C.

Human a Rabbit Bovine Pig RNase A a Human placental RNase b a Data from Ref. 19. b Data from Ref. 27.

ka'10 - s (M - ] s - l )

kd.107 (s -1)

Ki (fM)

1.8 3.4 + 0.4 6.4 + 1.0 5.5 + 1.0 3.4 1.9

1.3 0.5 22 12 150 1.8

0.71 0.15 3.4 2.2 44 0.9

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TIME (days) Fig. 4. Dissociation of bovine ( • ) , pig (o) and rabbit ( • ) angiogenins from the PRI complexes over time. Angiogenin was incubated with 1.5 equivalents PRI for 20 min, after which a 250-fold excess of RNase A was added as a scavenger. Samples were then incubated at 25°C and aliquots withdrawn periodically and injected onto a HPLC ion-exchange column to determine the amount of free angiogenin. Quantitation was based on comparison to standard samples containing only angiogenin and RNase A.

dissociation in a week or less, whereas human angiogenin undergoes only 10-12% dissociation after 10 days. The rate for rabbit angiogenin was slower yet; it dissociated only 6-7% in 10 days. Values of K~ for the different angiogenins, calculated from the individual rate constants, range from 0.15 to 3.4 fM (Table IV). It is noteworthy that the rate constants and K i for human angiogenin are more similar to those for human placental ribonuclease than to those for other species of angiogenin or RNase A (Table IV).

I Pyr: Tryp:

Sequencing studies The primary sequences of rabbit and pig angiogenins were determined by Edman degradation of fragments derived by chemical and enzymatic cleavages (Figs. 5 and 6; Supplemental Tables I-VI (see footnote on first page of this article)). Overlapping fragments were obtained for all but two of the peptide junctions in pig angiogenin and all but four in the rabbit. Nonoverlapping peptides were aligned by homology with the human and bovine proteins. One residue in rabbit angiogenin (Cys-92) and two in pig angiogenin (Cys-91 and Phe-99) yielded no detectable Edman degradation products but were identified from the amino-acid compositions of the peptides. The < Glu-1 of rabbit angiogenin is inferred from the amino-acid composition of tryptic peptide 1 and the requirement for pyroglutaminase treatment to unblock the amino-terminus. Calculated molecular weights are 14362 and 14059 for the rabbit and pig angiogenins, respectively. Partial sequence information was obtained for the mouse protein confirming its identity as an angiogenin. Edman degradation of 300 pmol of intact mouse angiogenin yielded no detectable products. This, coupled with its amino-acid composition and genomic DNA sequence, suggests that the mouse protein has an amino terminal < Glu. Three tryptic peptides were recovered in sufficient quantities for analysis (Supplemental Table VII), and the sequences obtained, Phe-Leu-Thr-GlnHis-His-Asp-Ala-Lys-Pro-Lys, Asp-Val-Asn-Thr-PheIle-His-Gly-Asn-Lys and Ala-Ser-Ala-Gly-Phe-Arg, are identical to residues 9-19, 41-50, and 95-100, respec-

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Fig. 5. Amino-acid sequence of rabbit angiogenin and the evidence used to assign it. The extent of sequencing of fragments derived from treatment with pyroglutaminase (Pyr), trypsin (Tr,~), cyanogen bromide, and hydroxylamine is indicated by capital letters. A dot in the fragments represents a region of a peptide that was not sequenced but has an amino-acid composition consistent with the proposed sequence; a dot in the full sequence denotes a peptide bond which was assigned by homology, not by sequencing through the bond.

183

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tively, of the protein encoded by the mouse angiogenin gene [16]. Discussion

The angiogenins from different species exhibit many common properties: They all are members of the RNase superfamily of proteins, highly basic with molecular weights near 14000 and angiogenic in the CAM assay (Table III) [1,21]. Like other RNases, they bind PRI but unlike most of them, their activities toward conventional RNA substrates are orders of magnitude lower (Table II). Moreover, all angiogenins that have been 1 Mouse Rabbit Pig Bovine H ....

