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• W2070294819 abstract "Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfatedN-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location. Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfatedN-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location. fibroblast growth factor antithrombin 2,5-anhydromannitol fibroblast growth factor receptor d-glucuronic acid glucosamine glucosamine 6-sulfatase N-acetylglucosamine N-sulfoglucosamine hexuronic acid high perfomance liquid chromatography heparan sulfate proteoglycan l-iduronic acid α-l-iduronidase iduronate 2-sulfatase strong anion exchange heparan sulfate heparan sulfate proteoglycan phosphate-buffered saline 2-N-(morpholino)ethanesulfonic acid The fibroblast growth factors (FGFs)1 belong to a family of about 20 related polypeptides. They display biological activity toward cells of mesenchymal, neuronal, and epithelial origin and are involved in processes such as cell growth, organ development, and angiogenesis (1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar). The biological effects of FGFs are exerted through interactions with FGF receptors (FGFRs). The receptor family consists of four known members, FGFR1–4, with many isoforms (2Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Crossref PubMed Scopus (1178) Google Scholar). Upon ligand binding the receptor is thought to be activated through dimerization and phosphorylation by the intracellular tyrosine kinase domains (3Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4619) Google Scholar). Heparan sulfate proteoglycans (HSPGs), abundant components of cell surfaces and the extracellular matrix, appear central to signaling through FGF·FGFR complexes (for reviews, see Refs. 4Zimmermann P. David G. FASEB J. 1999; 13: 91-100Crossref PubMed Google Scholar, 5Rapraeger A.C. Curr. Opin. Cell Biol. 1993; 5: 844-853Crossref PubMed Scopus (120) Google Scholar, 6Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-393Crossref PubMed Scopus (970) Google Scholar). Cells lacking endogenous HSPGs respond poorly to FGF, whereas the response can be readily restored by addition of exogenous heparin (7Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1292) Google Scholar,8Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2094) Google Scholar). Accumulated evidence points to formation of biologically active complexes involving FGF, FGFR, and HSPGs, in which heparan sulfate interacts with both the FGF and FGFR components of the complex (9Schlessinger J. Plotnikov A.N. Ibrahimi O.A. Eliseenkova A.V. Yeh B.K. Yaon A. Linhardt R.J. Mohammadi M. Mol. Cell. 2000; 6: 743-750Abstract Full Text Full Text PDF PubMed Scopus (974) Google Scholar, 10Pellegrini L. Burke D.F. von Delft F. Mulloy B. Blundell T.L. Nature. 2000; 407: 1029-1034Crossref PubMed Scopus (630) Google Scholar, 11Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar). A direct interaction between HSPGs and FGFRs appears critical for FGFR activation (12Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Crossref PubMed Scopus (476) Google Scholar). A heparin-binding domain identified in the second Ig-loop of the four FGFRs comprises sequence of about 20 amino acids toward the NH2 terminus of the loop (12Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Crossref PubMed Scopus (476) Google Scholar). Different splice variants of the receptors differ in affinity for heparin, such that the interaction may vary with the structure of the extracellular receptor domain (13Wang F. Kan M. Yan G. Xu J. McKeehan W.L. J. Biol. Chem. 1995; 270: 10231-10235Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Heparin/HS chains are initially synthesized as polymers of alternating glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) units (for reviews, see Refs. 14Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar, 15Salmivirta M. Lidholt K. Lindahl U. FASEB. J. 1996; 10: 1270-1279Crossref PubMed Scopus (397) Google Scholar, 16Rosenberg R.D. Shworak N.W. Liu J. Schwartz J.J. Zhang L. J. Clin. Invest. 