Specificities of Heparin-binding Sites from the Amino-Terminus and Type 1 Repeats of Thrombospondin-1

Archives of Biochemistry and Biophysics Vol. 374, No. 1, February 1, pp. 13–23, 2000 doi:10.1006/abbi.1999.1597, available online at http://www.idealibrary.com on

Specificities of Heparin-binding Sites from the AminoTerminus and Type 1 Repeats of Thrombospondin-1 Haini Yu,* ,1 David Tyrrell,† ,2 JoAnne Cashel,* Neng-hua Guo,* Tikva Vogel,* ,‡ John M. Sipes,* Lun Lam,† Howard M. Fillit,§ Jacob Hartman,‡ Simona Mendelovitz,‡ Amos Panel,‡ and David D. Roberts* ,3 *Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; †Glycomed, Inc., Alameda, California 94501; ‡BioTechnology General, Ltd., Rehovot, Israel; and §Institute for the Study of Aging, New York, New York 10153, and Department of Geriatrics, The Mount Sinai Medical Center, New York, New York 10029

Received July 21, 1999, and in revised form October 15, 1999

Interactions of heparin with intact human thrombospondin-1 (TSP1) and with two heparin-binding fragments of TSP1 were characterized using chemically modified heparins, a vascular heparan sulfate proteoglycan, and a series of heparin oligosaccharides prepared by partial deaminative cleavage. The avidity of TSP1 binding increased with oligosaccharide size, with plateaus at 4 to 6 and at 8 to 10 monosaccharide units. The dependence on oligosaccharide size for binding to the recombinant amino-terminal heparinbinding domain of TSP1 was the same as that of the intact TSP1 molecule but differed from that of a synthetic heparin-binding peptide from the type 1 repeats, suggesting that the interaction between intact TSP1 and heparin is primarily mediated by the aminoterminal domain. Based on activities of chemically modified heparins, binding to TSP1 depended primarily on 2-N- and 6-O-sulfation of glucosamine and to a lesser degree on 2,3-O-sulfation and the carboxyl residues of the uronic acids. In contrast, all of these modifications were required for binding of heparin to the type 1 repeat peptides. Affinity purification of heparin octasaccharides on immobilized TSP1 type 1 repeat peptides revealed a preference for oligosaccharides containing the disaccharide sequence IdoA(2OSO 3)␣1-4-GlcNS(6-OSO 3). Binding of these oligosaccharides to the peptide required the Trp residues.

These data demonstrate that the heparin-binding specificities of intact TSP1 and peptides from the type 1 repeats overlap with that of basic fibroblast growth factor (FGF2) and are consistent with the ability of these TSP1-derived molecules to inhibit FGF2-stimulated angiogenesis. © 2000 Academic Press Key Words: heparin-binding proteins; thrombospondins; peptides; affinity chromatography; oligosaccharides.

Thrombospondin-1 (TSP1 4) is the first identified member of the thrombospondin gene family of extracellular matrix glycoproteins (reviewed in (1, 2)). In addition to its interactions with several extracellular matrix proteins and cell surface receptors (3), TSP1 binds specifically to heparin, heparan sulfate proteoglycans, and some sulfated glycolipids (reviewed in (4, 5)). The heparin-binding sites of TSP1 mediate high affinity binding of TSP1 to several cell types (6) and may play roles in several biological activities of TSP1, including cell spreading (7), chemotaxis (8, 9), and internalization of TSP1 (10 –12). A binding site for heparin, sulfatide, and heparan sulfate proteoglycans is located in the amino-terminal pentraxin domain of TSP1 (11, 13, 14). However, the central region of TSP1 contains additional heparin binding sequences in the three type 1 or properdin repeats (15–17).

1

Present address: Department of Ophthalmology, Tulane University School of Medicine, New Orleans, LA 70112. 2 Present address: Kimberly-Clark Corporation, WRE, Neenah, WI 54956. 3 To whom correspondence should be addressed at National Institutes of Health, Building 10 Room 2A33, 10 Center Drive, MSC 1500, Bethesda, MD 20892-1500. Fax: (301) 402-0043. E-mail: [email protected]. 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

4 Abbreviations used: BSA, bovine serum albumin; FGF2, basic fibroblast growth factor; HSPG, heparan sulfate proteoglycan; SDS, sodium dodecyl sulfate; Tris–BSA, 50 mM Tris, pH 7.8, 110 mM NaCl, 2.5 mM CaCl 2, 0.02% NaN 3, 0.1 mM phenylmethane sulfonyl fluoride, 1% w/v bovine serum albumin; TSP1, human thrombospondin-1.