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tested (human, bovine, rabbit and pig) selectively cleave 18 S rRNA in intact ribosomes and are potent inhibitors of in vitro protein synthesis. Sequence identity between the rabbit, pig, bovine and mouse angiogenins - relative to the human protein - is 73, 66, 64 and 75%, respectively (Fig. 7). Among all five angiogenins, half of the residues (62 of 123) are invariant. None of the angiogenins undergo post-translational modification apart from spontaneous cyclization of the N-terminal Gin residue in the human, rabbit and mouse proteins. There is no evidence of O-linked glycosylation and there are no Asn-X-Thr/Ser sites present for potential N-linked glycosylation, even though such sites exist in the human, bovine, pig and mouse pancreatic RNases. The angiogenins contain a much higher percentage of basic residues than any of the other RNases. Arg and Lys residues constitute 16.3-17.9% of the total residues in the angiogenins, while for most pancreatic and other RNases they are 9-12% [7]. The highly basic nature of the angiogenins is shared by other physiologically active proteins such as platelet-derived growth factor, basic fibroblast growth factor and y-interferon, and may contribute to biological activity, perhaps by aiding interaction with cell membranes or extracellular matrices.

120

F E FFS L F E V FQQKVH RIFID[EISlF I I T S Q F E F I TPRR

Fig. 7. Alignment of mammalian angiogenin sequences from current and previous studies [4,5,15,16]. Numbering is based on the human protein; residues conserved in all five angiogenins are boxed.

Interaction of mammalian angiogenins with human PRI The interaction of human angiogenin with PRI, an intracellular inhibitor found in many mammalian tissues [28], occurs with an extremely low K i value, 0.7 fM. Previous experiments revealed that formation of the inhibitor complex involved both the catalytic site

184 and the region around Trp-89 of angiogenin [17,18]. The latter region was implicated by the 50% increase in Trp fluorescence that occurs upon binding of angiogenin to PRI. All of the angiogenins have very similar catalytic site sequences, but they differ around residue 89. Pig and bovine angiogenins contain neither Trp-89 nor Pro-88. Hence, their interaction with PRI was compared with that of human and rabbit angiogenins to further establish the nature of the fluorescence changes that accompany binding. The fluorescence spectrum of the rabbit angiogenin: PRI complex is identical to that observed with human angiogenin (Fig. 3). The intensity is 50-60% greater than the sum of the individual protein and inhibitor spectra, and the ~'max is shifted toward higher wavelength. In contrast, both pig and bovine angiogenin induce only a 23-30% increase in fluorescence and the }tmax shifts toward lower wavelength. It is clear that some, if not all, of the spectral changes observed for human and rabbit angiogenins derive from changes in the local environment of their Trp-89 residue. Since the pig and bovine angiogenins lack Trp-89, some of the change that occurs when they interact with PRI must arise from Trp residues present in the inhibitor as well. The absence of any substantial spectral change on formation of the PRI complex with RNase A [18] would suggest that in this case either the Trp residues of the inhibitor are not perturbed or that the presence of Tyr in place of Trp-89 somehow offsets the spectral effect. It may be pertinent, in this regard, that the PRI-RNase A binding constant, 44 fM, is 13-300-times greater than that for any of the angiogenins. However, human placental RNase, which has 37% sequence identity to RNase A and 27% to angiogenin, is inhibited by PRI with a K i of 0.9 fM, virtually identical to that of human angiogenin [27]. The equilibrium and rate constants of binding reveal further differences between the angiogenins. Human and rabbit angiogenins and human placental RNase have kd values 10-40-fold lower than those for the pig and bovine proteins (Fig. 4, Table IV). As a consequence, half-lives of dissociation of inhibitor-protein complexes are on the order of months for the former two angiogenins versus 7 days or less for the latter two. Similarly, the human and rabbit angiogenins and human placental RNase have the slowest association rates and the lowest K i values, by a factor of three or more. * The PRI binding data are consistent with the observation that Trp-89 (in human and rabbit angiogenins)

