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• W1967737567 abstract "The interaction of basic fibroblast growth factor (bFGF) with heparan sulfate (HS)/heparin has been shown to strongly enhance the activity of the growth factor although the mechanism of activation is unclear. We have addressed the issue of the minimal stoichiometry of an active HS oligosaccharide·bFGF complex by chemically cross-linking the two components to form novel covalent conjugates. The cross-linking procedure produced both monomeric and dimeric bFGF·oligosaccharide complexes, which were purified to homogeneity. Dimer conjugates were shown to have been formed as a result of disulfide bridging of monomer conjugates. These monomer conjugates were subsequently found to be biologically active in a mitogenesis assay. We therefore conclude that a monomeric bFGF·oligosaccharide complex is the minimal functional unit required for mitogenic stimulation. The interaction of basic fibroblast growth factor (bFGF) with heparan sulfate (HS)/heparin has been shown to strongly enhance the activity of the growth factor although the mechanism of activation is unclear. We have addressed the issue of the minimal stoichiometry of an active HS oligosaccharide·bFGF complex by chemically cross-linking the two components to form novel covalent conjugates. The cross-linking procedure produced both monomeric and dimeric bFGF·oligosaccharide complexes, which were purified to homogeneity. Dimer conjugates were shown to have been formed as a result of disulfide bridging of monomer conjugates. These monomer conjugates were subsequently found to be biologically active in a mitogenesis assay. We therefore conclude that a monomeric bFGF·oligosaccharide complex is the minimal functional unit required for mitogenic stimulation. Basic fibroblast growth factor (bFGF) 1The abbreviation used is: bFGF, basic fibroblast growth factor; CL, cross-linked; dp, degree of polymerization (i.e. disaccharide = dp2); EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; FGFR, fibroblast growth factor receptor; GlcNSO3, N-sulfated glucosamine; HS, heparan sulfate; IdceA(2S), iduronic acid 2-sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; S-NHS, N-hydroxysulfosuccinimide MES, 4-morpholineethanesulfonic acid.1The abbreviation used is: bFGF, basic fibroblast growth factor; CL, cross-linked; dp, degree of polymerization (i.e. disaccharide = dp2); EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; FGFR, fibroblast growth factor receptor; GlcNSO3, N-sulfated glucosamine; HS, heparan sulfate; IdceA(2S), iduronic acid 2-sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; S-NHS, N-hydroxysulfosuccinimide MES, 4-morpholineethanesulfonic acid. is one of a family of at least 18 polypeptides (reviewed in Ref. 1Coulier F. Pontarotti P. Roubin R. Hartung H. Goldfarb M. Birnbaum D. J. Mol. Evol. 1997; 44: 43-56Crossref PubMed Scopus (185) Google Scholar; also see Refs. 2McWhirter J.R. Goulding M. Weiner J.A. Chun J. Murre C. Development. 1997; 124: 3221-3232Crossref PubMed Google Scholar, 3Miyake A. Konishi M. Martin F.H. Hernday N.A. Ozaki K. Yamamoto S. Mikami T. Arakawa T. Itoh N. Biochem. Biophys. Res. Commun. 1998; 243: 148-152Crossref PubMed Scopus (135) Google Scholar, 4Hoshikawa M. Ohbayashi N. Yonamine A. Konishi M. Ozaki K. Fukui S. Itoh N. Biochem. Biophys. Res. Commun. 1998; 244: 187-191Crossref PubMed Scopus (89) Google Scholar, 5Ohbayashi N. Hoshikawa M. Kimura S. Yamasaki M. Fukui S. Itoh N. J. Biol. Chem. 1998; 273: 18161-18164Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), and has been shown to influence a variety of cellular processes such as proliferation, migration, and differentiation (6Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar, 7Baird A. Klagsbrun M. Cancer Cells. 1991; 3: 239-243PubMed Google Scholar, 8Mason I.J. Cell. 1994; 78: 547-552Abstract Full Text PDF PubMed Scopus (525) Google Scholar). It has been implicated in a number of disease states, including tumor growth, rheumatoid arthritis, and diabetic retinopathy, due to its ability to stimulate angiogenesis (9Folkman J. Klagsbrun M. Science. 