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• W2000521581 abstract "Fibroblast growth factor-2 (FGF-2), a potent angiogenic factor, requires heparin for dimerization and activation of the FGF receptor tyrosine kinase. The binding of multiple fibroblast growth factors by heparin may be necessary for dimerization of the FGF receptor. Analytical ultracentrifugation of FGF-2 in the presence of heparin-derived saccharides shows that both an active heparin octasaccharide and an inactive heparin-like disaccharide induce fibroblast growth factor-2 self-association. Analysis of the data indicates that the heparin octasaccharide induces a monomer-dimer-tetramer assembly of FGF-2 while the disaccharide induces a monomer-dimer equilibrium. Evidence is presented indicating that the dimer conformation induced by the heparin octasaccharide is a side by side dimer with the FGF-2 molecules cis to the heparin, while the disaccharide-induced dimer is a head to head dimer in which FGF-2 molecules are trans to the ligand. These results, combined with previous studies, support the model that formation of a specific side by side heparin-induced FGF-2 dimer is required for activation of the FGF receptor. Fibroblast growth factor-2 (FGF-2), a potent angiogenic factor, requires heparin for dimerization and activation of the FGF receptor tyrosine kinase. The binding of multiple fibroblast growth factors by heparin may be necessary for dimerization of the FGF receptor. Analytical ultracentrifugation of FGF-2 in the presence of heparin-derived saccharides shows that both an active heparin octasaccharide and an inactive heparin-like disaccharide induce fibroblast growth factor-2 self-association. Analysis of the data indicates that the heparin octasaccharide induces a monomer-dimer-tetramer assembly of FGF-2 while the disaccharide induces a monomer-dimer equilibrium. Evidence is presented indicating that the dimer conformation induced by the heparin octasaccharide is a side by side dimer with the FGF-2 molecules cis to the heparin, while the disaccharide-induced dimer is a head to head dimer in which FGF-2 molecules are trans to the ligand. These results, combined with previous studies, support the model that formation of a specific side by side heparin-induced FGF-2 dimer is required for activation of the FGF receptor. Fibroblast growth factor-2 (FGF-2) 1The abbreviations used are: FGF-2, fibroblast growth factor-2; HS, heparin or heparan sulfate; HSPG, heparan sulfate proteoglycan; FGFR, fibroblast growth factor receptor; FGF-1, fibroblast growth factor-1; HS-8, sulfated heparin octasaccharide; SOS, sucrose octasulfate; β-ME, β-mercaptoethanol; EGF, epidermal growth factor. is a cytokine whose biological activity is dependent on heparin or heparan sulfate (HS, collectively) (1Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (562) Google Scholar). The binding of FGF-2 and heparin to the FGF receptor (FGFR) activates a signal transduction cascade, eventually stimulating cell proliferation and differentiation. Cells that are activated by FGF-2 require two classes of receptors: low-affinity receptors which are heparan sulfate proteoglycans (HSPGs) and high-affinity receptors which are transmembrane receptor tyrosine kinases (2Klagsbrun M. Baird A. Cell. 1991; 67: 229-231Abstract Full Text PDF PubMed Scopus (498) Google Scholar). The heparin or HSPG low-affinity receptors are thought to bind to FGF initially and present FGF to the FGF receptor (3Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2094) Google Scholar), leading to a ternary FGF·HS·FGFR complex that can then activate the FGF signal transduction pathway (1Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (562) Google Scholar). It has been shown that activation of the FGF signal transduction pathway occurs upon dimerization of the FGF tyrosine kinase receptor and transphosphorylation of each kinase domain (4Bellot F. Crumley G. Kaplow J.M. Schlessinger J. Jaye M. Dionne C.A. EMBO J. 1991; 10: 2849-2854Crossref PubMed Scopus (163) Google Scholar). The role of heparin in activating the FGF signaling pathway is unclear. Heparin has been shown to interact with the FGF receptor directly (5Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Crossref PubMed Scopus (476) Google Scholar) and may facilitate the formation of a high-affinity FGF·FGFR complex by bridging the two proteins and binding to each (6Nugent M.A. Edelman E.R. Biochemistry. 