* It should be noted that these data were obtained with h u m a n PRI and a more comprehensive analysis of interspecies differences should include rabbit, pig, bovine and m o u s e PRIs.

or other residues in its immediate vicinity form contacts with the inhibitor. The involvement of Trp-89 itself might account for the differences in the rate constants of the human and rabbit versus the pig and bovine angiogenins. The slower kj values for the former might result from the extra free energy required to displace the Trp indole ring from a hydrophobic pocket in PRI, and their slower association rates could reflect the energy required to properly orient the large Trp side chain for interaction with the inhibitor. This type of involvement of exposed hydrophobic residues in protein-protein interactions has been observed elsewhere [29]. Alternatively, these differences could be accounted for by the fact that a positively charged residue, Arg, replaces Trp in the pig and bovine angiogenins (Fig. 7).

Functional implications of the angiogenin sequences Sequence homology and enzymatic activity identify the angiogenins as members of the superfamily of RNases which includes the pancreatic RNases, eosinophil-derived neurotoxin, eosinophil cationic protein, bovine seminal RNase, bovine brain RNase, and others (reviewed in Refs. 7 and 30). The three-dimensional structure of the most well-studied member of the superfamily, bovine pancreatic RNase A, has been determined by both crystallographic and NMR techniques. The overall structures of the other proteins in the superfamily are thought to be similar to that of RNase A, since the important active site and structural residues are completely conserved, as are three of the disulfide bonds. Moreover, all members cleave RNA preferentially on the 3' side of pyrimidines. Despite the basic structural similarities, differences do exist, and the activities and physiological roles of some of the members have clearly diverged. Angiogenin and eosinophil cationic protein, for example, display unique enzymatic activities that are 6 and 2 orders of magnitude lower than those of the other RNases, respectively. In addition, several members display potent biological activities. Both eosinophil proteins are neurotoxic and helminthotoxic; bovine seminal RNase is cytotoxic for tumor cells; angiogenin is a powerful inducer of neovascularization [7,30]. It seems likely that different members of the RNase family have undergone specific mutations that are responsible for their characteristic activities. One means to identify critical regions and/or residues for a given member, e.g., angiogenin, is to examine the amino-acid sequences of that protein across species. Residues that are involved in the physiological activities of angiogenin are likely to be conserved or replaced conservatively in different species. Moreover, these critical residues are more likely to be replaced radically in other members of the superfamily which display different biological activities.

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Fig. 8. Amino-acid sequences of human angiogenin [5,6] and bovine pancreatic RNase A [31]. Residues conserved (solid-line boxes) or conservatively replaced (dashed-line boxes) in human, bovine, rabbit, pig and mouse angiogenins are indicated, as are residues conserved in 42 pancreatic RNases from 40 mammalian species [7,12]. Asterisks indicate residues that are conserved in the angiogenins but differ from the corresponding amino acid in greater than 90% of the pancreatic RNases.