1987; 235: 442-447Crossref PubMed Scopus (4037) Google Scholar, 10Moses M.A. Langer R. Bio/Technology. 1991; 9: 630-634Crossref PubMed Scopus (84) Google Scholar). This family of growth factors act primarily through high affinity tyrosine kinase receptors (FGFRs) (11Givol D. Yayon A. FASEB J. 1992; 6: 3362-3369Crossref PubMed Scopus (398) Google Scholar), although in addition their activity is modulated by lower affinity heparan sulfate (HS) proteoglycan receptors (12Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1290) Google Scholar, 13Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2081) Google Scholar, 14Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (559) Google Scholar). However, the mechanism by which this occurs is far from understood. The FGFR family comprises four related molecules, which each contain a highly conserved tyrosine kinase domain (15Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Crossref PubMed Scopus (1175) Google Scholar), further diversity being provided by the existence of alternatively spliced isoforms (15Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Crossref PubMed Scopus (1175) Google Scholar, 16Werner S. Duan D.S. de Vries C. Peters K.G. Johnson D.E. Williams L.T. Mol. Cell. Biol. 1992; 12: 82-88Crossref PubMed Scopus (289) Google Scholar, 17Avivi A. Yayon A. Givol D. FEBS Lett. 1993; 330: 249-252Crossref PubMed Scopus (91) Google Scholar). Intracellular signaling is believed to be initiated by receptor dimerization and receptor transphosphorylation (18Bellot F. Crumley G. Kaplow J.M. Schlessinger J. Jaye M. Dionne C.A. EMBO J. 1991; 10: 2849-2854Crossref PubMed Scopus (163) Google Scholar, 19Ueno H. Gunn M. Dell K. Tseng Jr., A. Williams L. J. Biol. Chem. 1992; 267: 1470-1476Abstract Full Text PDF PubMed Google Scholar); however, signaling without receptor dimerization cannot be ruled out, as it has not yet been shown whether FGFRs are activated in their monomeric forms. Indeed it has been suggested that bFGF activates multiple signaling pathways by utilizing either monomeric FGFRs or FGFR dimers/multimers (20Krufka A. Guimond S. Rapraeger A.C. Biochemistry. 1996; 35: 11131-11141Crossref PubMed Scopus (59) Google Scholar). Several HS binding sites on bFGF have been identified with the major site comprising residues Asn-27, Lys-26, Lys-125, Lys-135, and Arg-120 (21Eriksson A.E. Cousens L.S. Matthews B.W. Protein Sci. 1993; 2: 1274-1284Crossref PubMed Scopus (68) Google Scholar, 22Thompson L.D. Pantoliano M.W. Springer B.A. Biochemistry. 1994; 33: 3831-3840Crossref PubMed Scopus (277) Google Scholar, 23Faham S. Hileman R.E. Fromm J.R. Linhardt R.J. Rees D.C. Science. 1996; 271: 1116-1120Crossref PubMed Scopus (736) Google Scholar). A second possible site was also identified using synthetic di- and trisaccharides and x-ray crystallography (24Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar). A HS binding site has also been identified on the FGFR and was found to be located between the receptors Ig loops I and II (25Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Crossref PubMed Scopus (474) Google Scholar). The primary high affinity FGFR binding domain has also been identified on bFGF and was found to consist of three hydrophobic amino acids (Tyr-103, Leu-140, and Met-142) and two polar amino acids (Arg-44 and Asn-101) (26Pantoliano 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, 27Springer B.A. Pantoliano M.W. Barbera F.A. Gunyuzlu P.L. Thompson L.D. Herblin W.F. Rosenfeld S.A. Book G.W. J. Biol. Chem. 1994; 269: 26879-26884Abstract Full Text PDF PubMed Google Scholar). A secondary low affinity FGFR binding site was also identified (on bFGF) and comprises amino acids Lys-110, Tyr-111, and Trp-144 (26Pantoliano 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, 27Springer B.A. Pantoliano M.W. Barbera F.A. Gunyuzlu P.L. Thompson L.D. Herblin W.F. Rosenfeld S.A. Book