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The heparin-binding sites of TSP1 may also play important roles in regulation of cell growth and survival (reviewed in (4)). Heparin-binding peptides from the type 1 repeats directly inhibit proliferation and trigger signals that modulate the function of ␣v␤3 integrin by binding to a heparan sulfate proteoglycan on melanoma cells (18, 19). TSP1 inhibits proliferation and motility of endothelial cells stimulated by basic fibroblast growth factor (FGF-2) (20, 21) and inhibits angiogenesis in several animal models of normal and pathological angiogenesis (21–24). Both CD36- and heparin-binding sequences from TSP1 may contribute to the anti-angiogenic activity of the intact protein (22, 25). The amino-terminal heparin-binding domain expressed as a recombinant fragment and synthetic heparin-binding peptides from the type 1 repeat region inhibit cell proliferation stimulated by FGF2 or serum (9). The type 1 repeat peptides also induce endothelial cell apoptosis (26) and inhibit angiogenic responses in vivo (22, 27). Inhibitory activities of synthetic analogs of these TSP1 peptides correlate with their ability to bind to heparin and to inhibit binding of FGF2 to immobilized heparin or to endothelial cells (9, 28). Because binding to the HSPG is essential for presentation of FGF2 to the high affinity FGF2 receptor (reviewed in (29, 30)), these results suggest that competition for binding of FGF2 to endothelial cell surface HSPGs accounts at least in part for the anti-angiogenic activities of TSP1 and its fragments. Endothelial cells synthesize both cell surface and extracellular matrix-associated HSPGs, including syndecans, perlecan, biglycan, and glypican (31–33). Previous data have demonstrated that TSP1 binds to syndecan-1 (34) and perlecan (33) and have shown that some of the interactions of TSP1 with endothelial cells are mediated by endothelial HSPGs (11, 12, 33, 35, 36). Direct competition between the TSP1 type 1 repeat peptides and FGF2 for binding to endothelial cells and heparin (9, 28) implies that the binding specificity of the TSP1 peptides overlap with that of FGF2. The heparin binding specificity of FGF2 has been defined using both heparin and heparan sulfates (reviewed in (29, 30)). Minimal sequences for FGF2 binding or receptor activation contain from 5 to 12 monosaccharide units. High affinity binding of FGF2 to heparin requires both 2N-sulfation of GlcN and 2-O-sulfation of IdoA residues (37). In addition, the negative charge of the carboxyl group of uronic acid residues is required (38). Other studies, however, demonstrate that nonsulfated oligosaccharides also bind to FGF2 and mediate receptor activation (39). Active binding oligosaccharides isolated from heparan sulfate were enriched in the disaccharide unit iduronate 2-sulfate-GlcNSO 3 (40, 41), but 6-sulfation also contributes to binding of heparan sulfate to FGF2 (42).

The heparin-binding specificity of TSP1 is not well defined. Earlier work demonstrated that an 8-mer was the minimal size oligosaccharide that inhibited heparin-dependent interactions of TSP1 with CHO cells, but the structural requirements for binding were not defined (43). Different size-dependencies were found for inhibiting TSP1 binding, degradation, or adhesion of cells on immobilized TSP1, but the activity of an 18-mer approached that of intact heparin in all assays. Affinity coelectrophoresis of TSP1 and FGF2 did not show binding specificity for a subpopulation of lowmolecular-weight heparin (44). This result indicated that if heparin binding is sequence specific, both proteins must recognize a structure common to a major population of heparin molecules. In contrast, the specificity for cross-competition between TSP1 or TSP1derived peptides and other heparin-binding proteins suggests that binding is sequence specific (10, 14). To better define the basis for TSP1 binding to heparin and the consequences of this binding to the biological responses of endothelial cells to TSP1, we have examined the structural requirements for binding to heparin of the synthetic peptides and recombinant fragments derived from TSP1. Equilibrium binding studies demonstrate that the recombinant amino-terminal heparin-binding domain and synthetic peptides containing the heparin-binding motif WSXW differ in their heparin binding specificities. The overlap of these binding specificities with that of FGF2 supports a role for the heparin-binding sites in antagonism of FGF2 by TSP1. MATERIALS AND METHODS Materials. TSP1 was purified from thrombin-stimulated human platelets (45). Recombinant heparin-binding fragments of TSP1 were expressed in E. coli strain A4255 F⫺ under the control of the thermoinducible ␭P L promoter and CII ribosomal binding site. The 28kDa fragment contains amino acids 1–242 of human TSP1 with a Met residue preceding the first amino acid. The 18-kDa fragment contains residues 1–175 with an initiating Met preceding the first residue and the sequence –Arg–Ser–Ala–Ser–Gln added to the carboxyl terminus (9). The recombinant proteins were purified from inclusion bodies by chromatography on DEAE–Sepharose, CM– Sepharose, and heparin–Sepharose. The 28-kDa fragment was oxidized in the presence of 100 ␮M oxidized glutathione. Both fragments were lyophilized from 1 mM NaHCO 3 at 200 ␮g/ml, pH 8.8, with 1 mM dithiothreitol for the 18-kDa fragment, and pH 10.5 for the 28-kDa fragment. The recombinant TSP1 fragments were readily soluble in water at 1 mg/ml. Following desalting on a Sephadex PD10 column (Amersham-Pharmacia) equilibrated in Dulbecco’s PBS, the fragments were stored in aliquots at ⫺20°C for up to 3 months. Synthetic peptides from the type 1 repeats of TSP1 were prepared as previously described (15). TSP1 and its fragments were iodinated using Iodogen (Pierce Chemical Co., Rockford, IL) or Bolton–Hunter reagent (Dupont NEN) as previously described (45). The heparin fragments from deaminative cleavage were prepared as described using porcine mucosal heparin (M r (weight average) ⫽ 12,000, Ming Han Co., Oakland, CA) (46). Chemically modified porcine mucosal heparins (preferentially 6-O-desulfated heparin (M r ⫽ 11,000), 2,3-O-desulfated heparin (M r ⫽ 10,000), low-molecular-