Thus, the residues of interest are those that are conserved across species yet differ in other members of the superfamily. Among the human, rabbit, pig, bovine and mouse angiogenins, 62 residues are strictly conserved and an additional 15 are conservatively replaced, a total of 77 invariant amino acids (Fig. 8). Of these, 52 also occur in the same position in at least some of the pancreatic RNases and presumably serve structural or catalytic roles that are shared by other RNases. Indeed for RNase A, many of them have been shown to be involved in maintaining structure (e.g., Phe-9, Asp-15, Tyr-25, Arg-33, Asn-43 and Leu-ll5), substrate binding (e.g., Gin-12, Thr-44, Glu-108, and Ser-ll8), catalysis (His-13, Lys-40, and His 114), or alignment of catalytic residues (Asp-ll6). These assignments are based on crystallographic, 2-D NMR and chemical investigations [13,14,32-34]. The remaining 25 conserved residues of the angiogenins differ from the corresponding ones in the pancreatic RNases, and these are the ones most likely to be involved in the unique activities of angiogenin. Their positions in the primary sequence are indicated in Fig. 8, and in the computed three-dimensional structure of angiogenin [35] in Fig. 9. Interestingly, these residues tend to cluster in certain regions of the tertiary structure, primarily in external loops and bends. In particular, residues 54, 62, 64, 69, 70, 109 and 110 are all spatially dose, with residues 62-69 occupying a large convoluted loop (Arg-70 likely is part of a fl-sheet) and residues 109 and 110 part of a compact bend. This area also contains four deletions with respect to RNase A (Fig. 9) and, hence, is significantly different from the

same area in the RNases. Other regions showing groups of conserved residues which differ from the RNases include positions 16-21, in which there are several bends or turns leading away from the active site; residues 5-11, in the N-terminal a-helix; and perhaps also residues 38-41, at the active site. Mutagenesis experiments are consistent with important roles for at least two of the above regions. In one study, residues 58-70 of human angiogenin were replaced with the corresponding segment from RNase A (residues 59-73), which inserted a fourth disulfide bond into angiogenin [24]. The ribonucleolytic activity of the mutant protein was increased while the angiogenic potency decreased suggesting that the product was more RNase-A-like and less angiogenin-like. In another study, residues 8-22 of human angiogenin were replaced with 7-21 of RNase A [35]. This mutation had no effect on activities toward tRNA, naked 18 S and 28 S rRNA, or dinucleotide substrates, but did decrease the capacity of the protein to inhibit in-vitro protein synthesis 30-fold. Both sets of data are consistent with the involvement of the altered regions in the biological, non-RNase-A-type activities of angiogenin. In addition, recent experiments concerning the effects of deamidation and limited proteolysis [37,38] indicate that the former region, at least, may be involved in the interaction of angiogenin with endothelial cells, perhaps by binding to a cellular receptor [39]. Thus, the experimental data support the participation of two of

60 Fig. 9. Ball-and-stick model of human angiogenin based on the computed structure of Palmer et al. [35]; residues 1 and 120-123 were not included in the structure. Black-filled circles show residues indicated by an asterisk in Fig. 8; X indicates a deletion in angiogenin relative to RNase A; gray-filled circles show positions of essential catalytic residues His-13, Lys-40 and His-ll4.

186 the regions indicated in the angiogenic activities of angiogenin. An additional inference from the angiogenin sequence data relates to the low level of ribonucleolytic activity displayed by human angiogenin and eosinophil cationic protein. This has been attributed to the lack of a positively-charged residue at either position 65 or position 117 which could be involved in substrate binding [40]. The mammalian pancreatic RNases, turtle pancreatic RNase, and eosinophil-derived neurotoxin all possess an Arg or Lys in one of these two positions, and all are relatively active toward conventional RNA substrates. Human angiogenin and eosinophil cationic protein, on the other hand, do not contain a basic residue in either position, and both are much less active. It is now apparent that the bovine, pig, rabbit and mouse angiogenins also lack basic residues here, and they, too, are inactive toward conventional RNA substrates. Therefore, the current data are consistent with the above postulate. It should be noted, however, that the inhibition of in-vitro protein synthesis by angiogenin also involves ribonucleolytic activity, i.e., hydrolysis of ribosomal RNA. In this system angiogenin appears to be at least as potent as RNase A [10]. Hence, the role of a positive charge in these two positions still requires investigation.

Acknowledgements We gratefully acknowledge the expert technical assistance of N. Nguyen, R. Ettling, the late N. Sarkissian and W. Brome. This work was supported by funds from Hoechst AG, under an agreement with Harvard University.

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