1992; 31: 8876-8883Crossref PubMed Scopus (205) Google Scholar). Another model is that heparin may activate the FGFR by binding several FGFs, thereby recruiting two cell-surface FGFRs and promoting transphosphorylation of the FGFR kinase domains (1Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (562) Google Scholar). Heparin has been shown to bind multiple FGF molecules (1Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (562) Google Scholar, 7Thompson L.D. Pantoliano M.W. Springer B.A. Biochemistry. 1994; 33: 3831-3840Crossref PubMed Scopus (279) Google Scholar, 8Mach H. Volkin D.V. Burke C.J. Middaugh C.R. Linhardt R.J. Fromm J.R. Loganathan D. Mattsson L. Biochemistry. 1993; 32: 5480-5489Crossref PubMed Scopus (190) Google Scholar, 9Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Abstract Full Text PDF PubMed Scopus (596) Google Scholar). These studies have shown that one FGF molecule can bind per every 4–5 saccharide units in heparin. Heparin-induced oligomerization of FGF-1 and FGF-2 has been observed by cross-linking studies (1Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (562) Google Scholar, 9Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Abstract Full Text PDF PubMed Scopus (596) Google Scholar, 10Ornitz D.M. Leder P. J. Biol. Chem. 1992; 267: 16305-16311Abstract Full Text PDF PubMed Google Scholar); cross-linking has also confirmed dimerization of FGF-2 by an active nonsulfated oligosaccharide analog (11Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.-M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar). These results support the model that heparin binds multiple FGFs to activate the receptor, but the question remains whether heparin is merely binding multiple FGFs nonspecifically along the long heparin chain, or whether heparin is involved in forming a specific dimer conformation of FGF-2 that is required for activation of the FGFR. Recently, the crystal structures of two biologically active, nonsulfated oligosaccharides bound to FGF-2 were determined (11Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.-M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar). These structures both showed three binding sites (labeled 1, 2, and 2′) for the oligosaccharides. Site 1 corresponded to the well characterized high-affinity heparin-binding site (7Thompson L.D. Pantoliano M.W. Springer B.A. Biochemistry. 1994; 33: 3831-3840Crossref PubMed Scopus (279) Google Scholar, 12Faham S. Hileman R.E. Fromm J.R. Linhardt R.J. Rees D.C. Science. 1996; 271: 1116-1120Crossref PubMed Scopus (740) Google Scholar, 15Zhu X. Hsu B.T. Rees D.C. Structure. 1993; 1: 27-34Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 22Eriksson A.E. Cousens L.S. Matthews B.W. Protein Sci. 1993; 2: 1274-1284Crossref PubMed Scopus (68) Google Scholar). Sites 2 and 2′, previously undescribed, brought together two FGF-2 molecules by simultaneously binding to a single saccharide ligand, suggesting that the biological activity of these small nonsulfated compounds may be due to ligand-mediated dimerization of FGF-2. This hypothesis was supported by a cross-linking experiment showing FGF-2 dimerization in the presence of one of these ligands (11Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.