G.W. J. Biol. Chem. 1994; 269: 26879-26884Abstract Full Text PDF PubMed Google Scholar, 28Baird A. Schubert D. Ling N. Guillemin R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2324-2328Crossref PubMed Scopus (280) Google Scholar). Various groups have identified sequences within HS/heparin that interact strongly with bFGF (29Turnbull J.E. Fernig D.G. Ke Y. Wilkinson M.C. Gallagher J.T. J. Biol. Chem. 1992; 267: 10337-10341Abstract Full Text PDF PubMed Google Scholar, 30Habuchi H. Suzuki S. Saito T. Tamura T. Harada T. Yoshida K. Kimata K. Biochem. J. 1992; 285: 805-813Crossref PubMed Scopus (163) Google Scholar, 31Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar, 32Ishihara 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, 33Maccarana M. Casu B. Lindahl U. J. Biol. Chem. 1993; 268: 23898-23905Abstract Full Text PDF PubMed Google Scholar, 34Pye D.A. Kumar S. Biochem. Biophys. Res. Commun. 1998; 248: 889-895Crossref PubMed Scopus (13) Google Scholar). These oligosaccharides were found to be enriched in IdceA(2S)α1,4GlcNSO3(± 6S) disaccharides, with their affinity for bFGF increasing with 2-O-sulfate content. In addition, no role was established for 6-O-sulfation in the interaction with the primary bFGF binding site (23Faham S. Hileman R.E. Fromm J.R. Linhardt R.J. Rees D.C. Science. 1996; 271: 1116-1120Crossref PubMed Scopus (736) Google Scholar). The minimum length of saccharide required to activate bFGF in mitogenic assays has been variously reported as ranging from di- to dodecasaccharide (14Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (559) Google Scholar, 24Ornitz D.M. Herr A.B. Nilsson M. Westman J. Svahn C.M. Waksman G. Science. 1995; 268: 432-436Crossref PubMed Scopus (270) Google Scholar, 31Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar, 32Ishihara 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,35Gambarini A.G. Miyamoto C.A. Lima G.A. Nader H.B. Dietrich C.P. Mol. Cell. Biochem. 1993; 124: 121-129Crossref PubMed Scopus (44) Google Scholar, 36Ishihara M. Shaklee P.N. Yang Z. Liang W. Wei Z. Stack R.J. Holme K. Glycobiology. 1994; 4: 451-458Crossref PubMed Scopus (91) Google Scholar, 37Walker A. Turnbull J.E. Gallagher J.T. J. Biol. Chem. 1994; 269: 931-935Abstract Full Text PDF PubMed Google Scholar). The investigations described above have led to a number of models being proposed by which HS·bFGF·FGFR interact to bring about a biological response. These include the simultaneous binding of two bFGF molecules to a HS oligosaccharide sequence within the HS chain so as to present bFGF dimers to the FGFRs, thereby enabling two high affinity FGFRs to be brought into close proximity to one another for receptor dimerization and transphosphorylation (14Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (559) Google Scholar, 22Thompson L.D. Pantoliano M.W. Springer B.A. Biochemistry. 1994; 33: 3831-3840Crossref PubMed Scopus (277) Google Scholar, 29Turnbull J.E. Fernig D.G. Ke Y. Wilkinson M.C. Gallagher J.T. J. Biol. Chem. 1992; 267: 10337-10341Abstract Full Text PDF PubMed Google Scholar, 38Mach H. Volkin D.B. 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, 39Miao H.Q. Ishai-Michaeli R. Atzmon R. Peretz T. Vlodavsky I. J. Biol. Chem. 1996; 271: 4879-4886Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). An alternative HS·bFGF monomer model has been suggested, in which HS acts as a bridge by simultaneously binding single molecules of bFGF and FGFR to form a signaling complex (25Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Crossref PubMed Scopus (474) Google Scholar, 31Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar). Another HS·bFGF monomeric model has also been proposed, in which a monomeric complex of HS and bFGF facilitates receptor dimerization through two FGFR-binding interfaces on the growth factor (26Pantoliano 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). Other models have also been proposed, with complexes as large as bFGF tetramers being implicated as an active complex (40Moy F.J. Safran M. Seddon A.P. Kitchen D. Bohlen P. Aviezer D. Yayon A. Powers R. Biochemistry. 