HEPARIN-BINDING SPECIFICITIES OF THROMBOSPONDIN-1 weight (LMW) 2,3-O-desulfated heparin (M r ⫽ 3500 – 4500), and carboxyl-reduced heparin (M r ⫽ 11,500)) were prepared as described (38). [Periodate-oxidized, borohydride reduced] heparin (NAC1) (M r ⫽ 11,000) was prepared as previously described (47). N-Deacetylated [periodate-oxidized, borohydride-reduced] heparin (NAC2, M r ⫽ 7500) was prepared by treating porcine mucosa heparin with 70% aqueous hydrazine containing 1% hydrazine sulfate as catalyst as previously described (48). The sample was extensively dialyzed against water and lyophilized. The polymer was dissolved in 0.25 M sodium bicarbonate, pH 8.3, and a solution of 0.4 M I 2 in 0.2 M KI was added dropwise to remove hydrazides formed during the reaction. Upon producing a yellow color, indicative of excess I 2, an aqueous solution of hydrazine was added dropwise until the solution turned colorless. The solution was then dialyzed against water and lyophilized. The N-deacetylated heparin was then subjected to periodate oxidation followed by borohydride reduction as described (49, 50) with some modifications (47). N-Acetylation of N-desulfated heparin was performed using a modification of the procedure reported by Reg (51). This involved treating N-desulfated heparin, prepared by solvolytic treatment of pyridinium heparin in DMSO:water (9:1) at 50 – 60°C for 1–1.5 h in sodium bicarbonate solution (52), with multiple aliquots of acetic anhydride and dimethylformamide over a period of 24 h. The pH was maintained at neutrality by adding solid sodium bicarbonate as necessary. The molecular weight of [N-desulfated, N-reacetylated] heparin was 12,000. Low molecular weight porcine mucosal heparin (M r ⫽ 6000) was obtained from Sigma Chemical Co. (St Louis, MO). Heparin–BSA conjugates were prepared by coupling bovine lung heparin (The Upjohn Co., Kalamazoo, MI) through the reducing termini to BSA by reductive amination in the presence of NaBH 3CN essentially as described (53) or obtained from Sigma. Chemically modified heparin chains were fractionated according to hydrodynamic size using a high-performance size exclusion chromatography method similar to that of van Dedem and Nielsen (54). Molecular weight profiles were obtained using tandem 7.8-mm ⫻ 30-cm Tosohaas Inc. TSK-GEL G3000SW XL and G2000SW XL columns preceded by a 6-mm ⫻ 4-cm guard column and a 0.22-␮m precolumn filter. The columns were eluted at 0.3 ml/min with 100 mM ammonium acetate, pH 7, and detected by monitoring refractive index. Molecular weight assignments were made on the basis of retention time relative to a series of heparin oligosaccharides of known molecular weight obtained from partial depolymerization of heparin with nitrous acid. Collected data were analyzed and the average molecular weight calculated as M r ⫽ ⌺(h iM i)/⌺h i, where h i is the refractive index detector response and M i is the corresponding molecular weight obtained from the calibration curve. Bovine kidney vascular HSPG was prepared as previously described (55). Biotinylated heparan sulfate chains were prepared as previously described for biotinylation of hyaluronic acid (56), using a benzoquinone intermediate, a spacer arm of diaminodipropylamine, and N-hydroxysuccinimidobiotin. Chemically deglycosylated protein core was isolated from purified HSPG as previously described by a modified trifluoromethane sulfonic acid digestion (57). Briefly, 4 mg of purified HSPG was desiccated and flushed with nitrogen. To the HSPG was added 0.4 ml of trifluoromethanesulfonic acid:anisole (2:1) and the mixture was vortexed and placed on ice for 15 min. The preparation was dialyzed overnight against 2 L of 1% SDS, 0.192 M glycine, and 0.025 M Tris buffer at 4°C for immunoblotting and further dialyzed against distilled water and lyophilized. The material was further purified by FPLC on a mono Q column and eluted at 0.6 M NaCl. The purified core was essentially free of contaminating proteins and intact HSPG by SDS page. Immunoblotting employing monoclonal antibodies revealed a single band with M r ⫽ 110,000 (58). Ligand binding assays. TSP1 and TSP1 fragment binding to heparin–BSA or HSPG were determined using a solid phase assay.

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Heparin–BSA (0.075 ␮g/well) or HSPG were adsorbed onto 96-well polyvinyl chloride microtiter plate wells by incubation in 50 ␮l of Dulbecco’s PBS for 16 h at 4°C. The wells were emptied and filled with Tris–BSA buffer. After 30 min, the wells were emptied, and 30 ␮l of various concentrations of inhibitors diluted in Tris–BSA buffer or buffer alone and 30 ␮l of [ 125I]TSP1 or [ 125I]TSP1 fragments (0.1– 0.2 ␮g/ml, 1–5 ␮Ci/␮g) were added to each well. After incubation for 4 h at 4°C, the wells were washed six times with 0.15 M NaCl and cut from the plate, and the bound radioactivity was counted. In the reverse of the assay described above, TSP1 or its fragments or peptides were immobilized on the microtiter plates. The concentration of the protein or peptides in each well were as follows: TSP1, 4 ␮g/well; 28-kDa recombinant fragment, 2.5 ␮g/well; 18-kDa recombinant fragment, 1.3 ␮g/well; and peptide 246 (KRFKQDGGWSHWSPWSS), 0.75 ␮g/well. [ 125I]Heparin–BSA (final concentration 9 ⫻ 10 ⫺3 ␮g protein per well, with a sp act of 22 ␮Ci/␮g protein) was added and incubated as above. Bound heparin–BSA was quantified as above. TSP1 and recombinant TSP1 fragment binding to sulfatide were determined using a solid phase assay with the glycolipid immobilized in a phosphatidylcholine/cholesterol monolayer on polyvinyl chloride microtiter plates as previously described (45). For equilibrium binding studies, wells were coated with a mixture of 200 ng of sulfatide, 50 ng of phosphatidyl choline, and 30 ng of cholesterol. Binding to isolated heparan sulfate chains conjugated to biotin was determined by a modification of the same method (56). Streptavidin, 50 ␮g/ml in PBS, was adsorbed onto microtiter plate wells by incubation for 16 h at 4°C. The wells were filled with Tris–BSA buffer and incubated for 1 h. Heparan sulfate– biotin (1 to 100 ␮g/ml for dose dependence or 50 ␮g/ml for determination of binding constants) was added and incubated for 2 h at 25°C. The wells were emptied and washed twice with Tris–BSA. [ 125I]TSP1 binding was determined as described above. For calculation of equilibrium binding constants, binding was determined in triplicate at each concentration to wells coated with the indicated ligands and corrected for nonspecific binding determined at each ligand concentration using uncoated wells blocked with the Tris–BSA buffer alone. Binding data were analyzed using the LIGAND program (59). The activity of the labeled TSP1 ligands were assessed on the day of each binding experiment by affinity chromatography on heparin agarose. The bound fraction was used to calculate the active labeled ligand concentration. Affinity chromatography of heparin fragments. Affinity chromatography of heparin oligosaccharides was performed on immobilized recombinant heparin binding domain. A 1-ml volume of Reacti-Gel CDI (Pierce), which was drained of acetone and washed with ice-cold water, was added to 0.5 ml of 0.02 M NaHCO 3 (pH 9) coupling buffer containing recombinant 18-kDa TSP1 fragment (1–2.5 mg/ml gel). The coupling was performed with gentle mixing for 22 h at room temperature. The coupling buffer was removed and the gel was washed twice with 0.02 M NaHCO 3. Remaining sites were blocked using 0.5 ml of 1 M ethanolamine under the same condition for 2 h. The coupled gel was equilibrated with 20 mM Tris–HCl buffer, pH 7.6, containing 0.2 mM CaCl 2, 150 mM NaCl, and 0.02% NaN 3 and packed into a 10 ⫻ 0.5-cm glass column with a water jacket to 4°C. Separation of 3H-labeled heparin oligosaccharides was performed on the column by isocratic elution with the same buffer. The TSP1 peptide KRFKQDGGWSHWSPWSS was coupled to Affigel 10 (BioRad). Briefly, 30 ␮mol of peptide was coupled to 6 ml of Affigel 10 in PBS by mixing overnight at 4°C. Unreacted groups were blocked by incubation for 4 h in 0.2 M Tris–HCl, pH 8.0. The gel was washed extensively and stored in PBS. Control columns were prepared by coupling the TSP1 peptides VTCGDGVITR or KRFKQDGGASHASPASS as above or by blocking with Tris without reacting with peptide. For analytical separations, 3H-labeled heparin oligosaccharides were applied in 20 mM Tris–HCl, pH 7.2, 150 mM NaCl, 0.2 mM CaCl 2, 0.02% NaN 3 to the column equilibrated in the same