-M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar). Venkataraman and colleagues (13Venkataraman G. Sasisekharan V. Herr A.B. Ornitz D.M. Waksman G. Cooney C.L. Langer R. Sasisekharan R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 845-850Crossref PubMed Scopus (102) Google Scholar) compared this FGF-2/nonsulfated oligosaccharide structure with six crystal structures of apo-FGF-2 and found that all of the structures exhibited identical side by side positioning of the FGF-2 monomers. The authors proposed that apo-FGF-2 has a weak dimerization interface close to the site 2/site 2′ region that promotes formation of a side by side dimer. Such a dimer would allow the binding of a single heparin chain of 8–10 saccharide units (corresponding to what has been reported as the shortest active heparin fragment (1Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (562) Google Scholar, 14Ishihara M. Tyrrell D.J. Stauber G.B. Brown S. Cousens L.S. Stack R.J. J. Biol. Chem. 1993; 268: 4675-4683Abstract Full Text PDF PubMed Google Scholar)) to the high-affinity heparin-binding site (site 1) contributed by each FGF-2 molecule (illustrated in Fig. 4 C). In their modeling studies, Venkataraman et al. (13Venkataraman G. Sasisekharan V. Herr A.B. Ornitz D.M. Waksman G. Cooney C.L. Langer R. Sasisekharan R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 845-850Crossref PubMed Scopus (102) Google Scholar) showed that the binding of a heparin dodecasaccharide to a side by side FGF-2 dimer (with both FGF-2 molecules cis to the heparin ligand) would stabilize the dimer by increasing the protein contact surface area from 761 Å2 in the apo-dimer to 2036 Å2 in the heparin-bound dimer. To better understand the role of heparin and heparin-like compounds in oligomerizing FGF-2, we have used analytical ultracentrifugation to analyze the association state of FGF-2 in the presence of the shortest reported biologically active heparin fragment, an octasaccharide (HS-8) (1Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (562) Google Scholar). For comparison, we also analyzed the association state of FGF-2 in the presence of sucrose octasulfate (SOS), an inactive sulfated disaccharide which binds to FGF-2 (20Arakawa T. Wen J. Philo J.S. J. Protein Chem. 1993; 12: 689-693Crossref PubMed Scopus (11) Google Scholar) but is too short to bridge the high-affinity heparin-binding sites of a putative side by side dimer. FGF-2 was generously provided by J. Abraham (Scios Nova) as a stock solution of 8.3 mg/ml in 20 mm sodium citrate, pH 5.0, 1 mm EDTA, and 9% sucrose. Heparin octasaccharide fragment HS-8 (derived from heparin by nitrous acid depolymerization) in phosphate-buffered saline was a gift from C. Svahn (Pharmacia). The sodium salt of SOS was a gift from Bukh Meditec. Concentration of protein was determined spectrophotometrically ( A2800.1% = 0.964 (16Pantoliano M.W. Horlick R.A. Springer B.A. Van Dyk D.E. Tobery T. Wetmore D.R. Lear J.D. Nahapetian A.T. Bradley J.D. Sisk W.P. Biochemistry. 1994; 33: 10229-10248Crossref PubMed Scopus (229) Google Scholar)). For all titration experiments involving HS-8, FGF-2 was dialyzed extensively against a buffer containing 20 mm sodium citrate, pH 6.9, 1 mm EDTA, 5 mmβ-mercaptoethanol (β-ME), and 140 mm NaCl. The solutions of FGF-2 and HS-8 were then diluted into “sedimentation buffer” containing 20 mm sodium citrate, pH 6.9, 1 mm EDTA, and 5 mm β-ME, to achieve a final NaCl concentration of 20 mm. For the study of the effect of salt, FGF-2 was dialyzed against sedimentation buffer, and NaCl was added to appropriate levels as indicated under “Results.” For titration experiments involving SOS, the stock solution of FGF-2 was initially diluted 100-fold into the sedimentation buffer and reconcentrated to approximately 10 mg/ml by ultrafiltration (Amicon centricon 10). However, because interference with sucrose was observed (see “Results”), later titration and salt effect experiments were carried out with FGF-2 extensively dialyzed in sedimentation buffer. Sedimentation equilibrium experiments were conducted using a Beckman XL-A Optima analytical ultracentrifuge equipped with an absorbance optical system. Runs were carried out at 20,000, 25,000, and 30,000 rpm, at 20 °C. Six-channel, charcoal-filled epon centerpieces with quartz windows were used in a four-hole Ti-60a titanium rotor. Sample volumes in the two inner sample wells of each centerpiece were 100 μl, and the outer well contained 90 μl. Each sample well contained 10 μl (inner two wells) or 30 μl (outer well) of FC43 (Beckman), an inert fluorocarbon