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Analysis of Putative Heparin-binding Domains of Fibroblast Growth Factor-1

1995; Elsevier BV; Volume: 270; Issue: 43 Linguagem: Inglês

10.1074/jbc.270.43.25805

1083-351X

Pauline Wong, Brian Hampton, Ewa Szylobryt, Anne Gallagher, Michael Jaye, Wilson H. Burgess,

Peptidase Inhibition and Analysis

The contribution of individual basic amino acids within three putative "consensus sequences" for heparin binding of fibroblast growth factor-1 have been examined by site-directed mutagenesis. The results indicate that a significant reduction in the apparent affinity of fibroblast growth factor-1 for heparin is only observed when basic residues in one of the three regions are mutated. Mutation in the other regions are without affect on heparin binding. The heparin binding properties of synthetic peptides based on the three "consensus sequences" paralleled the mutagenesis results. That is, synthetic peptides corresponding to regions of the protein that were affected by mutagenesis with respect to heparin binding exhibited a relatively high affinity for immobilized heparin, whereas those corresponding to regions of similar charge density that were unaffected by mutagenesis did not. In addition, amino acid substitution of a nonbasic residue in the heparin-binding peptide could abolish its heparin binding capacity. The heparin-binding peptide could antagonize the mitogenic activity of FGF-1, probably because of the heparin dependence of this activity. Together these data demonstrate that the heparin binding properties of fibroblast growth factor-1 are dictated by structural features more complex than clusters of basic amino acids. The results of these and other studies indicate that consensus motifs for heparin-binding require further definition. More importantly, the results provide a basis for the design of peptide-based inhibitors of FGF-1. The contribution of individual basic amino acids within three putative "consensus sequences" for heparin binding of fibroblast growth factor-1 have been examined by site-directed mutagenesis. The results indicate that a significant reduction in the apparent affinity of fibroblast growth factor-1 for heparin is only observed when basic residues in one of the three regions are mutated. Mutation in the other regions are without affect on heparin binding. The heparin binding properties of synthetic peptides based on the three "consensus sequences" paralleled the mutagenesis results. That is, synthetic peptides corresponding to regions of the protein that were affected by mutagenesis with respect to heparin binding exhibited a relatively high affinity for immobilized heparin, whereas those corresponding to regions of similar charge density that were unaffected by mutagenesis did not. In addition, amino acid substitution of a nonbasic residue in the heparin-binding peptide could abolish its heparin binding capacity. The heparin-binding peptide could antagonize the mitogenic activity of FGF-1, probably because of the heparin dependence of this activity. Together these data demonstrate that the heparin binding properties of fibroblast growth factor-1 are dictated by structural features more complex than clusters of basic amino acids. The results of these and other studies indicate that consensus motifs for heparin-binding require further definition. More importantly, the results provide a basis for the design of peptide-based inhibitors of FGF-1. INTRODUCTIONThe fibroblast growth factor (FGF) 1The abbreviations used are: FGFfibroblast growth factorHSPGheparan sulfate proteoglycansHPLChigh performance liquid chromatographyFmocN-(9-fluorenyl)methoxycarbonyl. family consists of at least nine structurally related proteins(1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar, 2Burgess W.H. Winkles J.A. Pusztai L. Lewis C.E. Yap E. Regulation of the Proliferation of Neoplastic Cells. Oxford University Press, Oxford, United Kingdom1995: 155-218Google Scholar, 3Miyamoto M. Naruo K.