(Received for publication, September 3, 1996, and in revised form, December 9, 1996)
From the Center for Biochemical and Biophysical
Sciences and Medicine and ¶ Department of Pathology, Harvard
Medical School, Boston, Massachusetts 02115
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 ~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.
Cell adhesion and angiogenesis are essential events in the establishment, growth, and dissemination of solid tumors. Molecules that are involved in these processes therefore constitute attractive targets for cancer therapy. Angiogenin (Ang),1 a 14.1-kDa single-chain, basic polypeptide in the pancreatic RNase superfamily (1), is both a potent inducer of angiogenesis (2) and an excellent substrate for adhesion of human tumor cells (3). Ang was first isolated from medium conditioned by HT-29 human adenocarcinoma cells (2), and recent findings strongly suggest that it plays a critical role in tumor formation. Thus, non-cytotoxic, anti-human Ang monoclonal antibodies and the Ang-binding protein actin prevent or delay the establishment of HT-29 and other human tumor xenografts in athymic mice (4, 5). This inhibition may reflect interference with the initial attachment of tumor cells to the substratum and/or suppression of subsequent angiogenesis required for nourishment of the growing tumor.
The precise molecular events involved in Ang-induced blood vessel formation and in cell adhesion to Ang remain to be determined. Results to date indicate that angiogenic activity requires both the ribonucleolytic action of Ang (6-8) and binding of the protein to endothelial cells (9-11). Adhesion of tumor cells to Ang is mediated by an as yet unidentified cell-surface heparan sulfate/chondroitin sulfate proteoglycan (3). Treatment of cells with heparinase or with inhibitors of proteoglycan synthesis or secretion decreases adhesion, as does the presence of soluble glycosaminoglycans (GAGs). Moreover, Ang binds tightly to heparin-Sepharose. These observations, apart from their implications for the nature of the adhesion receptor, raise the possibility that heparin itself may regulate or modulate some of the actions of Ang in vivo.
In the present study, physical and functional aspects of the interaction of Ang with heparin have been investigated further. We have examined the heparin recognition site on Ang and the stoichiometry of binding. Purified heparin fragments have been used to define the heparin size requirements for inhibition of tumor cell adhesion to Ang. The effects of heparin on the ribonucleolytic and angiogenic activities of Ang have also been determined.
Materials
<Glu-1 Ang and Met-(1) Ang were obtained from a recombinant
expression system in Escherichia coli (12). The two forms
differ only at their N termini (<Glu versus Met-Gln) and
are indistinguishable with respect to angiogenic and ribonucleolytic
activities. The <Glu-1 mutant derivatives R5A, R32A, R66A, and R70A,
and the Met-(
1) derivatives K40Q, H13A, R31A, and R33A were from
earlier studies (6, 7, 13). Porcine intestinal mucosa heparin (average Mr 16,500) and heparin disaccharides (I-A, I-B,
I-C, and I-D) were from Sigma. RNase A and Hitrap heparin-Sepharose
columns were from Pharmacia Biotech Inc. Protein concentrations were
determined by amino acid analysis with
6-aminoquinolinolyl-N-hydroxysuccinimidyl carbamate
precolumn derivatization (14).
Cell Culture
HT-29 human colon carcinoma cells (HTB38 from the American Type Culture Collection) were cultured in 75-cm2 culture flasks (Nunc) in Dulbecco's modified Eagle's medium (DMEM, Whittaker Bioproducts) containing 5% heat-inactivated fetal bovine serum (HyClone), 50 µg/ml gentamycin, and 200 ng/ml fungizone in a humidified atmosphere of 5% CO2, 95% air at 37 °C and routinely passaged at a 1/5 ratio.
Assays
Cell AdhesionBacteriological Petri dishes were coated with protein test samples as described (15). Subconfluent cell monolayers were harvested with 1 mM EDTA in Dulbecco's phosphate-buffered saline (DPBS), resuspended in DMEM containing 1 mg/ml bovine serum albumin (BSA fraction V, low endotoxin, Sigma; DMEM-BSA) and centrifuged for 5 min at 400 × g at room temperature. The cell pellet was washed twice with DMEM-BSA and resuspended in the same medium at 30,000 cells/ml. One ml of the cell suspension was seeded in the coated dishes and incubated in a humidified atmosphere of 5% CO2, 95% air at 37 °C for the indicated amount of time. The dishes were then washed and fixed, and the cell number was measured as described (15).