oil that displaces the sample solution in the outermost region of the well, thereby maximizing the data that can be collected. The reference wells were filled with 125 μl of the appropriate buffer. Radial scans at 280 nm were collected between 5.9 and 7.2 cm as the average of five measurements, with a step size of 0.001 cm. The samples were allowed to achieve sedimentation equilibrium over the course of 14–20 h, depending on the rotor speed. Samples were considered at equilibrium when sequential scans 2 h apart were superimposable. The partial specific volume of FGF-2 was calculated as 0.734 g/ml based on its amino acid composition, using values from Ref. 17Laue T.L. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. The Royal Society of Chemistry, London1992: 90-125Google Scholar. The contribution of ligand to the partial specific volume of FGF-2 was considered to be negligible, since the partial specific volumes of sugars are similar to proteins (17Laue T.L. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. The Royal Society of Chemistry, London1992: 90-125Google Scholar) and since the mass of FGF-2 is substantially larger than the masses of the ligands used. The buffer density was determined to be 0.995 g/ml, calculated from tables in Ref. 17Laue T.L. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. The Royal Society of Chemistry, London1992: 90-125Google Scholar and the density of β-ME. The raw data files were edited using the program REEDIT (D. Yphantis) and then analyzed by global nonlinear least-squares analysis using the program NONLIN (18Johnson M.L. Correira J.J. Yphantis D.A. Halvorson H.R. Biophys. J. 1981; 36: 575-588Abstract Full Text PDF PubMed Scopus (778) Google Scholar). All data files were first fitted to a single-species model to yield a value for the reduced buoyant molecular weight ς. The reduced molecular weight can be directly determined from the absorption curve, and it is related to the apparent monomer molecular weight (M) by the following equation, ς=∂lncr∂(r2/2)=sω2D=M(1−v¯ρ)ω2RTEquation 1 where c r is the concentration of the macromolecule at radial position r in the cell, sis the apparent sedimentation coefficient, D is the diffusion coefficient, v̄ is the partial specific volume of the macromolecule, ρ is the buffer density, ω2 is the square of the rotor's angular velocity (rpm π/30)2, R is the gas constant in g/mol K, andT is the absolute temperature. The data was then fitted to successively higher order assembly models (monomer-dimer, monomer-dimer-trimer, monomer-dimer-trimer-tetramer, monomer-dimer-tetramer, etc.), according to the equation, Yr=δj+exp[lnAO+ς(r2/2−ro2/2)]+Equation 2 ∑n=2mNexp[lnKN+lnAo+ς(r2/2−ro2/2)]where Y r is the absorbance at any radial point r, δ j is the baseline offset for the jth experiment, A o is the absorbance at reference point r o, K N is the association constant for the assembly reaction in which N monomers (M) form the assembled species M N, and ς is the reduced buoyant molecular weight as described above. Several models were analyzed to determine the simplest model that gave the best fit. A model was deemed to fit the data well when the residuals were small and randomly distributed, the square root of the variance was under 0.01, and the freely varying ς corresponded closely to the actual monomer molecular weight of FGF-2 (ς = 1.868 for apo-FGF-2; M r = 17,100). At high concentration gradients the refractive index of the sample solution may be affected, causing steps in the absorbance curve (19Burz D.S. Ackers G.K. Biochemistry. 