-I. Seko C. Matsumoto S. Kondo T. Kurokawa T. Mol. Cell. Biol. 1993; 13: 4251-4259Crossref PubMed Scopus (392) Google Scholar). Two of these proteins, FGF-1 and FGF-2, have been characterized under many different names, most often as acidic FGF and basic FGF, respectively. Although there is a large amount of overlap in the spectrum of biological activities and receptor-binding properties of the FGFs, the only known function shared by all members of the family is a relatively high affinity for heparin or heparan sulfate proteoglycans (HSPGs). It has been established that heparin can potentiate the mitogenic activity of FGF-1 (4Burgess W.H. Shaheen A.M. Ravera M. Jaye M. Donohue P.J. Winkles J.A. J. Cell Biol. 1990; 111: 2129-2138Crossref PubMed Scopus (91) Google Scholar, 5Mueller S.N. Thomas K.A. Di Salvo J. Levine E.M. J. Cell. Physiol. 1989; 140: 439-448Crossref PubMed Scopus (71) Google Scholar, 6Damon D.H. Lobb R.R. Damore P.A. Wagner J.A. J. Cell. Physiol. 1989; 138: 221-226Crossref PubMed Scopus (142) Google Scholar) and protect both FGF-1 and FGF-2 from proteolytic and heat inactivation (7Rosengart T.K. Johnson W.V. Friesel R. Clark R. Maciag T. Biochem. Biophys. Res. Commun. 1988; 152: 432-440Crossref PubMed Scopus (130) Google Scholar, 8Lobb R.R. Biochemistry. 1988; 27: 2572-2578Crossref PubMed Scopus (62) Google Scholar, 9Gospodarowicz D. Cheng J. J. Cell. Physiol. 1986; 128: 475-484Crossref PubMed Scopus (680) Google Scholar). In addition, heparin increases the apparent affinity of FGF-1 for high affinity FGF receptors(10Schreiber A.B. Kenney J. Kowalski W.J. Friesel R. Mehlman T. Maciag T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6138-6142Crossref PubMed Scopus (232) Google Scholar, 11Kaplow J.M. Bellot F. Crumley G. Dionne C.A. Jaye M. Biochem. Biophys. Res. Commun. 1990; 172: 107-112Crossref PubMed Scopus (29) Google Scholar). Recently, an obligatory role for heparin or HSPGs in mediating the binding of FGF-1 or FGF-2 to the high affinity, tyrosine kinase receptors has been suggested(12Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2073) Google Scholar, 13Rapraeger A.C. Krufka A. Olwin B.B. Science. 1991; 252: 1705-1708Crossref PubMed Scopus (1285) Google Scholar, 14Klagsbrun M. Baird A. Cell. 1991; 67: 229-231Abstract Full Text PDF PubMed Scopus (497) Google Scholar, 15Partanen J. Vainikka S. Korhonen J. Armstrong E. Alitalo K. Prog. Growth Factor. Res. 1992; 4: 69-83Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 16Ornitz D.M. Yayon A. Flanagan J.G. Svahn C.M. Levi E. Leder P. Mol. Cell. Biol. 1992; 12: 240-247Crossref PubMed Scopus (558) Google Scholar, 17Spivak-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 (590) Google Scholar). It has also been reported that cell surface HSPGs are capable of binding and internalizing FGF-2(18Moscatelli D. Flaumenhaft R. Saksela O. Ann. N. Y. Acad. Sci. 1991; 638: 177-181Crossref PubMed Scopus (20) Google Scholar). A direct role of HSPG-bound FGF in mediating the various functions of this growth factor family has not been established.We reported previously that a change of lysine 132 in FGF-1 to a glutamic acid (K132E) by site-directed mutagenesis reduced the apparent affinity of the recombinant protein for heparin(4Burgess W.H. Shaheen A.M. Ravera M. Jaye M. Donohue P.J. Winkles J.A. J. Cell Biol. 1990; 111: 2129-2138Crossref PubMed Scopus (91) Google Scholar). The K132E mutant is fully capable of binding to and activating the high-affinity tyrosine kinase FGF receptors and can induce transcription of a variety of immediate-early genes(19Burgess W.H. Shaheen A.M. Hampton B. Donohue P.J. Winkles J.A. J. Cell. Biochem. 