1997; 36: 4782-4791Crossref PubMed Scopus (104) Google Scholar), as in cytokines such as interferon γ and platelet factor 4 (41Mayo K.H. Chen M.J. Biochemistry. 1989; 28: 9469-9478Crossref PubMed Scopus (85) Google Scholar, 42Lortat-Jacob H. Turnbull J.E. Grimaud J.A. Biochem. J. 1995; 310: 497-505Crossref PubMed Scopus (119) Google Scholar). However, conditions used to observe these complexes differ considerably from the component ratios found to be active in cell assay systems. One of the major issues still to be resolved in the bFGF mitogenic signaling mechanism is the exact nature of the signaling complex and, in particular, the stoichiometry of the HS·bFGF complex. In this study, we have produced novel covalently cross-linked conjugates of bFGF and HS oligosaccharides, which no longer bind any further HS but are fully active biologically. These conjugates have enabled us to determine the minimal bFGF·HS oligosaccharide stoichiometry required for initiating mitogenic activity. Horse serum and RPMI medium were obtained from Life Technologies, Inc. (Biocult, Paisley, United Kingdom (UK)). BaF3 (clone F32) and WEHI 3b cells were supplied by the Klagsbrun Laboratory (Boston, MA). Human recombinant bFGF was supplied by R&D Systems (Abingdon, Oxon, UK). Porcine mucosal HS was obtained from Organon (Oss, The Netherlands). Heparinase III (Flavobacterium heparinum; EC 4.2.2.8) was obtained from Grampian Enzymes (Orkney, UK). EDC and S-NHS were supplied by Pierce (Chester, UK). Bovine serum albumin, MES, Tween 20, and rabbit anti-human bFGF antibody were supplied by Sigma-Aldrich (Poole, Dorset, UK). Horseradish peroxidase-conjugated swine anti-rabbit IgG antibody was supplied by Dako (Cambridge, UK). ECL reagents were obtained from Amersham Pharmacia Biotech (Little Chalfont, Bucks, UK). [methyl 3H]Thymidine was supplied by NEN Life Science Products (Hounslow, UK). Superose 12 columns were purchased from Amersham Pharmacia Biotech (St. Albans, Herts, UK). Bio-Gel P6 was supplied by Bio-Rad (Hemel Hempstead, Herts, UK). Microscint O was obtained from Packard (Pangbourne, Berks, UK). All other reagents were obtained from BDH-Merk Ltd (Lutterworth, Leics, UK) and were of AnalaR grade. 100 mg of porcine mucosal HS in 0.5 ml of heparinase buffer (100 mmsodium acetate, 0.1 mm calcium acetate, pH 7.0), was incubated initially with 0.25 IU of heparinase III followed by two further additions after 24 and 48 h. The digest was monitored by absorbance at 232 nm until no further increase occurred. Oligosaccharides produced by heparinase III digestion were then resolved using a Bio-Gel P6 column (1.5 × 170 cm) in 0.5m NH4HCO3 at a flow rate of 6 ml/h. Fractions of 1 ml were collected and oligosaccharides detected by monitoring the absorbance at 232 nm. Size-defined oligosaccharides dp2–16 were pooled, freeze-dried, and stored at −80 °C until required. Cross-linking was carried out essentially as described previously by Grabarek and Gergely (43Grabarek Z. Gergely J. Anal. Biochem. 1990; 185: 131-135Crossref PubMed Scopus (724) Google Scholar); however, conditions were chosen so as to optimize yields of cross-linked products. Briefly, for preparative reactions, dp12 oligosaccharides in coupling buffer (0.1 m NaCl, 0.1m MES, pH 6.0) were incubated for 15 min at 25 °C with 6 mm EDC and 15 mm S-NHS. The reaction was terminated by addition of β-mercaptoethanol to a final concentration of 20 mm. Activated oligosaccharides were then added to a 50 μm solution of bFGF in coupling buffer, to give a bFGF:dp12 molar ratio of 1:4 and incubated for 2 h at 25 °C. Products were analyzed by standard SDS-PAGE (12% gel) and Western blotting, followed by enhanced chemiluminescence (ECL) immunodetection prior to purification. Western blots of SDS-PAGE gels on nitrocellulose membranes were blocked overnight with 1% (w/v) bovine serum albumin in PBS and then washed three times with PBS, 0.05% (v/v) Tween 20. The