1996; 35: 3341-3350Crossref PubMed Scopus (23) Google Scholar). This causes poor fits to the data in the high absorbance range, regardless of the assembly model used. When this occurred, data files exhibiting such steps were slightly truncated to remove the steps in the absorbance curve and allow satisfactory fitting. The final results are reported for fits where ς was fixed at 1.9 (FGF-2/SOS) or 2.0 (FGF-2/HS-8), corresponding to a liganded FGF-2 monomer consistent with the particular model, to maintain consistency. Data files from three different speeds (20,000, 25,000, and 30,000 rpm) were simultaneously fit for each experimental run to better resolve the fitted parameters. This allows for the most accurate determination of association constants. The association constants reported by NONLIN are in absorbance concentration units, and were converted to molar association constants by the following equation, KM=Kabs([M0] n−1/n)(εb) n−1Equation 3 where K m is the molar association constant inm−1, K abs is the absorbance association constant in inverse absorbance units as reported by NONLIN, M o is the monomer molecular weight in g/mol, n is the stoichiometry of association, ε is the extinction coefficient, and b is the path length (1.2 cm for the cells used) (19Burz D.S. Ackers G.K. Biochemistry. 1996; 35: 3341-3350Crossref PubMed Scopus (23) Google Scholar). FGF-2 (585 nm) was cross-linked in the presence of varying amounts of SOS and HS-8. Cross-linking and electrophoresis were done as described in Ref. 11Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.-M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar. Dimer band intensities were quantified by scanning densitometry. The density of each dimer band was corrected by subtracting the density observed in the absence of saccharide; this corrects for nonspecific random cross-linking in the absence of saccharide. Each experiment consisted of a control sample of apo-FGF-2 as well as samples of FGF-2 in the presence of a saccharide ligand. Apo-FGF-2 consistently sedimented as a single species whose molecular weight clearly corresponded to that of a monomer (calculatedM r = 17,100) (data not shown). To determine whether heparin-derived compounds induce FGF-2 self-association in solution, sedimentation experiments with FGF-2 in the presence of HS-8 or sucrose octasulfate were carried out. Sedimentation equilibrium experiments were carried out with FGF-2 and various concentrations of saccharide ligand. The first set of experiments included solutions of 11.9 μm FGF-2 with 5.95, 11.9, 17.9, 23.8, 47.6, 119, or 238 μm HS-8. The data at HS-8 concentrations below 47.6 μm fit best to a monomer-dimer-tetramer association scheme, as shown in Fig.1, A and B. Fits to monomer-dimer-trimer or monomer-dimer-trimer-tetramer assembly models resulted in more non-random residuals and higher square roots of variance. The data for HS-8 concentrations of 47.6 μm or above fit best to a monomer-dimer equilibrium, as shown in Fig.1 B. The highest levels of association were seen at 5.95 μm HS-8; the monomer-dimer dissociation constant at this HS-8 concentration was 4.42 μm (K2 in Fig.4 B), and the dimer-tetramer stepwise dissociation constant was 109 μm (Kx in Fig. 4 B). The dimerization association constants showed a slight decrease with increasing HS-8 concentration, as shown in Fig. 1 B. The tetramerization (monomer-tetramer) association constants K4 also decreased with increasing HS-8 concentration, and tetramerization fell off to undetectable levels at HS-8 concentrations of 47.6 μm or above. The negative slope implies that although HS-8 is required for FGF-2 dimerization under these conditions, higher concentrations of HS-8 interfere with FGF-2 assembly. This is presumably due to saturation of the heparin-binding site on FGF-2, which would shift the equilibrium toward FGF-2 monomers, each bound to a single HS-8 molecule. To compare the action of HS-8 on FGF-2 with that of another sulfated saccharide, sedimentation experiments were also carried out with FGF-2 and SOS. These experiments included solutions of 11.9, 17.9, 23.8, 119, 238, 476, 714, or 952 μm SOS in 11.9, 17.9, 23.8, or 47.6 μm FGF-2. All solutions containing FGF-2 and SOS exhibited an FGF-2 monomer-dimer equilibrium, as shown in Fig.2 A for a representative set of data. However, the data from the FGF-2/SOS experiments showed no clear trend with increasing concentrations of SOS, which we suspected was due to interference from residual sucrose in the FGF-2 storage buffer (sucrose may interfere with binding of SOS, as another nonsulfated oligosaccharide (11Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.