1997; 99: 2062-2070Crossref PubMed Scopus (258) Google Scholar). In HS biosynthesis, the polymer is first modified by partialN-deacetylation/N-sulfation of GlcNAc residues. The further modification reactions, C5-epimerization of GlcA to iduronic acid (IdoA) units and O-sulfation at various positions (C2 of IdoA and GlcA, C3 and C6 of GlcN units), all occur in the vicinity of previously incorporated N-sulfate groups. Heparin, a highly specialized product of mast cells, is more extensively modified than HS and the modifications are more evenly distributed along the polymer (14Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar). The heparin/HS structures required for FGFR binding are poorly defined. However, both IdoA(2-OSO3) and GlcNSO3(6-OSO3) residues appear to be required for the FGF2 induced activation of FGFR1, whereas 2-O-sulfate groups alone are sufficient to mediate binding to FGF2 (11Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar, 17Rusnati M. Coltrini D. Caccia P. Dell'Era P. Zoppetti G. Oreste P. Valasina B. Presta M. Biochem. Biophys. Res. Commun. 1994; 203: 450-458Crossref PubMed Scopus (76) Google Scholar, 18Pye D.A. Romain R.V. Turnbull J.E. Hyde P. Gallagher J.T. J. Biol. Chem. 1998; 273: 22936-22942Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). While these findings suggest a role for 6-O-sulfated GlcNSO3 residues in the interactions with FGFR1, it would seem likely that different FGFRs may bind structurally distinct HS species. Neuroepithelial HSPG thus preferentially bound FGFR1 at the cell surface although FGFR3 was also present (19Brickman Y.G. Ford M.D. Small D.H. Bartlett P.F. Nurcombe V. J. Biol. Chem. 1995; 270: 24941-24948Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The importance of polysaccharide-FGFR interaction was underpinned by the finding that heparin could alone induce phosphorylation of FGFR4 in the absence of an FGF ligand (20Gao G. Goldfarb M. EMBO J. 1995; 14: 2183-2190Crossref PubMed Scopus (94) Google Scholar). In the present paper we describe FGF-independent binding of heparin and HS to the extracellular domain of FGFR4. We show that the interaction is mediated by N-sulfated octasaccharides that contain both IdoA(2-OSO3) and GlcNSO3(6-OSO3) residues, and provide sequence data for FGFR4-binding HS domains. All studies were performed using the soluble extracellular domain of FGFR4. The expression and purification of recombinant human FGFR4, containing the three extracellular Ig domains (Ser25-Arg366), were as described earlier (21Loo B.-M. Darwish K. Vainikka S. Saarikettu J. Vihko P. Hermonen J. Goldman A. Alitalo K. Jalkanen M. Int. J. Cell Biol. Biochem. 2000; 32: 489-497Crossref PubMed Scopus (7) Google Scholar). Briefly, the His-tagged protein was expressed in Sf9 insect cells and purified directly from the culture medium by nickel and heparin affinity chromatography. Heparin from pig intestinal mucosa (stage 14, Inolex Pharmaceutical Division, Park Forest South, IL), was purified as previously described (22Lindahl U. Cifonelli J.A. Lindahl B. Rodén L. J. Biol. Chem. 1965; 240: 2817-2820Abstract Full Text PDF PubMed Google Scholar). It was used either unlabeled or radiolabeled by 3H-acetylation of free amino groups (specific activity 75,300 dpm/nmol) as described (23Höök M. Riesenfeld J. Lindahl U. Anal. Biochem. 