1991; 45: 131-138Crossref PubMed Scopus (36) Google Scholar). This mutant is, however, an extremely poor mitogen for all cells tested(4Burgess W.H. Shaheen A.M. Ravera M. Jaye M. Donohue P.J. Winkles J.A. J. Cell Biol. 1990; 111: 2129-2138Crossref PubMed Scopus (91) Google Scholar). Together these results indicate that modulation of the heparin or HSPG binding properties of the FGFs could result in the development of specific agonists or antagonists of their functions. For example, FGF-1 is highly dependent on the presence of exogenous heparin for its mitogenic activity(19Burgess W.H. Shaheen A.M. Hampton B. Donohue P.J. Winkles J.A. J. Cell. Biochem. 1991; 45: 131-138Crossref PubMed Scopus (36) Google Scholar). In contrast, exogenous heparin is not required for FGF-1-induced mesoderm formation in Xenopus animal cap assays(20Slack J.M.W. Isaacs H.V. Darlington B.G. Development. 1988; 103: 581-590PubMed Google Scholar).To date there are no reports on the identification of a heparin-binding domain of any member of the FGF family using direct assays. We reported previously that a peptide corresponding to residues 49-71 of human FGF-1 could compete with full-length FGF-1 for binding of 125I-fluorescein-heparin(21Mehlman T. Burgess W.H. Anal. Biochem. 1990; 188: 159-163Crossref PubMed Scopus (20) Google Scholar). These studies suffered from the use of modified heparin in an indirect assay; furthermore, we were not able to demonstrate direct binding of this peptide to immobilized heparin. Baird et al.(22Baird A. Schubert D. Ling N. Guillemin R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2324-2328Crossref PubMed Scopus (279) Google Scholar) were able to demonstrate binding of [3H]heparin to certain peptides derived from the sequence of FGF-2 after the peptides were baked onto nitrocellulose filters. Two regions of heparin binding activity were identified corresponding to residues 32-76 and 101-128 of the human FGF-2 sequence.Several consensus sequences of heparin-binding regions in a variety of proteins have been proposed(23Cardin A.D. Weintraub H.J.R. Atherosclerosis. 1989; 9: 21-32Google Scholar, 24Jackson R.L. Busch S.J. Cardin A.D. Physiol. Rev. 1991; 71: 481-539Crossref PubMed Scopus (955) Google Scholar). These include the motifs XBBXBX and XBBBXXBX, where B is a basic amino acid and X is a hydropathic residue. Analysis of the primary sequence of FGF-1 revealed three regions that are in good agreement with the proposed consensus sequences(23Cardin A.D. Weintraub H.J.R. Atherosclerosis. 1989; 9: 21-32Google Scholar). These include residues 22-27, 113-120, and 124-131. Lysine 132 falls just outside of the latter sequence, yet its modification reduces significantly the apparent affinity of FGF-1 for immobilized heparin(19Burgess W.H. Shaheen A.M. Hampton B. Donohue P.J. Winkles J.A. J. Cell. Biochem. 1991; 45: 131-138Crossref PubMed Scopus (36) Google Scholar, 25Harper J.W. Lobb R.R. Biochemistry. 1988; 27: 671-678Crossref PubMed Scopus (58) Google Scholar). A basic residue in this position following the XBBBXXBX consensus is not common among known heparin-binding proteins(23Cardin A.D. Weintraub H.J.R. Atherosclerosis. 1989; 9: 21-32Google Scholar, 24Jackson R.L. Busch S.J. Cardin A.D. Physiol. Rev. 1991; 71: 481-539Crossref PubMed Scopus (955) Google Scholar). Whereas the existence of multiple heparin-binding domains centered around clusters of basic amino acids seems consistent with the fact that heparin is able to protect the majority of the FGF-1 protein from digestion with trypsin(7Rosengart T.K. Johnson W.V. Friesel R. Clark R. Maciag T. Biochem. Biophys. Res. Commun. 1988; 152: 432-440Crossref PubMed Scopus (130) Google Scholar), it does not appear to be consistent with the dramatic reduction in heparin affinity exhibited by the lysine 132 mutant(19Burgess W.H. Shaheen A.M. Hampton B. Donohue P.J. Winkles J.A. J. Cell. Biochem. 1991; 45: 131-138Crossref PubMed Scopus (36) Google Scholar). Margalit et al.