-M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar) has been shown to bind to the same site to which SOS binds (15Zhu X. Hsu B.T. Rees D.C. Structure. 1993; 1: 27-34Abstract Full Text PDF PubMed Scopus (84) Google Scholar)). Therefore, in a second set of experiments, solutions were prepared after the FGF-2 had been extensively dialyzed in sedimentation buffer (20 mm sodium citrate, pH 6.9, 1 mm EDTA, 5 mm β-ME), and sedimentation equilibrium experiments were carried out. As before, all the data fit best to a monomer-dimer equilibrium. The highest levels of association were seen at 952 μm SOS; the monomer-dimer dissociation constant at this SOS concentration was 92.91 μm. The dimerization constant increased with increasing SOS in an approximately linear fashion between 119 and 952 μm SOS, as shown in Fig. 2 B. This shows that binding of SOS in this range of concentration is positively linked to FGF-2 dimerization. However, in the lower range (below 119 μm) of SOS concentration studied, although fits to monomer-dimer equilibria were excellent, no clear trend in the value of the association constant was observed (data not shown). Fig. 2 B shows that the data for the sucrose-containing solutions with SOS concentrations equal to or higher than 476 μm (open squares) coincided with the data from the sucrose-free solutions. High concentrations of SOS would appear to compete effectively with sucrose for binding to FGF-2. The observation of a SOS-induced dimer was intriguing and could be interpreted as a mere artifact of the relatively high FGF-2 concentrations used. Therefore, FGF-2 dimerization in the presence of SOS at two concentrations (4 and 12 μm) was monitored by cross-linking, a more sensitive technique. FGF-2 concentration was 585 nm, well below the concentrations used for the sedimentation equilibrium experiments. A control experiment with HS-8 was also performed. Dimerization of FGF-2 was clearly observed in the presence of both SOS and HS-8. Addition of 4.0 or 12 μmSOS reproducibly increased FGF-2 dimerization by 22.2 or 22.3%, respectively, over nonspecific cross-linking (in the absence of saccharide), and addition of equivalent concentrations of HS-8 increased FGF-2 dimerization by 49.9 and 25.5% over nonspecific levels. Results for HS-8 were similar to previously published data for a 16-saccharide heparin fragment (11Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.-M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar). These results verify that both SOS and HS-8 are capable of inducing dimerization of FGF-2 at lower concentrations of FGF-2 than can be reliably observed using most biophysical techniques, and in a different buffer system (22 mm NaPO4, pH 7.4, 150 mm NaCl) than that used in the sedimentation studies. To further characterize this system, sedimentation experiments were also carried out with varying salt concentrations. Solutions of 17.9 μm FGF-2 with 17.9 μm HS-8 were prepared in sedimentation buffer containing increasing concentrations of NaCl (0, 0.05, 0.1, 0.25, 0.5, and 1 m NaCl). Solutions of FGF-2/HS-8 at low salt concentrations (0 and 0.05 m NaCl) showed the monomer-dimer-tetramer equilibrium described above. However, at NaCl concentrations above 50 mm, the equilibrium shifted to a monomer-dimer equilibrium and subsequently to a single monomeric species at 1 m NaCl, as shown in Fig.3 A. Sedimentation experiments were also carried out for FGF-2·SOS complexes; solutions of 17.9 μm FGF-2 and 17.9 μm SOS were prepared in sedimentation buffer containing 0, 0.05, 0.1, 0.25, 0.5, and 1m NaCl. A monomer-dimer equilibrium was observed at NaCl concentrations between 0 and 0.25 m NaCl, but at NaCl concentrations of 0.5 and 1 m NaCl, only a single monomeric species was observed, as shown in Fig. 3 B. The sedimentation equilibrium experiments described above reveal that HS-8 induces a monomer-dimer-tetramer assembly of FGF-2 at low HS-8 concentrations. The proposed self-association process for FGF-2 induced by HS-8 is illustrated in Fig.4 B. The HS-8-induced monomer-dimer assembly of FGF-2 is consistent with the information currently known about FGF-heparin interactions. Calorimetric studies using low M r heparin (M r∼ 3000 or approximately 10 saccharide units) have shown that two FGF-2 molecules bind per heparin molecule (7Thompson L.D. Pantoliano M.W. Springer B.A. Biochemistry. 