1982; 119: 236-245Crossref PubMed Scopus (85) Google Scholar). The selectively desulfated heparin preparations and oligosaccharides of bovine lung heparin (24Spillmann D. Witt D. Lindahl U. J. Biol. Chem. 1998; 273: 15487-15493Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 25Feyzi E. Lustig F. Fager G. Spillmann D. Lindahl U. Salmivirta M. J. Biol. Chem. 1997; 272: 5518-5524Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) were a kind gift from Dr. Dorothe Spillmann (Uppsala University, Uppsala, Sweden). Heparan sulfate preparations from bovine aorta, kidney, lung, and intestine were generously provided by Dr. Keiichi Yoshida (Seikagaku Corp., Tokyo, Japan). N-Sulfated HS oligosaccharides were prepared from bovine intestinal mucosa heparan sulfate (a gift from Kabi AB, Stockholm, Sweden) and3H-labeled as described previously (26Kreuger J. Prydz K. Petterson R.F. Lindahl U. Salmivirta M. Glycobiology. 1999; 9: 723-729Crossref PubMed Scopus (78) Google Scholar). Briefly, HS wasN-deacetylated by hydrazinolysis followed by treatment with nitrous acid at pH 3.9, resulting in cleavage at theN-unsubstituted GlcN residues. The resistentN-sulfated oligosaccharides were recovered, reduced with NaB3H4 (28 Ci/mmol, Amersham Pharmacia Biotech, Uppsala, Sweden), and separated by gel chromatography. The column materials, Sephadex G-15 and CH-Sepharose-4B, were obtained from Amersham Pharmacia Biotech, as were the PD-10 desalting and Superdex 30 columns. The Partisil-10 HPLC column (4.6 × 250 mm) was from Whatman Inc., Clifton, NJ, and the Propac PA1 HPLC column was from Dionex, Surrey, United Kingdom. In the filter-trapping assay (27Maccarana M. Lindahl U. Glycobiology. 1993; 3: 271-277Crossref PubMed Scopus (110) Google Scholar), radiolabeled glycosaminoglycans were incubated with FGFR4 in 8.1 mm Na2HPO4, 1.5 mmKH2PO4, 2.7 mm KCl, and 140 mm NaCl, pH 7.4 (PBS), containing 0.1 mg/ml bovine serum albumin, in a total volume of 200 μl for 2 h at room temperature. The mixtures were rapidly passed through nitrocellulose filters (Sartorius, diameter 25 mm, pore size 0.45 μm) using a vacuum suction apparatus, followed by washing with PBS. Proteins and protein bound saccharides remain on the filter whereas unbound saccharides pass through. The bound saccharides were released by 2 m NaCl and quantified by a β-scintillation counter. Binding studies were also performed using a CH Sepharose-4B column, with immobilized FGFR4, that was prepared according to the instructions of the manufacturer. For preparation of 1 ml of the affinity matrix, ∼0.5 mg of FGFR4 was used. Heparin (0.5 mg) was included in the immobilization reaction to protect the heparin-binding site on FGFR4. To avoid immobilization of heparin to the matrix, the heparin preparation used had been treated with HNO2 at pH 3.9 (28Shively J.E. Conrad H.E. Biochemistry. 1976; 15: 3932-3942Crossref PubMed Scopus (667) Google Scholar) to destroy any N-unsubstituted GlcN residues, followed by recovery of the high molecular weight fraction by gel chromatography on Superdex 30. Samples of 3H-labeled heparin/HS were applied to the column in PBS, with or without CaCl2supplementation, followed by washing with PBS and elution of the bound material with a linear gradient of NaCl (0.14–1.0 m) in PBS at a flow rate of 0.5 ml/min. Fractions of 1 ml were collected and measured for radioactivity. The NaCl gradient was monitored by measuring the conductivity of every third fraction. A control column was prepared without immobilized FGFR4. This column did not bind any of the tested glycosaminoglycans (data not shown). To study the interaction of the antithrombin (AT) binding heparin domain with FGFR4,3H-labeled heparin decasaccharides were subjected to affinity chromatography on antithrombin-Sepharose as described (29Höök M. Björk I. Hopwood J. Lindahl U. FEBS Lett. 1976; 66: 90-93Crossref PubMed Scopus (303) Google Scholar). Bound decasaccharides were eluted with a step gradient of NaCl (0.14, 0.5, and 2.0 m NaCl in 50 mm Tris-HCl, pH 7.4). The high affinity (∼2% of total saccharide) and non-binding