(26Margalit H. Fischer N. Ben-Sasson S.A. J. Biol. Chem. 1993; 268: 19228-19231Abstract Full Text PDF PubMed Google Scholar) reported a more stringent approach to the analysis of heparin-binding domains concentrating on sequences of heparin-binding proteins with established three-dimensional structures. They concluded that basic residues in human FGF-1 corresponding to positions 126 and 133 of the full-length sequence satisfied the spatial requirements of basic amino acids at opposite ends of a β-strand fold in their model. This result implicates residues 126 and 133 as crucial to heparin binding. It should be noted, however, that residue 133 in bovine and chicken FGF-1 is occupied by leucine(27Burgess W.H. Friesel R. Winkles J.A. Mol. Reprod. Dev. 1994; 39: 56-61Crossref PubMed Scopus (15) Google Scholar).We examined the role of additional basic amino acids in these three putative heparin binding domains by site-directed mutagenesis. The apparent affinities of the FGF-1 mutants were compared with that of wild-type protein by affinity-based chromatography. Synthetic peptides corresponding to regions of the wild-type and mutant sequences were synthesized, and their apparent affinities for heparin were determined. The results of these studies indicate that 1) a specific peptide with relatively high apparent affinity for heparin can be identified within the sequence of FGF-1 and 2) the role of clusters of basic amino acid residues in heparin binding is more subtle than predicted by the consensus sequence models. The results are consistent with predictions based on the crystal structure of FGF-1 (28Zhu X. Komiya H. Chirino A. Faham S. Fox G.M. Arakawa T. Hsu B.T. Rees D.C. Science. 1991; 251: 90-93Crossref PubMed Scopus (329) Google Scholar) and suggest a mechanism by which heparin protects the protein from degradation by trypsin. In addition, the identification of a heparin-binding domain in FGF-1 can serve as a basis for the development of peptide based antagonists of its function.EXPERIMENTAL PROCEDURESMaterialsHeparin-Sepharose, the pKK233 expression vector, and low molecular weight protein markers were purchased from Pharmacia Biotech Inc. All reagents for polyacrylamide gel electrophoresis and the Mighty Small Electrophoresis and transfer apparati were from Hoefer Scientific Instruments (San Francisco, CA). The heparin Econo cartridges (5 ml) were purchased from Bio-Rad. Reagents for reversed-phase HPLC, amino acid sequencing and peptide synthesis were from Applied Biosystems Inc. (Foster City, CA). The rabbit polyclonal FGF-1-specific antibody was provided by R. Friesel (Holland Laboratory, Rockville, MD). Isotopes and the in vitro mutagenesis system were from Amersham Corp. Chloramine T and sodium metabisulfate were from Sigma. Bovine serum albumin and endoproteinases Asp-N, Lys-C, and Glu-C were from Boehringer Mannheim. Reagents for amino acid analysis were from Waters Associates (Medford, MA). Eagle's minimal essential medium, Dulbecco's modified Eagle's medium, calf serum, pen-strep, L-glutamine, Ham's F-12 media, and dialyzed fetal bovine serum were from Biofluids (Rockville, MD). G418 sulfate was purchased from Life Technologies, Inc. Transferrin was from Intergen (Purchase, NY). Human epidermal growth factor was obtained from UBI (Lake Placid, NY), and selenium was from Sigma. Balb MK cells were provided by Dr. J. Rubin (National Cancer Institute, Bethesda, MD). Heparin (6.15 μg/unit) was purchased from Upjohn (Kalamazoo, MI). [3H]Thymidine and Na125I were from Amersham Corp. Other chemicals were reagent grade.Construction of Wild-type and Mutant FGF-1 Prokaryotic Expression PlasmidsThe plasmids expressing wild-type or mutant human FGF-1 were constructed exactly as described previously(19Burgess W.H. Shaheen A.M. Hampton B. Donohue P.J. Winkles J.A. J. Cell. Biochem. 1991; 45: 131-138Crossref PubMed Scopus (36) Google Scholar). The plasmid expressing wild-type bovine FGF-1 was constructed as follows. First, a bovine FGF-1 cDNA fragment was isolated using the reverse transcriptase polymerase chain reaction technique. RNA was isolated from bovine heart tissue (Pel Freeze, Roger, AR) using RNAzol (Tel Test, Inc., Friendswood, TX) according to the manufacturer's instructions. One μg of RNA was converted into cDNA as described previously(29Winkles J.A. Gay C.G. Cell Growth & Differ. 