Angiogenic ActivityThe effect of heparin on the angiogenic
activity of Ang was assessed with the chicken embryo chorioallantoic
membrane (CAM) assay (2, 16). Five-µl aliquots of Ang (2 µg/ml)
with or without heparin (10 mg/ml) in DPBS were applied to Thermanox
disks and air-dried. The disks were then implanted on the CAM, and
angiogenesis was evaluated after 68 ± 2 h. The numbers
of positive and negative responses for each sample from two sets of
assays were combined, and 2 values were calculated from
an outcome contingency table by comparing the test sample with a water
control; the associated probabilities, p, were then obtained
(11). A value of p < 0.05 identifies a sample as
active.
The effect of heparin on the
ribonucleolytic activity of Ang was examined with both dinucleotide
(CpA) and polynucleotide (tRNA) substrates. In the former case, <Glu-1
Ang (2-4 µM) was incubated with 100 µM
substrate in 0.2 M Hepes, pH 7, at 25 °C in the presence
or absence of heparin. After 15.5-18 h, the concentrations of CpA and
the products cytidine cyclic 2,3
-phosphate and adenosine in the
mixtures were determined by C18 HPLC and
kcat/Km values were
calculated (6, 17). Assays with tRNA as substrate were performed as
described (18), except that the buffer was 0.1 M Hepes, pH
7, and incubations were for 2 h.
Oligonucleotide-directed Mutagenesis
The gene for the Ang triple mutant <Glu-1 R31A/R32A/R33A was
prepared by the polymerase chain reaction overlap extension method of
Ho et al. (19) as described (11). The template for
polymerase chain reaction was the expression plasmid pAng3 (13), and
the mutagenic oligonucleotides were
5-GCGAATCGATTATGGGGTTAACTAGTCC and
5
-GGACTAGTTAACCCCATAATCGATTCGC. DNA
sequencing confirmed the presence of the expected mutations and the
absence of any spurious changes.
Expression and Purification of R31A/R32A/R33A
R31A/R32A/R33A (<Glu-1 form) was expressed in E. coli and purified to homogeneity by Mono S and C18 HPLC (13). The mutant protein eluted 11 min earlier than native Ang during Mono S chromatography and 3 min after Ang during C18 HPLC. Amino acid analysis demonstrated the loss of three arginines, an increase of three alanines, and no other significant changes compared with native Ang.
Heparin-Sepharose Chromatography
Protein was loaded onto a 1-ml Hitrap heparin-Sepharose column, which had been equilibrated with DPBS at room temperature. The column was then washed with 3 ml of DPBS, and a 35-min linear gradient from 0.14 to 1 M NaCl in DPBS was applied. The flow rate was 0.66 ml/min. One-min fractions were collected and their ionic strength measured by conductimetry. Human Ang elutes from the Hitrap column at 0.64 M NaCl, 0.14 M lower than the concentration required with heparin-Sepharose CL4B (3).
Effect of Heparin on Cleavage of Ang by Trypsin
Ang (28 µg) was incubated with 0.5 µg of HPLC-purified
trypsin (20) at 37 °C in 8.2 µl of DPBS in the presence or absence of 70 µg of heparin. Reactions were stopped by addition of 1 µl of
1 M Mes, pH 2.2. The digests were dried directly onto
arylamine membranes (Sequelon, Millipore) and coupled to the membranes
by a carbodiimide reaction (21). After washing with water and methanol, membranes were placed in a Millipore ProSequencer and sequencing was
performed for five cycles by the TFA100 protocol. PTH-derivatives were
quantitated by HPLC (22). The Ang preparation employed in these
experiments consisted of a 15:1 mixture of the <Glu-1 and Met-(1)
forms. The use of the
-NH2 blocked Ang as the primary species facilitated detection of internal sequences that opened up
during the early stages of digestion; the presence of a trace quantity
of the unblocked protein provided a basis for normalizing yields
obtained in the presence and absence of heparin (see legend to Table
III).