fractions recovered in the 2.0 and 0.14 m NaCl eluates, respectively, were tested for FGFR4 binding by affinity chromatography. Surface plasmon resonance analysis on a BiacoreX instrument (BiaCore AB, Uppsala, Sweden) was a third means of studying saccharide:FGFR4 binding. Heparin (0.5–1 mg) was biotinylated by incubation in 0.1m MES buffer (pH 5.5 with 50 mm biotin hydrazide (Calbiochem, San Diego, CA) and 10 mm N-ethyl-N′(dimethylaminopropyl)carbodiimide (Pierce Chemical Corp., Rockford, IL) for 5–6 h at room temperature. Biotinylated heparin was separated from excess reagent on a PD-10 column and immobilized to streptavidin-coated sensor chips (BiaCore AB). FGFR4 was incubated with saccharides for at least 10 min prior to injection over the heparin-coated surface. The running buffer used was PBS supplemented with 0.005% Tween 20. The concentrations of the interactants and flow rates were as indicated in the figure legends. A streptavidin surface without immobilized heparin was used as a control. The response from this surface was subtracted from the response of the heparin surface. N-Sulfated oligosaccharides from bovine intestinal HS were fractionated by binding to the FGFR4 affinity matrix. To deplete the unbound pool (∼80% of the total saccharide) of any remaining FGFR4 binding components, it was rechromatographed twice on the FGFR4 column (<4% of the material was bound to the matrix upon the second rechromatography step). The disaccharide composition of HS samples was determined as described (30Kusche M. Torri G. Casu B. Lindahl U. J. Biol. Chem. 1990; 265: 7292-7300Abstract Full Text PDF PubMed Google Scholar, 31Bienkowski M.J. Conrad H.E. J. Biol. Chem. 1985; 260: 356-365Abstract Full Text PDF PubMed Google Scholar). Briefly, saccharides were treated with nitrous acid (HNO2) at pH 1.5, leading to deaminative cleavage of the saccharide chain at GlcNSO3 units (28Shively J.E. Conrad H.E. Biochemistry. 1976; 15: 3932-3942Crossref PubMed Scopus (667) Google Scholar). The resultant terminal anhydromannose units were radiolabeled by reduction with NaB3H4 (0.25–0.5 mCi/reaction) yielding3H-labeled 2,5-anhydromannitol ([3H]aManR) residues. The labeled disaccharides were recovered by gel chromatography and further separated by anion-exchange HPLC on a Partisil-10 SAX column eluted with a step gradient of KH2PO4. The disaccharide peaks were identified by comparing their elution positions to those of standard heparin disaccharides. The proportions of non-O-sulfated disaccharides were determined by high voltage paper electrophoresis of the total labeled disaccharides in 0.83 m pyridine, 0.5 m acetic acid buffer, pH 5.3 (32Kusche M. Lindahl U. Enerbäck L. Rodén L. Biochem. J. 1988; 253: 885-893Crossref PubMed Scopus (23) Google Scholar). FGFR4 binding HS oligosaccharides, containing a reducing terminal [3H]aManRresidue, were prepared for sequence analysis by anion-exchange HPLC on a Propac PA1 column in H2O, pH 3 (adjusted with HCl). The bound oligosaccharides were eluted with a linear gradient of NaCl (up to 1.5 m). The fractions containing the octasaccharides of interest were pooled, desalted, dried in a centrifugal evaporator, and sequenced through a combination of chemical and enzymatic degradation procedures as described (33Vivès R.R. Pye D.A. Salmivirta M. Hopwood J.J. Lindahl U. Gallagher J.T. Biochem. J. 1999; 339: 767-773Crossref PubMed Scopus (86) Google Scholar). Samples were first subjected to partial HNO2 (pHNO2) cleavage (34Turnbull J.E. Hopwood J.J. Gallagher J.