1991; 2: 531-540PubMed Google Scholar). An aliquot was used for the polymerase chain reaction. These reactions were performed using human FGF-1 sense and antisense primers as described previously(29Winkles J.A. Gay C.G. Cell Growth & Differ. 1991; 2: 531-540PubMed Google Scholar). Samples were subjected to 35 cycles of amplification using a Perkin-Elmer 9600 thermocycler. Each cycle included denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and primer extension at 72°C for 30 s.An aliquot of the amplification mixture was subjected to agarose gel electrophoresis, and the 489-base pair bovine FGF-1 DNA fragment was excised and purified by Geneclean (Bio 101, La Jolla, CA). BamHI and NdeI restriction sites were introduced at the 5′ end, and a BamHI site at the 3′ end of the cDNA fragment using polymerase chain reaction with the following primers: 5′-ACCTGGGATCCCATATGAATTACAAGAAG-3′ (sense), 5′-CAACAGGGATCCTTAATCAGAGGAGAC-3′ (antisense).The resulting fragment was digested with BamHI, separated by agarose gel electrophoresis, excised from the gel, purified by Geneclean, and subcloned into the BamHI site of pBluescript sk+ (Stratagene, La Jolla, CA). After ligation and transformation, 10 colonies were isolated. Plasmid DNA from each colony was purified, and the sequence of the cDNA inserts was obtained by the Sanger dideoxy sequencing method using a Sequenase version 2.0 sequencing kit (U. S. Biochemical Co.). The bovine FGF-1 cDNA was then subcloned into the expression vector pET3c(30Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5987) Google Scholar). This construct was used to transform BL21(DE3)pLysS Escherichia coli cells(30Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5987) Google Scholar). Production and purification of the recombinant proteins was performed as described previously(31Jaye M. Burgess W.H. Shaw A.B. Drohan W.N. J. Biol. Chem. 1987; 262: 16612-16617Abstract Full Text PDF PubMed Google Scholar).Heparin-binding Properties of Wild-type and Mutant FGF-1sCultures of the E. coli strain JM103 bearing recombinant plasmids were grown at 37°C in Luria broth containing 100 μg/ml ampicillin. A fresh overnight culture was diluted and grown until the absorbance reached 0.2 at 550 nm. Isopropylthiol β galactoside was then added to 1 mM, and the cultures were incubated at 37°C for an additional 2 h. Cell pellets were collected by centrifugation and frozen at −80°C. Frozen pellets from 2 liters of culture were resuspended in 50 ml of 50 mM Tris, 10 mM EDTA, 50 mM glucose, pH 7.4. Egg lysozyme was added to 10 μg/ml. The cells were incubated at 4°C for 45 min and then sonicated at maximum intensity for 30 s using the large probe of a Heat System 380 sonicator. Lysates were clarified by centrifugation at 6,000 × g for 30 min at 4°C. The supernatants were diluted to 100 ml with 50 mM sodium phosphate, pH 7.5 (buffer A) and applied to a Bio-Rad heparin cartridge using a Waters Associates HPLC system. Samples were eluted with a linear gradient of buffer A and buffer A containing 1.2 M NaCl. Flow was 1 ml/min, and 1-min fractions were collected.Western Blot AnalysisAliquots of the fractions eluted from the heparin cartridge were subjected to electrophoresis using the SDS-polyacrylamide gel electrophoresis system of Laemmli(32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar). A 15% acrylamide, 0.4% N,N'-methylenebisacrylamide solution was polymerized in a Hoefer Mini-gel apparatus, and electrophoresis was carried out at 200 V. Proteins were transferred from the gel to nitrocellulose, and FGF-1 containing fractions were identified using rabbit polyclonal FGF-1-specific antibodies and 125I-protein A as described previously(19Burgess W.H. Shaheen A.M. Hampton B. Donohue P.J. Winkles J.A. J. Cell. Biochem. 