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Light Scattering
Successive aliquots of <Glu-1 Ang (5.5 µl each, containing 2 nmol) were mixed with 2 nmol of heparin in 1 ml of DPBS in a quartz cuvette, and light scattering at 90° was measured at 320 nm with a Perkin-Elmer MPF-3 fluorescence spectrophotometer 1-5 min after each addition. Scattering was constant during this time interval. The observed scattering was corrected for the effect of dilution (in all cases < 10%). Scattering of the individual components (Ang at 30 µM; heparin at 2 µM) was negligible.
Heparin Fragments
One gram of heparin was depolymerized by treatment with nitrous acid as described (23). The product mixture was desalted on a 2.6 × 50-cm Sephadex G-10 column in 10% ethanol, concentrated to 5 ml by rotary evaporation, and fractionated by gel filtration on a Sephadex G-50 superfine column (1.6 × 200 cm) in 0.5 M ammonium bicarbonate at a flow rate of 15-20 ml/h. Fractions of 4.5 ml were collected and assayed for their uronic acid content by the carbazole reaction (24) and for their effects on HT-29 cell adhesion to Ang. Selected fractions were analyzed by PAGE with Azure A staining (25), which revealed size heterogeneity in all cases. Various pools were lyophilized, dissolved in 10% ethanol, and rechromatographed on Sephadex G-50 as above. Fractions that eluted in the region of interest were assayed for their uronic acid content, and selected fractions were then lyophilized and resuspended in water for further analysis.
Mass Spectrometry
The molecular masses of heparin fragments were measured by matrix-assisted laser desorption ionization mass spectrometry with Ang as a complexing agent and sinapinic acid as the matrix (26, 27).
Heparin-binding Sites on Angiogenin
Effects of Ang Mutations on Affinity for Heparin-SepharoseThe location of the heparin-binding site on Ang
was investigated initially by measuring the affinity for
heparin-Sepharose of eight Ang mutants available from previous studies.
The derivatives selected contained replacements of basic residues,
primarily arginines, from several different regions of the molecule.
Basic amino acids are known to be important components of
heparin-binding sites in general, and arginine has been shown to have
particularly high affinity for sulfate groups on GAGs (28). Some of the
mutant proteins had been produced as Met-(1) derivatives, whereas
others were in the natural <Glu-1 form (see "Experimental
Procedures"). Heparin-Sepharose chromatography of unmutated Met-(
1)
Ang and <Glu-1 Ang revealed that these two forms elute from
heparin-Sepharose at different NaCl concentrations (0.68 M
and 0.64 M, respectively; Table I). Thus the
behavior of each mutant was compared with that of its corresponding
parent. Four of the derivatives examined (R31A, R32A, R33A (Fig.
1A), and R70A) had significantly decreased affinity for heparin-Sepharose: they eluted at NaCl concentrations 0.05-0.10 M lower than for Ang (Table I). Three other
mutants with similarly decreased basicity (R5A, K40Q, and R66A) had
virtually unaltered affinity, as did the one His mutant examined, H13A. RNase A, a highly basic Ang homologue, eluted at a substantially lower
NaCl concentration, 0.25 M (Fig. 1A).
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To assess the overall contribution of the basic cluster Arg-31, -32, and -33, a triple mutant (R31A/R32A/R33A) was prepared by oligonucleotide-directed mutagenesis. It eluted from heparin-Sepharose at a NaCl concentration 0.15 M lower than for the parent (<Glu-1) protein (Fig. 1B). This decrease in affinity is greater than that resulting from any of the three individual mutations, and somewhat less than that expected if their effects are independent and additive.