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buffer and eluted with a pH gradient to 7.6 in the same buffer. For preparative separation of oligosaccharides, the column was equilibrated in 0.15 M ammonium formate, pH 6.1. Samples were applied in the same buffer and eluted using a step gradient to 0.15 M ammonium formate, pH 9.0. Disaccharide composition of 3H-labeled octasaccharides. The 3Hlabeled octasaccharides prepared previously by reduction of the anhydromannose reducing ends with NaB 3H 4 (468.7 mCi/mmol) were applied to a TSP1 type 1 repeat peptide column as described above. The 3H-oligosaccharides (i.e., samples applied, bound, and unbound fractions) were depolymerized to disaccharides and other smaller oligosaccharides by oxidative deamination at N-sulfated GlcN residues with nitrous acid treatment at pH 1.5 (48). In addition, these samples were depolymerized to their substituent disaccharides by N-deacetylation using 70% aqueous hydrazine reagent containing 1% hydrazine sulfate as catalyst followed by oxidative deamination at N-sulfated and GlcN residues with sequential nitrous acid treatments at pH 1.5 and 4.0 (48). The anhydromannose termini of the disaccharides were reduced with NaB 3H 4 (359.8 mCi/mmol) (60). The 3 H-labeled disaccharides were quantitatively analyzed by reversedphase ion-pairing HPLC (RPIP-HPLC) as previously described (61). Disaccharides from porcine mucosal heparin, average molecular weight 12,000 (Ming Han CO, Oakland, CA), were used as a reference in each analysis. Analytical PEI ion-exchange HPLC of 3H-labeled octasaccharides. The 3H-labeled octasaccharides (bound and unbound fractions from the TSP1 peptide column as well as the sample applied to the column (total)) were analyzed for charge heterogeneity by PEI ion-exchange HPLC. A single column (10 ⫻ 250 mm) with a 0.22-␮m precolumn filter was equilibrated in deionized water, loaded at 0.1 ml/min with 3 H-labeled octasaccharides (bound, 3.8 ⫻ 10 6 cpm; unbound, 3.4 ⫻ 10 5 cpm; and total, 2.3 ⫻ 10 7 cpm, 117 mCi/mmol), and eluted at 0.5 ml/min with a 178-ml linear gradient of 0 –1000 mM aqueous ammonium bicarbonate. Aliquots of 50 ␮l from the 1-ml fractions were quantified for 3H by liquid scintillation counting. The CPM for the bound and unbound fractions were normalized to the total CPM of the 3H-labeled octasaccharides applied to the affinity column to compare profiles. Total CPM refers to the sum of the CPM measured from 10 to 200 min.

RESULTS

Two recombinant forms of the amino-terminal heparin-binding domain of TSP1 and synthetic peptides derived from the second type 1 repeat were used to compare the binding specificities of two identified heparin-binding sites in TSP1 (13, 15). Intact TSP1 and two recombinant fragments from the amino terminal heparin-binding domain were compared for binding to immobilized heparin, a vascular HSPG, and sulfatide (Table I). In all cases, intact TSP1 was the most active ligand. The 18-kDa fragment was slightly more active than the 28-kDa fragment for binding to heparin and the vascular HSPG, whereas the 28-kDa fragment was 2-fold more active for binding to sulfatide. Binding of intact TSP1 to heparin was 25- to 40-fold more avid than binding to HSPG or sulfatide, based on its ability to compete with the radiolabeled 28-kDa fragment for binding to the respective ligands. Binding constants for the two recombinant heparinbinding fragments of TSP1 were determined by selfdisplacement of the respective labeled fragments (Fig. 1). Binding constants were determined using the

TABLE I 125

Inhibition of [ I]28-kDa TSP1 Fragment Binding by Thrombospondin and Fragments Ligand Competitive inhibitor

Sulfatide (K i, nM)

Heparin (K i, nM)

HSPG (K i, nM)

TSP1 18 kDa rTSP1 28 kDa rTSP1

34 1500 880

1.3 85 170

52 600 1700

Note. Inhibition of [ 125I]28-kDa recombinant TSP1 (6 nM) binding to the indicated immobilized ligands by varying concentrations of TSP1, 18-kDa, or 28-kDa recombinant fragments derived from the amino-terminal heparin-binding domain of TSP1 was determined at equilibrium at 4°C. Apparent inhibition constants (K i) were determined using the LIGAND program.