1991; 45: 131-138Crossref PubMed Scopus (36) Google Scholar).Peptide Synthesis and CharacterizationPeptides were synthesized using an Applied Biosystems (Foster City, CA) model 431A peptide synthesizer and small scale Fmoc cycles supplied by the manufacturer. Peptides were purified by reversed-phase HPLC, and the ratio of their absorbance at 280 and 215 nm was monitored to evaluate removal of side chain protection groups. Purified peptides were analyzed further by amino acid analysis using a Waters Associates Pico Tag system and amino acid sequencing using an Applied Biosystems model 477A protein sequencer with on-line phenylthiohydantoin derivative analyzer as described previously(33Hampton B.S. Marshak D.R. Burgess W.H. Mol. Biol. Cell. 1992; 3: 85-93Crossref PubMed Scopus (87) Google Scholar). Aliquots of each peptide were dried in a Savant Speed Vac resuspended in water, mixed with buffer A, and applied to a heparin column. The relative affinities of the synthetic peptides for immobilized heparin were determined as described above for recombinant FGFs using UV absorption to monitor the eluants. The peptides corresponding to residues 122-137 with substitutions at residue 131 were made using a cartridge containing a mixture of 10 different Fmoc amino acids at the appropriate position in each of two syntheses.Mitogenic AssaysBalb MK cells were grown to ˜80% confluence at 37°C in 48-well plates containing Eagle's minimal essential medium, 10% dialyzed fetal bovine serum, and 5 ng/ml epidermal growth factor. The cells were serum starved in a 1:1 mix of Ham's F-12 and Eagle's minimal essential medium containing 5 μg/ml transferin and 3 × 10-8M selenium for 72 h. Growth factors were added directly to the starvation media. After 20 h, the cells were pulsed with [3H]thymidine (1 μCi/ml). The cells were harvested 4 h later, and [3H]thymidine incorporation into DNA was measured as described previously(19Burgess W.H. Shaheen A.M. Hampton B. Donohue P.J. Winkles J.A. J. Cell. Biochem. 1991; 45: 131-138Crossref PubMed Scopus (36) Google Scholar). NIH 3T3 cells were grown to ˜80% confluence at 37°C in 48-well plates containing Dulbecco's modified Eagle's medium, 10% calf serum, penicillin (100 units/ml), streptomycin (100 μg/ml), and L-glutamine (2 mM). The cells were starved for 24 h in the same media containing 0.5% calf serum. Stimulation of DNA synthesis was assayed as described above.Receptor Binding AssayNIH 3T3 cells overexpressing FGF receptor-1 (flg) were prepared as described previously(11Kaplow J.M. Bellot F. Crumley G. Dionne C.A. Jaye M. Biochem. Biophys. Res. Commun. 1990; 172: 107-112Crossref PubMed Scopus (29) Google Scholar). Cells were grown to ˜80% confluence at 37°C in Dulbecco's modified Eagle's medium, 10% calf serum, penicillin (100 units/ml), streptomycin (100 μg/ml), L-glutamine (2 mM), and 500 μg/ml G418. Cells were starved in the same media except the serum was reduced to 0.5% and G418 was omitted. FGF-1 was iodinated using chloramine T. Labeled protein was isolated by heparin-Sepharose chromatography. Cells were incubated with 125I-FGF-1 and increasing concentrations of unlabeled wild-type or mutant proteins for 90 min at 4°C in binding buffer (Dulbecco's modified Eagle's medium, 25 mM Hepes, pH 7.4, 0.5% bovine serum albumin, and 5 units/ml heparin). Cells were washed 3 times with binding buffer and solubilized in 0.5 N NaOH, and bound FGF was quantitated by γ counting.RESULTS AND DISCUSSIONThe apparent affinities of wild-type and various mutants of FGF-1 for immobilized heparin were examined by chromatography using defined NaCl gradient elution and Western blot analysis of the eluted fractions. Lysine residues corresponding to positions in putative consensus sequences for heparin binding (23Cardin A.D. Weintraub H.J.R. Atherosclerosis. 1989; 9: 21-32Google Scholar, 24Jackson R.L. Busch S.J. Cardin A.D. Physiol. Rev. 1991; 71: 481-539Crossref PubMed Scopus (955) Google Scholar) were changed to glycine residues by site-directed mutagenesis (Fig. 1). The range of mutants covered every dibasic cluster in FGF-1 as well as other residues predicted to play a role in heparin binding(23Cardin A.D. Weintraub H.J.R. Atherosclerosis. 