Effects of Heparin on Cleavage of Angiogenin by TrypsinIncubation of Ang with 2-3% (w/w) trypsin at 37 °C for 18 h results in cleavage of all 19 theoretically susceptible peptide bonds (1). The capacity of heparin to protect these various sites from trypsin was examined by sequencing the unseparated peptide mixtures produced during the early stages of digestion. Consistent with previous findings (29), the predominant site of cleavage in the absence of heparin was Lys-60: i.e. the major products obtained during the five cycles analyzed were PTH-Asn, -Gly, -Asn, -Pro, and -His, respectively (the sequences of all possible tryptic peptides are shown in Table II). Approximately 5% of the Ang had been hydrolyzed at this site after 2 min (Table III). Three- to 5-fold lower quantities of products indicative of cleavages at Arg-5, Arg-21, Lys-54, Arg-70, Arg-95, Arg-101, and Arg-122 were also found. The most striking effect of heparin at this time was on the reaction at Lys-60, which was suppressed by a factor of about 4, as indicated by the decreases in the yields of Asn, Gly, Asn, Pro, and His in cycles 1-5, respectively. A less pronounced (<2-fold) decrease in cleavage at Arg-101 (yielding Asn-Val-Val-Val-Ala) was also evident. Hydrolysis at the other sites appeared to be virtually unchanged, indicating that heparin did not inhibit the protease itself. The results for 5 min tryptic digests (not shown) were similar. Again, heparin primarily decreased cleavage at Lys-60 (by ~3-fold) and to a lesser extent reduced hydrolysis at Arg-101. In this case, however, cleavage at Arg-31 (producing Arg-Arg) also seemed to be diminished by 2-fold.
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Adhesive Properties of Angiogenin Mutants
Since human HT-29 cell adhesion to Ang is mediated by a heparan
sulfate proteoglycan (3), the relationship between the heparin-binding
properties of the different Ang mutants and their capacities to support
cell adhesion was investigated. At a plating concentration of 0.1 µg/cm2, the four single-site mutants that have lower
affinity for heparin-Sepharose (R31A, R32A, R33A, and R70A), supported
the adhesion of fewer cells than did Ang (Fig.
2A). Indeed, adhesion to these derivatives was no greater than for RNase A. Among the mutants with unchanged heparin affinity, R5A, H13A, and K40Q supported adhesion nearly as well
as Ang, but R66A was substantially less effective (see "Discussion"). At higher plating concentrations (0.2
µg/cm2), all six mutants were indistinguishable from Ang.
The triple mutant R31A/R32A/R33A was less effective than any of the
single-site derivatives (Fig. 2B); adhesion at 0.2 µg/cm2 was 42% of that with Ang, and the plateau reached
at concentrations
0.6 µg/cm2 was 17% lower than
that for Ang. RNase A supports the adhesion of only 26% as many cells
as Ang, even at plating concentrations as high as 1 µg/cm2 (3).
Effects of Heparin on the Enzymatic and Angiogenic Activities of Angiogenin
The Ang-heparin interaction was characterized further by examining
the effects of heparin on the ribonucleolytic and angiogenic activities
of Ang. Heparin was shown previously to inhibit Ang-catalyzed cleavage
of tRNA at pH 5.5 with an IC50 value of 700 µg/ml (30). The present study used both tRNA and the dinucleotide CpA as substrates at the more physiological pH of 7. With tRNA, 0.5 mg/ml heparin inhibited by 48%. However, at concentrations of 1.0 and 2.0 mg/ml inhibition increased only to 55% and 63%, respectively, suggesting that heparin acts as a partial inhibitor. The effect on cleavage of CpA
was even smaller; both 0.25 and 0.75 mg/ml heparin decreased the
kcat/Km value by 15%, from
3.3 M1 s
1 to 2.8 M
1 s
1.
Heparin did not alter significantly the angiogenic response to Ang on the chicken embryo CAM. In the absence of heparin, 10 ng of Ang produced 51% positive responses (27/53; p = 0.00005). When 50 µg of heparin were included, 48% of the eggs were positive (24/50; p = 0.00014). A water control sample assayed simultaneously yielded 10% positive responses (4/39).
Stoichiometry of the Ang-Heparin Interaction
The stoichiometry of the interaction between angiogenin and
heparin was studied by measuring the light scattering of mixtures containing 2 µM heparin and various concentrations of Ang
(Fig. 3) (31). Although no scattering was detected in
the presence of 1 or 2 molar eq of Ang, a significant amount was
observed with 3 eq. Scattering then increased in a nearly linear manner
up to 8 eq and reached a maximum intensity at 9 eq. Further additions of Ang up to 15 eq had no effect.