membrane was then incubated with a 1:200 dilution of rabbit anti human bFGF monoclonal antibody for 2 h at 4 °C. The antibody solution was then removed and the membrane washed three times with PBS, 0.05% (v/v) Tween 20. The membrane was then further incubated with a 1:1000 dilution of horseradish peroxidase-conjugated swine anti-rabbit IgG for 1 h. The membrane was then finally washed eight times in succession with PBS, 0.05% (v/v) Tween 20 and the presence of bFGF visualized by ECL (Amersham Pharmacia Biotech) following the manufacturer's protocol. Cross-linked samples were applied to two coupled Superose 12 columns linked to a Gilson HPLC system, equilibrated in 50 mm phosphate buffer containing 2.0 m NaCl, pH 7.4. Samples were eluted at a flow rate of 0.5 ml/min, fractions (250 μl) were collected, and elution profile monitored by absorbance at 280 nm. Fractions containing protein were desalted by microdialysis (M r cut-off 12,000) against 5 mm Tris-HCl, pH 7.4, at a flow rate of 1.5 ml/min overnight at 4 °C. Desalted samples were analyzed for purity by SDS-PAGE, followed by Western blotting and ECL immunodetection. Fractions containing cross-linked products were pooled and reapplied to the columns and the procedure repeated as above. Finally, purified components were lyophilized and stored at −80 °C until required. BaF3 cells transfected with FGFR1 (designated F32 cells) (14Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (559) Google Scholar) were routinely maintained in RPMI 1640 medium, 10% horse serum supplemented with interleukin-3 conditioned medium (prepared from WEHI 3b cells) at 37 °C, 5% CO2. For the assay system, F32 cells were plated into 96-well plates at a density of 50,000 cells/well in 100 μl of RPMI 1640 medium, 10% horse serum supplemented with cross-linked conjugates. Cells were incubated for 46 h before addition of [3H]thymidine (0.3 μCi/well) for another 2 h. Incorporation of [3H]thymidine was stopped by harvesting cells on a Filtermate-196 cell harvester. Cells were allowed to air-dry before addition of 25 μl of Microscint O to each well, and incorporated radioactivity counted on a top count system. The assay was performed using a membrane filtration apparatus (Millipore, Watford, Herts, UK). Briefly, 4 μg of bFGF or bFGF·HS complex was applied to the filter in binding buffer (10 mm Tris-HCl, pH 7.3). Filters were then washed with 10 ml of 2.0 m NaCl to remove any non-cross-linked oligosaccharide that may have been present, and then re-equilibrated by washing with binding buffer. Metabolically labeled endothelial cell [3H]HS, prepared as described previously (44Pye D.A. Kumar S. Biochim. Biophys. Acta. 1995; 1266: 235-244Crossref PubMed Scopus (8) Google Scholar), was applied in 5 ml of binding buffer and cycled through the filter three times. Filters were then washed twice with 5 ml of binding buffer to remove unbound [3H]HS. Bound [3H]HS was then removed from the growth factor by washing, first with three 5-ml aliquots of 0.3 m NaCl, followed by further washing with three 5-ml aliquots of 2.0m NaCl in binding buffer. Fractions (5 ml) were collected and eluted material quantified by scintillation counting. In order to produce potentially active HS oligosaccharide·bFGF conjugates, we have employed a specific method of zero length cross-linking. As no spacer is introduced using this method, cross-linking should only occur between the oligosaccharide and amino acid side chains within the actual HS binding site of bFGF. The cross-linking process is a two-step procedure, initially involving a brief incubation of HS oligosaccharide with EDC in the presence of NHS. This results in the conversion of some of the oligosaccharide carboxyls into succinimide esters. This oligosaccharide activation reaction is then terminated by neutralizing the EDC with β-mercaptoethanol prior to addition of growth factor. Cross-linking arises from the reaction of the succinimide esters with the lysine ε-amino groups of the growth factor. A two-step process has the