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2698-2703Crossref PubMed Scopus (140) Google Scholar) by treatment with 2 mm NaNO2 in 20 mm HCl on ice. After incubation for various periods of time (30, 60, 90, 120, and 180 min), aliquots were removed and the reaction was stopped by addition of 200 mm sodium acetate, pH 6. The aliquots, containing the cleavage products from the different time points, were combined and subjected to enzyme digestion. The exoenzymes used for sequence analysis were iduronate-2-sulfatase (IdoA2Sase), α-l-iduronidase (IdoAase), and glucosamine-6-sulfatase (GlcN6Sase) (Oxford GlycoSciences, Abingdon, U.K.). The enzymes are recombinant human (IdoA2Sase and IdoAase) or caprine (GlcN6Sase) proteins produced in Chinese hamster ovary K1 cells. IdoA2Sase removes ester sulfates at C2 of nonreducing terminal IdoA(2-OSO3) residues of heparin/heparan sulfate whereas IdoAase cleaves the α1–4 linkage between IdoA and GlcNR in heparin/heparan sulfate (Ris -COCH3, -SO 3−, or -HS 3). GlcN6Sase cleaves ester sulfates at C6 of nonreducing GlcNR residues. The substrate specificities of the enzymes were confirmed using known heparin structures as substrates. IdoA2Sase and IdoAase were shown to be active on all potential substrates including heparin disaccharide deamination products. Notably, 6-O-sulfated aManR is not a substrate for GlcN6Sase. 2J. Kreuger and U. Lindahl, unpublished data. The amounts of enzyme used were 1 milliunit/reaction of IdoA2Sase/IdoAase, and 0.2 milliunits/reaction of GlcN6Sase. The samples were incubated with a single enzyme or combinations of the enzymes in a total volume of 25 μl, in the incubation buffer provided with the enzymes (50 mm sodium acetate, pH 5.0, and 0.1 mg/ml bovine serum albumin) at 37 °C for 13–15 h. In digestions involving several enzymes, the enzymes were added at 2-h intervals, in the order of action. The enzyme-treated saccharides were analyzed by Propac ion-exchange chromatography as described above. Sequence information was obtained by detecting shifts in the elution positions of the enzyme-treated subfragments (33Vivès R.R. Pye D.A. Salmivirta M. Hopwood J.J. Lindahl U. Gallagher J.T. Biochem. J. 1999; 339: 767-773Crossref PubMed Scopus (86) Google Scholar, 34Turnbull J.E. Hopwood J.J. Gallagher J.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2698-2703Crossref PubMed Scopus (140) Google Scholar). To assess the binding of the recombinant extracellular domain of FGFR4 to heparin, increasing amounts of [3H]heparin were incubated with FGFR4 at physiological ionic strength. The protein-polysaccharide complexes formed were trapped on nitrocellulose filters and the bound saccharide was quantified by scintillation counting (27Maccarana M. Lindahl U. Glycobiology. 1993; 3: 271-277Crossref PubMed Scopus (110) Google Scholar). The results indicated that heparin bound FGFR4 in a dose-dependent and saturable manner (Fig. 1 A), whereas the binding was completely abolished by addition of excess cold heparin (data not shown). A KD value of 0.3–0.4 μm was determined for the FGFR-heparin interaction (assuming an average molecular weight of 10 kDa for heparin) by fitting a hyperbolic function using nonlinear regression analysis to the data visualized by a Scatchard plot (inset, Fig. 1 A). The interaction was also studied by affinity chromatography of [3H]heparin on immobilized FGFR4, as well as by surface plasmon resonance (BiaCore) measurements of soluble FGFR4 binding to immobilized heparin. In affinity chromatography, [3H]heparin was found to require 0.25–0.50 mNaCl for elution from the FGFR4 column (Fig. 1 B). Surface plasmon resonance analysis showed saturable binding of FGFR4 to the heparin-coated surface whereas little or no binding was seen to a control surface without heparin (data not shown). Together, these data indicate that the FGFR4 ectodomain is capable of binding heparin in a FGF-ligand independent fashion, in agreement with previous results (20Gao G. Goldfarb M. EMBO J. 1995; 14: 2183-2190Crossref PubMed Scopus (94) Google Scholar,21Loo B.-M. Darwish K. Vainikka S. Saarikettu J. Vihko P. Hermonen J. Goldman A. Alitalo K. Jalkanen M. Int. J. Cell Biol. Biochem. 