Identification of Inhibitory Heparin Fragments
Intact heparin (50 µg/ml) inhibits adhesion of HT-29 cells to
Ang by 60% (3). In contrast, four heparin disaccharides (see "Experimental Procedures") tested individually at up to 100 µg/ml did not inhibit HT-29 cell adhesion to Ang (data not shown). To determine the minimum size required for a heparin fragment to inhibit
effectively, heparin was depolymerized with nitrous acid and the
products were separated by gel filtration and assayed for their effect
on adhesion. In the chromatographic system used, intact heparin elutes
with a Kav value of 0.15, whereas disaccharides elute with a Kav value of 0.85. Heparin
fragments that eluted with Kav < ~0.6
retained strong inhibitory activity. PAGE analysis of fractions with
Kav values between 0.48 (60% inhibition) and 0.67 (no significant inhibition) in all cases showed multiple discrete
bands. Rechromatography of various pools of this material yielded
several fractions that were composed primarily of a single size species
as judged by PAGE (Fig. 4A). Mass
spectrometry of these fractions revealed average molecular masses
consistent with the presence of 8-, 10-, 12-, 14-, and 16-saccharide
products (lanes 2-6, respectively) (Table
IV). When tested for their effects on HT-29 cell
adhesion to Ang at 50 µg/ml (Fig. 4B), only the dodecamer,
tetradecamer, and hexadecamer inhibited appreciably: by 46%, 53%,
and 63%, respectively.
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Heparin binds tightly and specifically to a vast array of disparate proteins, including angiogenesis factors (the fibroblast growth factors (FGFs) (32), vascular endothelial growth factor (33), and Ang (3)), protease inhibitors (antithrombin III (34), heparin cofactor II (35), and mucus protease inhibitor (36)), proteases (thrombin (37)), lipases (lipoprotein lipase (38)), and extracellular matrix molecules (fibronectin (39), laminin (40), and tenascin (41)). Despite long-standing interest in these interactions, some of which have well established physiological significance, direct information on the molecular details of heparin binding has been obtained thus far only for a single protein, basic FGF (bFGF). The crystal structure recently reported by Faham et al. (42) for the complex of bFGF with a heparin-derived hexasaccharide shows 13 polar contacts between 8 FGF residues and, primarily, sulfate groups on the GAG. The FGF residues are located on three elements of primary structure, and all but one lie on surface loops. About half of the interactions are ionic and involve Arg or Lys side chains, whereas the others utilize Asn or Gln side chains or main-chain NH groups. This picture of the heparin binding site on FGF agrees substantially with previous proposals based on chemical modification, mutagenesis, and thermodynamic results (43-45), and the crystal structures of FGF complexes with small anions (46, 47).
Analogous structure-function, kinetic, and modeling studies with other heparin-binding proteins suggest that many features of the heparin-bFGF complex are widely shared. Thus, the heparin-binding sites on antithrombin III (see Refs. 48 and 49), thrombin (50, 51), fibronectin (52), and lipoprotein lipase (53) also appear to be assembled from multiple discrete segments and to be rich in arginines and lysines. (Only these two residue types have been closely scrutinized as potential candidates.) The effects of ionic strength on Kd values for the complexes examined vary widely, indicating that the energetic contributions of salt linkages range, minimally, from ~40% (antithrombin (54)) to >80% (thrombin (55) and mucus proteinase inhibitor (37)).
In the present study, the heparin binding site on Ang was investigated
by measuring the effects of mutating various basic residues of Ang on
affinity for heparin-Sepharose (Table I, Fig. 1) and by determining
which basic residue (i.e. tryptic) cleavage sites on the
native protein are protected by heparin (Table III). Four single-site
mutants (R31A, R32A, R33A, and R70A) bound more weakly than Ang to
heparin-Sepharose, and the triple mutant R31A/R32A/R33A had an affinity
lower than for any of the individual
mutants.2 In the three-dimensional
structure of Ang (56), residues 31-33 lie on -helix 2 whereas
residue 70 is part of
-strand 3 more than 30 Å away (Fig.
5). The side chains of Arg-31 and -32 extend toward the
exterior of the molecule and are fully accessible for interactions. In
contrast, the side chain of Arg-33 already engages in multiple hydrogen
bonds with the main-chain carbonyl oxygens of Thr-11 and Tyr-14 on
-helix 1 (56) and is available only for a single additional bond
unless the intramolecular linkages are broken upon heparin binding.