1994; 33: 3831-3840Crossref PubMed Scopus (279) Google Scholar). Cross-linking studies with a 16-saccharide heparin have also detected FGF-2 dimers and trimers (1Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (562) Google Scholar, 11Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.-M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar). In the study presented here, an active heparin fragment of minimal size (8 saccharide units) was used; yet, this short heparin fragment promotes the formation of FGF-2 dimers with a moderately high association constant. Since the site size for heparin binding is ∼4–5 saccharide units per FGF molecule (1Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (562) Google Scholar,7Thompson L.D. Pantoliano M.W. Springer B.A. Biochemistry. 1994; 33: 3831-3840Crossref PubMed Scopus (279) Google Scholar, 8Mach H. Volkin D.V. Burke C.J. Middaugh C.R. Linhardt R.J. Fromm J.R. Loganathan D. Mattsson L. Biochemistry. 1993; 32: 5480-5489Crossref PubMed Scopus (190) Google Scholar, 9Spivak-Kroizman T. Lemmon M.A. Dikic I. Ladbury J.E. Pinchasi D. Huang J. Jaye M. Crumley G. Schlessinger J. Lax I. Cell. 1994; 79: 1015-1024Abstract Full Text PDF PubMed Scopus (596) Google Scholar), this implies that in a complex with HS-8, FGF-2 molecules are in close proximity. There are three possibilities for the configuration of the FGF-2 molecules bound to HS-8: 1) the FGF-2 molecules are located in trans relative to the heparin; 2) the FGF-2 molecules are bound to neighboring sites on heparin but are not in contact with each other; 3) the FGF-2 molecules are bound to neighboring sites (incis) on heparin and are in contact with each other. Venkataraman et al. (13Venkataraman G. Sasisekharan V. Herr A.B. Ornitz D.M. Waksman G. Cooney C.L. Langer R. Sasisekharan R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 845-850Crossref PubMed Scopus (102) Google Scholar) have argued that the third configuration is the most likely. Indeed, the crystal structures of FGF-2 bound to heparin fragments or to nonsulfated heparan analogs (11Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.-M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar,12Faham S. Hileman R.E. Fromm J.R. Linhardt R.J. Rees D.C. Science. 1996; 271: 1116-1120Crossref PubMed Scopus (740) Google Scholar) suggest that HS-8 could bind across two adjacent FGF-2 monomers, stabilizing an FGF-2 “side by side” dimer cis to the heparin as illustrated in Fig. 4, B and C. Heparin has been reported by both NMR and x-ray diffraction studies to be a helical polysaccharide in solution with a 2-fold screw symmetry between successive disaccharide units (23Mulloy B. Forster M.J. Jones C. Davies D.B. Biochem J. 1993; 293: 848-849Crossref Scopus (369) Google Scholar). As a result, every fourth saccharide residue has approximately the same orientation with a translation of 16.5 to 16.7 Å along the heparin chain. An octasaccharide of heparin therefore consists of two side by side near-identical orientations of a tetrasaccharide, with an overall length of 33.0–33.4 Å. This corresponds closely to the width of the FGF-2 molecule (11Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.-M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar, 13Venkataraman G. Sasisekharan V. Herr A.B. Ornitz D.M. Waksman G. Cooney C.L. Langer R. Sasisekharan R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 845-850Crossref PubMed Scopus (102) Google Scholar) and would be long enough to partially bind to the high-affinity heparin-binding sites on two neighboring FGF-2 monomers, potentially stabilizing an FGF-2 dimer (13Venkataraman G. Sasisekharan V. Herr A.B. Ornitz D.M. Waksman G. Cooney C.L. Langer R. Sasise" @default.
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