2000; 32: 489-497Crossref PubMed Scopus (7) Google Scholar, 35Kan M. Wu X. Wang F. McKeehan W.L. J. Biol. Chem. 1999; 274: 15947-15952Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). An important role for divalent cations such as Ca2+ in the binding of heparin to FGFR1, another member of the FGFR family, were proposed by McKeehan and co-workers (36Kan M. Wang F. Kan M. To B. Gabriel J.L. McKeehan W.L. J. Biol. Chem. 1996; 271: 26143-26148Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 37Patstone G. Maher P. J. Biol. Chem. 1995; 271: 3343-3346Abstract Full Text Full Text PDF Scopus (34) Google Scholar). To examine whether calcium ions affect the heparin-FGFR4 interaction, we tested the binding of [3H]heparin to the FGFR4 affinity matrix in the presence of 1.3 mm Ca2+, which corresponds to the physiological Ca2+ concentration of extracellular fluids. Under these conditions, the peak elution of [3H]heparin occurred at ∼0.40 m NaCl, as compared with a peak elution position corresponding to ∼0.35m NaCl in the absence of Ca2+ ions (Fig.1 B). These results suggest that Ca2+ ions may slightly enhance, but are not required for, the heparin-FGFR4 interaction. To identify the minimal size of the FGFR4 binding heparin domain, even numbered, 3H-end-labeled heparin oligosaccharides were incubated with FGFR4 in solution, after which the binding was assessed by the filter-trapping method (see “Experimental Procedures”). Octasaccharides were the shortest oligosaccharides with appreciable FGFR4 binding capacity (Fig.2 A). The binding of decasaccharides and longer fragments to FGFR4 increased gradually with increasing fragment length, but without any striking differences in binding between the consecutive fragments of the series. Surface plasmon resonance was also used to define the minimal FGFR4-binding heparin domain by assessing the ability of heparin oligosaccharides to inhibit binding of FGFR4 to biotinylated full-length heparin, immobilized on the chip surface (Fig. 2 B). Octasaccharides were the smallest fragments with substantial inhibitory capacity, whereas decasaccharides and longer fragments had still higher inhibitory effect. Taken together, the above data implicate a minimal FGFR4-binding site within a sequence encompassing eight monosaccharide units. We next studied the importance of the N-, 2-O-, and 6-O-sulfate groups of heparin in FGFR4 binding, by testing the ability of selectively desulfated heparin preparations to inhibit binding of [3H]heparin to FGFR4 in solution (Fig.3 A). Whereas low concentrations (1–5 μg/ml) of unlabeled, native heparin blocked the binding almost completely, corresponding amounts of the various selectively desulfated heparin preparations showed little inhibitory capacity. However, each of the preparations resulted in 50–75% inhibition at high concentrations (50–100 μg/ml) (Fig.3 A). In Biacore studies (Fig. 3 B), FGFR4 was incubated with the desulfated heparin preparations prior to injection of the mixture over the heparin-coated surface. The results were in agreement with the data from filter trapping assays, such that each of the selective desulfation treatments led to a dramatic decrease in inhibitory capacity (Fig. 3 B). Collectively, these results suggest that the N-, 2-O-, and 6-O-sulfate groups of heparin all contribute to binding FGFR4. Recently, the AT-binding pentasaccharide motif of heparin, containing a critical 3-O-sulfated GlcNSO3 residue was implicated in binding to FGFRs (38McKeehan W.L. Wu X. Kan M. J. Biol. Chem. 1999; 274: 21511-21514Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). We decided to reassess this proposal by separating 3H-labeled heparin decasaccharides with regard to affinity for AT, and then test the resultant high and low affinity fractions for ability to bind