LIGAND program, fitting the data to a single class of binding sites—18-kDa fragment, K a ⫽ 2.8 ⫾ 0.5 ⫻ 10 6 M ⫺1, R ⫽ 1.8 ⫾ 0.2 ⫻ 10 ⫺8 M; 28-kDa fragment, K a ⫽ 3.5 ⫾ 0.7 ⫻ 10 6 M ⫺1, R ⫽ 1.45 ⫾ 0.19 ⫻ 10 ⫺8 M. The data in Fig. 1A suggest that a second class of binding site with lower affinity may be present for the 18-kDa fragment, but could not be characterized using the available ligand concentrations. Binding of TSP1 to the vascular HSPG was mediated primarily by the heparan sulfate chains, as biotinylated heparan sulfate chains released from the proteoglycan were 23% as active on a mass basis, but the core protein prepared by chemical deglycosylation was only 4% as active as the intact HSPG (data not shown). This result is consistent with the relatively minor contribution of core proteins to the binding of TSP1 to syndecan-3 from brain (62). Equilibrium binding analyses demonstrated heterogeneity in binding to the intact HSPG or to free heparan sulfate chains (Fig. 2). Binding to the biotinylated heparan sulfate chains (Fig. 2A) could be described by two classes of sites with association constants of 2.7 ⫾ 0.7 ⫻ 10 8 M ⫺1 and 3.4 ⫾ 2 ⫻ 10 6 M ⫺1, respectively. Although binding to the intact proteoglycan was also heterogeneous (Fig. 2B), it was not possible to determine a binding constant for the minor high affinity component. TSP1 bound to the major class of sites with an apparent association constant of 1.27 ⫾ 0.16 ⫻ 10 7 M ⫺1. Several heparin oligosaccharides of defined size were tested for inhibition of intact TSP1 binding to heparin. The apparent inhibition constants observed depended on whether the heparin conjugate or TSP1 was used as the detecting ligand (Table II). Using labeled soluble TSP1 and immobilized heparin, the activities of oligosaccharides markedly increased with their size, with no plateau evident whether the inhibition constant was expressed on a weight basis or normalized on a molar basis assuming a constant weight per disaccha-

HEPARIN-BINDING SPECIFICITIES OF THROMBOSPONDIN-1

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weaker inhibitors than for inhibiting binding to intact TSP1. This contrasts with the 40-fold lower activity of intact heparin for inhibiting binding to the peptide versus intact TSP1. The increase in binding activities of the larger oligosaccharides may result in part from their ability to interact with more than one molecule of immobilized peptide. Several chemically modified analogs of heparin were tested for inhibitory activity (Tables III and IV). Two periodate-modified heparins, lacking anticoagulant activity, were very active inhibitors of TSP1 binding to heparin. Using either TSP1 or the recombinant fragment, the [periodate-oxidized, borohydride-reduced] heparin (NAC1) was as active or slightly more active than unmodified heparin. Thus, unsubstituted uronic acid residues are not required for interaction with TSP1 or the amino-terminal recombinant fragment. The N-deacetylated, periodate-oxidized, borohydridereduced heparin (NAC2) was about fivefold less active.

FIG. 1. Equilibrium binding of 18- and 28-kDa recombinant TSP1 fragments to heparin. Microtiter plate wells were coated with 0.6 ␮g of heparin– bovine serum albumin conjugate. Heparin-coated and uncoated wells were incubated for 30 min in Tris–BSA buffer and washed. [ 125I]18-kDa TSP1 fragment (A) or [ 125I]28-kDa TSP1 fragment (B) at 7.6 nM to 5600 nM was added in Tris–BSA buffer and incubated for 3 h at 2°C. The wells were washed and the bound radioactivity was counted. Net binding was determined at each concentration based on triplicate determinations and corrected for nonspecific binding determined to wells without heparin–BSA at each ligand concentration. The data are presented as Scatchard plots.

ride unit. Using larger fragments, this trend continued (data not shown). In contrast, the apparent inhibition constants plateaued at tetra- to hexasaccharides and octa- to decasaccharides when TSP1 was immobilized and labeled soluble heparin conjugate was the detection ligand. Since use of trivalent labeled TSP1 in solution introduces a statistical contribution to the apparent inhibition constants, using labeled heparin as the detected ligand may give a better estimate of the intrinsic affinities for interaction of the oligosaccharides with a single subunit. The oligosaccharide binding data for immobilized intact TSP1 and the recombinant heparin-binding domain from the amino terminus were in good agreement (Table III). Disaccharides were much less active than tetrasaccharides or hexasaccharides, and octa- and decasaccharides exhibited similar activities. Hexasaccharides and shorter oligosaccharides were poor inhibitors of heparin binding to the peptide KRFKQDGGWSHWSPWSS, but decasaccharides were only 4-fold

FIG. 2. Equilibrium binding of TSP1 to bovine endothelial cell heparan sulfate and heparan sulfate proteoglycan. Wells were coated with the indicated ligands and incubated for 30 min in Tris–BSA buffer to reduce nonspecific binding and washed. Binding of [ 125I]TSP1, 0.2 to 350 ␮g/ml, was determined to biotinylated heparan sulfate immobilized on streptavidin (1.5 ␮g/well, A) or HSPG (0.6 ␮g/well, B). Triplicate determinations were averaged and corrected for nonspecific binding determined at each ligand concentration using duplicate wells without proteoglycan or biotinylated heparan sulfate. The wells were washed, and the bound radioactivity was counted. Binding constants were determined using the LIGAND program. Results are presented as Scatchard plots.