Such a disruption seems unlikely, however, since removal of these
interactions (by Ala substitution) decreases enzymatic activity by
7-fold (13, 56) and yet heparin inhibits only slightly. Arg-70 is also
not completely free; its NH1 group hydrogen bonds to both OD1 of Asn-59
and the carbonyl O of Gly-62. NH2, however, is accessible. The N
terminus of Met-(
1) Ang appears to interact with heparin as well,
since the Met-(
1) derivative binds more tightly to heparin-Sepharose
than does the <Glu-1 form. This part of the protein is not seen in the
crystal structure owing to its high flexibility, but is likely to be on the surface.
Four single-site mutations (Arg-5 Ala, Arg-66
Ala, His-13
Ala, and Lys-40
Gln) do not affect binding to heparin-Sepharose, although all of the residues substituted are accessible. Arg-5 and
Arg-66 in native Ang are entirely free of interactions. His-13 and
Lys-40 each form a hydrogen bond with another Ang residue but are
available for additional ones. Indeed, they are components of the
enzymatic active site that are predicted to interact directly with
phosphate moieties on RNA (7, 8, 56). The failure of mutations of these
four accessible basic Ang residues to decrease binding to
heparin-Sepharose, together with the low heparin affinity of the
related basic protein RNase A, strongly suggests that binding of Ang to
this GAG is specific. Thus, the weakened binding of the Arg-31, -32, -33, and -70 mutants can be presumed to reflect the loss of specific
interactions rather than a change in the ion-exchange properties of the
protein.
The effects of these replacements on binding to heparin-Sepharose
([NaCl] = 0.04-0.10) are similar to some of those reported for
mutations of putative heparin-binding arginines and lysines in
fibronectin (52), lipoprotein lipase (53), and thrombin (57). Much
larger changes (
[NaCl] up to 0.75; Ref. 44) have been observed for
mutations of bFGF residues known (42) to be part of the contact
surface. Thus, the individual interactions may not be as well optimized
in Ang and these other proteins as in bFGF, which binds heparin much
more tightly. Alternatively, other residues, as yet unidentified, may
make much larger contributions to heparin binding.
Consistent with the involvement of Arg-31, -32, -33, and -70 in heparin binding, R31A, R32A, R33A, and R70A are less effective than Ang as substrates for adhesion of HT-29 cells (Fig. 2), a process shown previously to be mediated by a cell-surface heparan sulfate/chondroitin sulfate proteoglycan (3). Mutation of Arg-66 also diminishes cell adhesion, although in this case there is no parallel decrease in binding to heparin-Sepharose. The dissociation of these two effects may reflect chemical differences between the heparin attached to the Sepharose resin versus the heparan sulfate chains on the proteoglycan. Alternatively, replacement of Arg-66 may affect binding to the chondroitin sulfate or protein portion of the proteoglycan rather than the heparan sulfate chains.3
Heparin decreases tryptic cleavage of Ang at Lys-60 by severalfold, suggesting that it also binds to the region of Ang that contains this residue. Since a free portion of the heparin chain might sterically hinder the action of trypsin, it is unclear from this finding whether Lys-60 itself interacts with heparin. We note, however, that this residue is on a highly accessible surface loop. Heparin also produces more modest effects on tryptic cleavage at Arg-31 and Arg-101. Failure of heparin to influence digestion at any other sites may reflect the loss of structural integrity of Ang and its capacity to bind heparin once the first few sites have been cleaved.
The present results identify the cluster Arg-31/Arg-32/Arg-33 as a major site of interaction with heparin, and indicate that the GAG chain may extend from this region in the direction of Lys-60, Arg-70, and the N terminus (Fig. 5). The N terminus and the Lys-60 loop are on the same face of Ang as the arginine cluster and, although these various sites are separated by as much as 37 Å, a heparin molecule 8 or more saccharides in length could bridge such a distance. However, Arg-70 is on the opposite face of Ang and cannot contact the same heparin chain as residues 31-33. The weakened binding of R70A Ang to heparin-Sepharose may therefore be due to the loss of interactions between the Arg-70 guanidino group and the loop that contains Lys-60 (see above). It is also possible that Arg-70 may form part of a second heparin-binding site, although simultaneous attachment of two heparin molecules to a single Ang would seem unlikely due to the strong electrostatic repulsion between the two highly charged GAG chains.