advantage that only the oligosaccharide comes into contact with the active cross-linking reagents; hence, only oligosaccharide protein cross-links are formed. This eliminates possible complications arising from protein-protein cross-linking, which could perturb further interaction of the conjugates with FGFRs. Preliminary experiments were carried out using the zero length cross-linking procedure, in order to assess the type of conjugates formed and to optimize the yield of products. Oligosaccharides of six disaccharides in length (dp12) were chosen, as these have previously been shown to be the minimum size saccharide fragments that have maximal bFGF stimulating activity, when compared with intact chains (31Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar, 37Walker A. Turnbull J.E. Gallagher J.T. J. Biol. Chem. 1994; 269: 931-935Abstract Full Text PDF PubMed Google Scholar). Fig. 1A shows an SDS-PAGE and ECL immunodetection analysis of native bFGF (approximate mass of 18 kDa) and the products obtained by cross-linking of dp12 oligosaccharides to bFGF. It can be seen that the products of cross-linking include both dimer (CL-dp12·bFGF dimer) and monomer (CL-dp12·bFGF monomer) conjugates of oligosaccharide and growth factor. Increasing the ratio of oligosaccharide to growth factor resulted in higher yields of both CL-dp12·bFGF monomers and CL-dp12·bFGF dimers, with the most striking increase seen with the CL-dp12·bFGF monomers. However, the majority of the growth factor remained in the native form. This is most likely due to a combination of the half-life of the active oligosaccharide esters being only 1–2 h, and the slow off-rate of the oligosaccharide when bound to the growth factor. Control experiments in the absence of cross-linking reagents showed no conjugate bands and no increase in disulfide-bonded bFGF dimers (Fig. 1A,lanes 1 and 2). In order to optimize yields of CL-dp12·bFGF dimers and CL-dp12·bFGF monomers within a single reaction, the ratio of bFGF:dp12 oligosaccharide was varied from 4:1 to 1:32 (results not shown). Additionally, the ratio of EDC to S-NHS has been shown to effect coupling efficiency (43Grabarek Z. Gergely J. Anal. Biochem. 1990; 185: 131-135Crossref PubMed Scopus (724) Google Scholar); in light of this, the ratio of EDC:S-NHS was also varied (results not shown). As a result of these experiments, optimal cross-linking conditions for production of both monomer and dimer conjugates were chosen to be a 1:4 ratio of bFGF:dp12 and EDC/S-NHS concentrations of 6/15 mm,respectively. A typical distribution of reaction products is shown in Fig. 1B. The lack of cross-linked product when denatured bFGF was used (results not shown) ruled out the possibility of the process being the result of nonspecific random collision of active oligosaccharide-succinimide esters and primary amines on the growth factor. Products of the cross-linking reaction were separated by gel-filtration chromatography using two Superose 12 columns connected in series, as described under “Experimental Procedures,” and a typical elution profile is shown in Fig. 2A. This clearly shows the conjugates (56–61 min) eluting prior to the free oligosaccharide (62–65 min as identified previously by absorbance at 232 nm), and much earlier than the native growth factor (results not shown) (73–76 min for native bFGF disulfide dimer and 77–80 min for the native bFGF monomer). This early elution of bFGF conjugates (just prior to free dp12 oligosaccharide) may be due to the rigid extended nature of the oligosaccharide dominating the conjugate's hydrodynamic shape. In previous work (results not shown), we have noticed that, due to the strength of the binding of oligosaccharide to bFGF, non-cross-linked complexes of bFGF and oligosaccharide also elute at a position just prior to the free oligosaccharide peak. This indicates that the cross-linked conjugates resemble at least in size/shape non-cross-linked dp12·bFGF oligosaccharide complexes. Indeed examination of the fractions by SDS-PAGE