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TABLE IV

Inhibition of TSP1 Binding to Heparin by Heparin Oligosaccharides

Inhibition of Heparin Binding to TSP1 or Type 1 Repeat Peptide from TSP1 by Chemically Modified Heparin Analogs

Inhibitor

[ 125I]TSP1 3 heparin

[ 125I]Heparin 3 TSP1

Disaccharide Tetrasaccharide Hexasaccharide Octasaccharide Decasaccharide

1000 (1.00) 325 (0.16) 320 (0.11) 120 (0.03) 51 (0.010)

4500 (1.00) 470 (0.052) 620 (0.046) 270 (0.015) 340 (0.015)

Binding assay

Note. Concentrations of inhibitor required for 50% inhibition of labeled ligand binding are expressed on a weight basis (␮g/ml) and in the parentheses on a molar basis relative to the equivalent effective concentration of the disaccharide, which is arbitrarily assigned the value 1.00. The data represent the mean of three independent determinations. Data in IC 50 (␮g/ml (molar equivalents)).

The lower activity of the latter may result from positive charges introduced by the exposure of free amino groups. Other modifications that reduced the charge on heparin strongly decreased its ability to inhibit intact TSP1 binding: de-N-sulfation with reacetylation (700fold), 6-O-desulfation (300-fold), 2,3-O-desulfation (130-fold), or carboxyl-reduction (60-fold). The periodate-modified heparins were somewhat less inhibitory of labeled heparin binding to immobilized peptide KRFKQDGGWSHWSPWSS, but the order of activities was the same as for intact TSP1. NAC2 was about fivefold less active than the unmodified heparin. Desulfation of any position, however, abolished inhibitory activity for binding of labeled heparin to the peptide. Only minimal inhibition was observed with 2,3-O and 6-O desulfated heparins at the highest conTABLE III 125

Inhibition of [ I]Heparin-BSA Binding to Immobilized TSP1, Recombinant TSP1 Fragment, or Synthetic Type 1 Repeat Peptide from TSP1 Immobilized ligand Inhibitor

TSP1

28-kDa rTSP1

peptide 246

Heparin NAC2 NAC1 Disaccharide Tetrasaccharide Hexasaccharide Octasaccharide Decasaccharide

0.0057 ⫾ .0006 0.037 ⫾ .012 0.0035 ⫾ .013 4500 470 ⫾ 180 620 ⫾ 230 270 ⫾ 30 340 ⫾ 160

0.009 ⫾ .004 0.042 ⫾ .010 0.0036 ⫾ .033 2300 ⫾ 400 370 ⫾ 30 500 ⫾ 140 320 ⫾ 30 450 ⫾ 70

0.22 ⫾ .07 1.2 ⫾ .3 0.43 ⫾ .15 ⬎5000 4250 ⫾ 350 5000 2500 ⫾ 0 1600 ⫾ 100

Note. Concentrations of ligand required to give 50% inhibition of [ 125I]heparin–BSA binding (IC 50) were determined from at least two independent experiments and are presented as mean ⫾ SD. NAC2 is N-deacetylated [periodate-oxidized, borohydride-reduced] heparin. NAC1 is [periodate-oxidized, borohydride-reduced] heparin. Data in IC 50 (␮g/ml, mean ⫾ SD).

[ Inhibitor

125

I]Heparin to TSP1

Heparin LMW heparin 2,3-O-desulfated LMW 2,3-O-desulfated 6-O-Desulfated N-Desulfated/N-Ac COOH-reduced

2 11 900 1000 1000 1000 800

[ 125I]TSP1 to heparin

[ 125I]Heparin to peptide 246

0.027 1.6 3.5 7 7 18.3 1.6

3 15 10,000 ⬎⬎10,000 10,000 ⬎⬎10,000 ⬎⬎10,000

Note. Data in IC 50 (␮g/ml).

centration tested. N-desulfated/N-reacetylated heparin and carboxyl-reduced heparin were inactive at the highest concentration tested, 10 mg/ml. Affinity chromatography was used to identify subpopulations of heparin octasaccharides that bound to the type 1 repeat peptides or to the amino terminal heparin-binding domain (Fig. 3). The octasaccharide heparin fraction was 3H-labeled and subjected to affinity chromatography on a recombinant 18-kDa fragment column (Fig. 3A). Approximately 50% of the [ 3H]octasaccharide eluted unretarded. Approximately 40% of the oligosaccharide eluted as a retarded peak in isotonic buffer. The amount of bound oligosaccharide (0.12 nmol) was less than the number of available protein binding sites on the column (0.67 nmol). Each fraction eluted as a single peak at the respective positions upon rechromatography, indicating purity and lack of saturation of the affinity column. Oligosaccharides did not bind to a control column prepared in the same manner without the 18-kDa fragment. Insufficient quantities of the bound oligosaccharide fractions were obtained from the 18-kDa fragment column for compositional analysis. A portion of the labeled heparin octasaccharides also bound to the type 1 repeat peptide column. Binding was observed at 4°C but not at 25°C. Binding was optimal when the oligosaccharides were applied in isotonic buffer containing 1 mM CaCl 2 (Fig. 3B). The retarded shoulder on the unbound peak in Fig. 3B was also observed using an Affigel column containing the control peptide VTCGDGVITR or a control column without a peptide (results not shown), but the bound [ 3H]octasaccharide fraction eluting in the pH gradient was specific for a column containing peptide 246. Initial attempts to elute the bound oligosaccharides using salt gradients yielded shallow peaks that did not differ significantly from that obtained by isocratic elution,