FGFR4. About 2% of the starting material bound with high affinity to immobilized AT, in good agreement with previous findings (29Höök M. Björk I. Hopwood J. Lindahl U. FEBS Lett. 1976; 66: 90-93Crossref PubMed Scopus (303) Google Scholar) and this fraction was quantitatively retained by the FGFR4 column (Fig.4 A), in accord with the proposal by McKeehan et al. (38McKeehan W.L. Wu X. Kan M. J. Biol. Chem. 1999; 274: 21511-21514Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, a major portion of the fraction with low affinity for AT also bound to the immobilized FGFR4, and elution of this material from the FGFR4 column required the same NaCl concentration as that needed to displace the decasaccharide with high affinity for AT (Fig. 4 A). This finding is in disagreement with the notion that the AT binding sequence is essential for FGFR binding. Indeed, isolation of the two FGFR4-binding fractions followed by analytical AT-Sepharose chromatography confirmed that one of the fractions, as expected, showed high affinity for AT whereas the other did not (Fig. 4 B). The major physiological polysaccharide ligand for FGFR4 is presumably HS rather than heparin, that is essentially confined to the mast cell. The interactions of3H-labeled HS samples from bovine lung, aorta, and kidney with FGFR4 were studied by affinity chromatography (Fig.5). All HS species tested were retained by the column and required 0.2–0.3 m NaCl for elution. Generally, the binding was somewhat weaker than that of heparin, judging from the higher NaCl concentration required to displace heparin compared with HS. The FGFR4 binding profiles of the various HS species differed such that a substantial portion of kidney HS emerged at NaCl concentrations >0.35 m, whereas aorta HS contained only minor amounts of such high-affinity material. We next proceeded to characterize HS domains with affinity toward FGFR4. The experiments with selectively desulfated heparin suggested that the binding of HS to FGFR4 would require highly sulfated structures, of the type represented by theN-sulfated domains of the polysaccharide. These domains are composed of consecutive N-sulfated disaccharide units and contain most of the O-sulfate groups of the HS chain. To isolate such domains, we used bovine intestinal HS that bound to the FGFR4 affinity column (data not shown) similar to the lung HS preparation shown in Fig. 5. N-Sulfated oligosaccharides were prepared as described under “Experimental Procedures” and3H-end-labeled by reduction with NaB3H4. Affinity chromatography of the [3H]decasaccharide on the FGFR4 column yielded 20% of bound material. After two reapplications of the unbound fraction, less than 4% of the material bound to the matrix, indicating depletion of FGFR4-binding species (data not shown). Analysis of the bound and unbound decamer pools (see “Experimental Procedures”) indicated that the bound fraction was markedly enriched in 6-O-sulfate groups, that were almost twice as abundant as in the unbound fraction (Table I, Fig. 6). The 6-O-sulfate groups occurred mainly in trisulfated -IdoA(2-OSO3)-GlcNSO3(6-OSO3)- disaccharide units. Similar results were obtained upon compositional disaccharide analysis of FGFR4 bound and unbound dodecasaccharides (not shown). Together, these findings suggest that the FGFR4-HS" @default.
• W2070294819 created "2016-06-24" @default.
• W2070294819 creator A5003103449 @default.
• W2070294819 creator A5014461710 @default.
• W2070294819 creator A5059845626 @default.
• W2070294819 creator A5078179214 @default.
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• W2070294819 date "2001-05-01" @default.
• W2070294819 modified "2023-10-11" @default.
• W2070294819 title "Binding of Heparin/Heparan Sulfate to Fibroblast Growth Factor Receptor 4" @default.
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