and ECL immunodetection, showed some contamination of the conjugates by both native bFGF disulfide dimer and monomer (Fig. 2B) as a result of the strong native bFGF·dp12 interaction described above. In order to finally remove all traces of free growth factor from the samples, fractions containing CL-dp12·bFGF dimers and CL-dp12·bFGF monomers were pooled separately and reapplied to the Superose 12 column in the presence of 2 m NaCl. This resulted in the recovery of conjugates with no free growth factor (by SDS-PAGE) or oligosaccharide contamination (as detected by absorbance at 232 nm results not shown). Fig. 3A shows a typical Superose 12 elution profile for the CL-dp12·bFGF monomer conjugate, with SDS-PAGE and ECL immunodetection analysis (Fig. 3B) confirming the absence of native growth factor. Purification of the CL-dp12·bFGF dimer conjugates was found to require several additional cycles of gel filtration in order to obtain an homogeneous preparation.Figure 3Purification of CL-dp12·bFGF monomer conjugates. A, typical gel-permeation elution profile of the CL-dp12·bFGF monomer conjugates when reapplied to two Superose 12 columns attached in series and run as described in Fig. 2.B, SDS-PAGE and ECL immunodetection analysis as described under “Experimental Procedures” of CL-dp12·bFGF monomer containing fractions.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A number of possible structures were envisaged for the CL-dp12·bFGF dimer conjugates. These were: 1) individual cross-linking of two separate bFGF molecules to a single oligosaccharide, as may expected in the case of the dimer activation model which has been previously proposed (14Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (559) Google Scholar, 22Thompson L.D. Pantoliano M.W. Springer B.A. Biochemistry. 1994; 33: 3831-3840Crossref PubMed Scopus (277) Google Scholar, 29Turnbull J.E. Fernig D.G. Ke Y. Wilkinson M.C. Gallagher J.T. J. Biol. Chem. 1992; 267: 10337-10341Abstract Full Text PDF PubMed Google Scholar, 38Mach H. Volkin D.B. 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, 39Miao H.Q. Ishai-Michaeli R. Atzmon R. Peretz T. Vlodavsky I. J. Biol. Chem. 1996; 271: 4879-4886Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar); 2) disulfide-bridged dimers comprising one CL-dp12·bFGF monomer conjugate, and a molecule of native bFGF; and 3) disulfide-bridged dimers of two CL-dp12·bFGF monomers. To clarify this, we ran non-reduced and reduced samples of the CL-dp12·bFGF dimer conjugate on SDS-PAGE (Fig. 4). This clearly shows that the dimer product consists of disulfide-bridged CL-dp12·bFGF monomers. Initial experiments also showed that small amounts of hybrid disulfide dimers were formed, which degraded to CL-dp12·bFGF monomers and unmodified bFGF monomers on reduction. However, more extensively purified dimer preparations such as that used in Fig. 4 were devoid of these hybrids. The fact that no true cross-linked dimer (two bFGFs to one dp12 oligosaccharide) is formed provides evidence that the binding of two bFGF molecules to a single oligosaccharide via identical growth factor binding sites is not an accurate model for the activation of signal transduction by HS·bFGF. As a result of this experiment, only the CL-dp12·bFGF monomer conjugate remained as a potential minimal mitogenic activation complex. The BaF3 cells are a lymphoblastoid line that do not express FGFRs and fail to respond to bFGF. These cells were transfected with FGFR1 to produce the F32 cell clone. The F32 cells, which are devoid of functional cell surface heparan sulfate proteoglycans, will respond to bFGF only in the presence of exogenous heparin or HS (14Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (559) Google Scholar" @default.
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• W1967737567 title "Monomer Complexes of Basic Fibroblast Growth Factor and Heparan Sulfate Oligosaccharides Are the Minimal Functional Unit for Cell Activation" @default.
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