HEPARIN-BINDING SPECIFICITIES OF THROMBOSPONDIN-1

19

but efficient elution was obtained by increasing the pH (Fig. 3B). The role of the WSXW motifs in the pH-dependent binding to immobilized TSP1 peptide KRFKQDGGWSHWSPWSS was examined using a column substituted with KRFKQDGGASHASPASS (Fig. 3C). Specific binding to the latter peptide (595 cpm) was only 14% of that obtained with the native TSP1 peptide (4292 cpm). Therefore, the heparin oligosaccharides recovered using these elution conditions represent a population that requires the WSXW motif for binding. Based on these results, the volatile ammonium formate buffer at pH 9 was used to preparatively elute heparin octasaccharides for recovery and composition analysis. Tetrasaccharides were also applied to the peptide column (Fig. 3D). In three experiments, 4 to 17% of the applied tetrasaccharide bound and were eluted when the pH of the eluent was increased to 9. Based on the PEI anion exchange HPLC analyses of the fractionated [ 3H]octasaccharides (Fig. 4), the bound and unbound heparin fractions differed in composition. The 3H-labeled octasaccharide fractions were subjected to nitrous acid treatment at pH 1.5 or to hydrazinolysis and nitrous acid treatments at pH 1.5 and 4.0. The resulting oligosaccharides were reduced with sodium borotritide, and the 3H-labeled disaccharides were analyzed by RPIP-HPLC (Table V). Following nitrous acid treatment at pH 1.5, analysis of the bound disaccharides showed a similar composition to that of the applied octasaccharides and the reference heparin, indicating that the regions enriched in Nsulfation have a high affinity for the peptides. The substituent disaccharides of the bound fraction generated after treatment with nitrous acid at pH 1.5 and 4.0 contained a 3.4-fold enrichment of the disulfated disaccharide, iduronic acid (2-OSO 3) ␣1-4 anhydromannitol(6-OSO 3), similar contents of the monosul-

FIG. 3. Affinity chromatography of heparin octasaccharides on TSP1 type 1 peptides and recombinant amino-terminal heparinbinding domain. (A) Chromatography of [ 3H]heparin octasaccharides

on recombinant 18-kDa TSP1 heparin-binding fragment immobilized on Reacti-gel. The column was equilibrated and eluted in 20 mM Tris–HCl, pH 7.6, containing 150 mM NaCl, 0.2 mM CaCl 2, and 0.02% NaN 3. (B) Chromatography of [ 3H]heparin octasaccharides on TSP1 peptide KRFKQDGGWSHWSPWSS immobilized on Affigel 10. Bound octasaccharides were eluted using a pH 7.2–7.6 gradient (---) of 20 mM Tris–HCl buffer containing 150 mM NaCl and 1 mM CaCl 2 at 4°C. (C) Specificity of heparin octasaccharide binding to the TSP1 peptide KRFKQDGGWSHWSPWSS under the conditions used for preparative affinity chromatography was assessed using 1-ml Affigel 10 columns substituted with 5 ␮mol of KRFKQDGGWSHWSPWSS (peptide 246, F), KRFKQDGGASHASPASS (peptide 388, E), or unsubstituted gel (Œ). Equal volumes of octasaccharides were applied to each column, and unbound oligosaccharides were eluted in 0.15 M ammonium formate, pH 6.1. Elution was initiated at fraction 40 by a step gradient to the same buffer, pH 9.0. (D) Chromatography of [ 3H]heparin tetrasaccharides on TSP1 peptide KRFKQDGGWSHWSPWSS immobilized on Affigel 10 equilibrated in 150 mM ammonium formate, pH 6.1. Bound tetrasaccharides were eluted using a pH step gradient to ammonium formate, pH 9.0.

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YU ET AL.

FIG. 4. Characterization by PEI ion-exchange HPLC of heparin octasaccharides fractionated on a TSP1 type 1 repeat peptide affinity column. 3H-labeled octasaccharides affinity fractionated on immobilized KRFKQDGGWSHWSPWSS as in Fig. 3C were analyzed for charge heterogeneity by elution from PEI HPLC with linear gradients of ammonium bicarbonate (0 –1000 mM). (A) 3H-labeled octasaccharides applied to the affinity column; (B) unbound fraction of 3 H-labeled octasaccharides; (C) bound fraction of 3H-labeled octasaccharides. Dashed line, ammonium bicarbonate gradient.

fated disaccharides, and a 6.5-fold depletion of the unsulfated disaccharides relative to the unbound fraction. DISCUSSION

The high avidity interaction of intact TSP1 with heparin depends on multivalent binding to heparin,

since intact TSP1 was 60- to 130-fold more active than two monovalent recombinant forms of the heparinbinding domain. The ratios of activities were less using an endothelial HSPG in place of heparin, ranging from 10- to 30-fold. This may reflect a lower density of high affinity TSP1 binding sites on the vascular HSPG than on heparin, which reduces the probability of simultaneous binding of TSP1 to more than one site on the glycosaminoglycan and thus decreases the statistical advantage of trivalent TSP1 over the monovalent heparin-binding domains. This is also consistent with the report that TSP1 bound more avidly to heparin than to heparan sulfate from brain (62). Similarly, the twodimensional array of binding sites presented by a monolayer of sulfatide in a phosphatidylcholine/cholesterol monolayer could allow multivalent interactions, indicated by the 25- to 45-fold higher avidity of TSP1 relative to the 18- and 28-kDa recombinant fragments measured using sulfatide as the immobilized ligand. The heparin binding activities of the 18- and 28-kDa fragments are comparable to that of a previously described recombinant heparin-binding fragment of TSP1, containing residues 1 to 229, which bound heparin with a dissociation constant of 71 nM (63). Multivalent binding probably also accounts for the greater progressive increase in activities of heparin fragments for inhibiting TSP1 binding to immobilized heparin than in the reverse assay. Based on oligosaccharide inhibition, the minimal heparin sequence for avid binding to TSP1 is a tetrasaccharide. This contrasts with a previous report that oligosaccharides shorter than 10 mers did not compete for binding of iodinated heparin to immobilized TSP1 (43). Larger oligosaccharides were also required for inhibiting TSP1 binding to Chinese hamster ovary cells. It is unlikely that the amino terminal domain of a single subunit of TSP1 has a binding site that interacts with such a long span of heparin sequence, with a predicted length of 42 Å for a decasaccharide (64). Thus, the minimal active size observed in these assays may reflect their lower sensitivity to inhibition by heparin analogs. The enhanced activity of the higher oligomers is probably due to their ability to interact simultaneously with more than one subunit of TSP1. The size-dependence for heparin oligosaccharide binding to recombinant amino-terminal fragments of TSP1 is identical to that of the intact protein, indicating that the major interaction between intact TSP1 and heparin is mediated by the amino-terminal domain. This is an important result since we and others have identified at least two additional heparin binding sites in other regions of the protein (15, 17, 65). This result, however, does not establish whether the secondary heparin-binding sites are functional. The ability of the WSXW motif in native type 1 repeats to bind heparin is controversial, since some of the TSP1 fragments