The binding density of ~9 Ang/heparin chain determined by measuring light scattering after addition of Ang to heparin translates into 1 Ang molecule/5 or 6 monosaccharide units. Given the heterogeneity of heparin, this rules out the possibility that Ang, like antithrombin III (23), recognizes only a single, highly specific structure. At the same time, it contrasts with the finding that heparin fragments of at least 12 saccharides are required to inhibit HT-29 cell adhesion to Ang (Fig. 4B). This implies that heparin sequences that bind sufficiently tightly to prevent the interaction of Ang with the adhesion receptor may occur relatively infrequently or that they are significantly larger than 5-6 saccharide units. In this regard, it should be noted that Ang is long enough (43 Å) to form contacts over an 8-10-saccharide portion of heparin (i.e. a 10-12-saccharide nitrous acid digestion product of heparin, since the terminal unit is converted to 2, 5-anhydromannitol-6-sulfate).
The partial map of the heparin binding site on Ang here determined, together with the observed functional effects of this GAG, suggests that the interaction of Ang with heparin does not modulate the enzymatic or angiogenic activities of Ang. The active site mutants tested (H13A, K40Q, and R5A) all have unchanged affinity for heparin, and heparin has essentially no influence on Ang-catalyzed cleavage of dinucleotide substrates. It is a somewhat more effective, but still partial, inhibitor of tRNA cleavage, indicating that it may contact some peripheral subsite for RNA binding outside the catalytic center. Heparin has no detectable effect on the capacity of Ang to induce new blood vessels on the CAM, although it interacts with the cluster Arg-31-Arg-33 that is involved in translocation of Ang to the nucleolus of endothelial cells (58), thought to be an essential step in the angiogenic mechanism (59). Moreover, it binds near or within the putative cell-binding site of Ang, which is required for angiogenic activity. This site is distinct from the enzymatic active center and includes Asn-109 plus residues in the segment 61-67 (10, 11, 13); proteolytic cleavage of the 60-61 or 67-68 peptide bond, deamidation of Asn-61 or Asn-109, and mutation of Arg-66 to Ala all abolish angiogenic activity without influencing ribonucleolytic activity appreciably. Although heparin does not contact Arg-66 in this site, it does hinder the action of trypsin at Lys-60, which is either adjacent to or part of this region. Thus the heparin recognition site may overlap the cell-binding site. In this case, the failure of heparin to affect activity in the CAM assay might indicate that the local concentration of heparin maintained during the 2-3-day time course of the assay is not sufficient to prevent binding to cell-surface receptors involved in the angiogenic response. It therefore remains possible that, under different circumstances, heparin may indeed inhibit the angiogenic activity of Ang.
Whatever the answer to this question ultimately turns out to be, the inhibitory effects of heparin and heparin fragments on the adhesion of tumor cells to Ang raise the possibility that heparin-related compounds may have therapeutic potential as antagonists of Ang-dependent tumor formation and metastasis. As noted above, Ang has been demonstrated to play a key role in the establishment of some human tumors in an athymic mouse model (4, 5), and part of this role may involve the critical early event of tumor cell attachment. HT-29 cells in culture adhere to Ang remarkably quickly, in fact, much more rapidly than to the common extracellular matrix molecules fibronectin, laminin, and collagen (3). Further studies on the in vivo importance of Ang-mediated tumor cell attachment and on the potential utility of heparin derivatives for inhibiting this process therefore seem warranted at this time.
Mass spectrometry data were kindly provided by Dr. P. Juhasz at the MIT Mass Spectrometry Facility, which was supported by National Institutes of Health Grant RR 00317 (to Prof. K. Biemann). We thank Dr. K. R. Acharya for providing Fig. 5, Dr. J. F. Riordan for valuable discussions, and Kerrin Green for excellent technical assistance. The support and advice of Dr. B. L. Vallee are gratefully appreciated.