21

HEPARIN-BINDING SPECIFICITIES OF THROMBOSPONDIN-1 TABLE V 3

Disaccharide Composition of [ H]octasaccharides Fractionated by Affinity Chromatography on Immobilized Thrombospondin Peptide KRFKQDGGWSHWSPWSS Nitrous acid treatment pH 1.5

Nitrous acid treatments pH 1.5 and 4.0

Disaccharide a

Heparin

Total

Bound

Heparin

Bound

Unbound

GM IM Unknown ISM GMS ⫹ GSM IMS GMS 2 ISMS GSMS

— — — 8.5 10.8 2.9 3.2 74.5 —

— — — 9.6 7.7 4.8 2.9 75.0 —

— — — 9.5 7.3 6.1 3.0 74.1 —

2.7 2.3 2.1 11.0 12.0 6.0 6.7 54.8 2.3

3.4 2.2 7.9 8.9 9.2 6.9 4.5 55.3 1.7

27.5 9.3 20.1 7.0 7.0 6.3 6.5 16.3 nd b

Note. [ 3H]Octasaccharides were fractionated by affinity chromatography on KRFKQDGGWSHWSPWSS-Affigel. Porcine mucosal heparin and the applied (total), bound, and unbound octasaccharide fractions were depolymerized, labeled with NaB 3H 4, and analyzed by RPIP-HPLC as described under Materials and Methods. a GM, glucuronic acid␤1-4-anhydromannitol; IM, iduronic acid␣1-4-anhydromannitol; ISM, iduronic acid(2-OSO 3)␣1-4-anhydromannitol; GMS, glucuronic acid␤1-4-anhydromannitol(6-OSO 3); GSM, glucuronic acid(2-OSO 3)␤1-4-anhydromannitol; IMS, iduronic acid␣1-4-anhydromannitol(6-OSO 3); GMS 2, glucuronic acid␤1-4-anhydromannitol(3,6-diOSO 3); ISMS, iduronic acid(2-OSO 3)␣1-4-anhydromannitol(6OSO 3); GSMS, glucuronic acid(2-OSO 3)␤1-4-anhydromannitol(6-OSO 3). b nd, not detected.

that have been prepared containing this sequence bind heparin, but others do not (17, 66). At least part of the type 1 repeat sequence is exposed in intact TSP1, since TGF␤ binds to the second type 1 repeat and to peptide 246 derived from this repeat (67), and polyclonal antibodies to this peptide also bind to native TSP1 (J. A. Cashel and D. D. Roberts, unpublished data). The heparin-binding specificity of the type 1 repeat peptide differs from that of the amino-terminal domain of TSP1. Tetrasaccharides were 10-fold less active, and decasaccharides were 4-fold less effective at inhibiting heparin binding to this peptide than for inhibiting heparin binding to the amino-terminal domain or to intact TSP1. However, high affinity binding to both sites from TSP1 requires modification by N-sulfation. Sulfation at the 6 position and possibly the 3 position of glucosamine or the 2 position of iduronic acid, as well as an intact carboxyl group on the uronic acid residues, are more important for binding to the peptide than to the intact TSP1, since heparin analogs modified at these sites were weak inhibitors of intact TSP1 binding but inactive for inhibiting binding to the peptide. The binding specificities of the type 1 repeat and amino terminal heparin-binding sites overlap with that of FGF2 in that both require 2-O-sulfation and N-sulfation and prefer an intact carboxylate on the uronic acid residues (37, 38, 68). 2-O- and 6-O-sulfation are also required for the “site B” in heparin that may interact with the FGF tyrosine kinase receptor or participate in dimer formation (68). Thus, the binding specificities support the observed antagonism between the TSP1 peptides and FGF2 for binding to heparin

and endothelial cells (9, 28). Although affinity coelectrophoresis did not detect differences in specificities for binding of TSP1 and FGF2 to subpopulations of heparin (44), the inhibition studies show that TSP1 has a requirement for 6-O-sulfation that is not observed for FGF2 binding to heparin (37), although 6-O-sulfation is required for promoting the mitogenic activity of FGF2 by heparan sulfate (42). This specificity is consistent with the observed antagonist activity of these TSP1 peptides for FGF2-dependent endothelial cell proliferation and chemotaxis (9, 28). Although the mechanism for binding of peptides containing the WSXW motifs to heparin remains to be defined, these studies provide evidence for a structural specificity in the recognition of heparin by this peptide. A control peptide containing the flanking basic amino acids but lacking the Trp residues was only 14% as efficient for binding heparin octasaccharides as the native TSP1 peptide. Since the dissociation rate from heparin of WSXW peptides lacking a basic amino acid motif is too fast to extend the present studies to simple WSXW peptides, the specificities of the basic and WSXW motifs cannot be completely resolved at present. However, the present data are consistent with the previous observation that intact heparin did not bind to type 1 repeat peptide analogs that had the basic amino acid motif but lacked the WSXW motif (16). This conclusion is also supported by the recent reports that a peptide from lipoprotein lipase containing the related sequence FSWSDWWS bound strongly to heparin (69) and that the side chain of Trp[69] forms part of the

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