1989; 9: 21-32Google Scholar, 24Jackson R.L. Busch S.J. Cardin A.D. Physiol. Rev. 1991; 71: 481-539Crossref PubMed Scopus (955) Google Scholar). We reported previously that a change of lysine 132 to a glutamic acid residue significantly reduced the apparent affinity of FGF-1 for immobilized heparin(19Burgess W.H. Shaheen A.M. Hampton B. Donohue P.J. Winkles J.A. J. Cell. Biochem. 1991; 45: 131-138Crossref PubMed Scopus (36) Google Scholar). A similar reduction in affinity is observed when lysine 132 is changed to a glycine residue (Fig. 2). Of the eight lysines replaced in the present study, only a change of lysine 127 resulted in a similar reduction in heparin affinity (Fig. 2). A change in the adjacent lysine 126 to a glycine residue does not produce a large reduction in heparin affinity, and changes in lysines 114 or 115 results in a small reduction in heparin affinity. Changes of lysines 23, 24, or 26 to glycine residues has no effect on the heparin-binding activity of FGF-1 (Fig. 2). Together these results indicate that of the three putative heparin-binding domains of FGF-1 predicted by consensus sequence analysis(23Cardin A.D. Weintraub H.J.R. Atherosclerosis. 1989; 9: 21-32Google Scholar, 24Jackson R.L. Busch S.J. Cardin A.D. Physiol. Rev. 1991; 71: 481-539Crossref PubMed Scopus (955) Google Scholar), changes of the lysine residues in one region (residues 22-27) have no effect on heparin binding, whereas changes in another (residues 113-120) result in a minor reduction in heparin-binding, and whereas changes in a third (residues 124-132) can have significant and varying effects on heparin-binding. We cannot rule out the possibility that the drastic loss of heparin affinity exhibited by the residue 127 and residue 132 mutations is not due to an alteration in the folding or stability of the mutant protein. Both mutants are, however, able to compete equally with wild-type FGF-1 for binding to cells overexpressing FGF receptor-1 (Fig. 3). The binding was done in the presence of exogenous heparin to inhibit binding to cell surface HSPGs.Figure 2Heparin-binding properties of wild-type and sitedirected point mutants of human FGF-1. Recombinant wild-type and mutant proteins were generated as described under "Experimental Procedures." Lysates of E. coli producing the various FGF-1 forms were subjected to heparin affinity-based chromatography using a linear gradient of 0-1.2 M NaCl. Eluted fractions were examined for FGF-1 immunoreactivity by Western blot analysis. Autoradiograms of selected fractions for the indicated proteins (wild-type or mutant) are shown along with the range of NaCl concentrations sufficient for FGF-1 elution.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Ability of wild-type and mutant FGF-1 to compete with 125I-labeled wild-type protein for binding to NIH 3T3 cells overexpressing human FGF receptor-1 (flg). Binding was performed as described under "Experimental Procedures." The ability of unlabeled wild-type (□), unlabeled 132 mutant (○), or unlabeled 127 mutant FGF-1 to displace labeled wild-type protein is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We examined the heparin-binding properties of these putative heparin-binding domain sequences further using synthetic peptides. The site-directed mutagenesis studies described above indicated that a change of lysines 126, 127, or 132 resulted in the largest reduction in apparent affinity of FGF-1 for heparin. A peptide corresponding to residues 122-137 of the human sequence (peptide 1 in Fig. 1) was synthesized, and its apparent affinity for immobilized heparin was examined. As shown in Fig. 4, this peptide exhibited a reasonably high apparent affinity for heparin (elution at ˜0.3 M NaCl). In contrast, peptide 2 (residues 15-29), which exhibits a similar distribution of basic residues as peptide 1, did not bind to the heparin column at all (data not shown). This result was consistent with the site-directed mutagenesis that showed substitution of lysines 23, 24, or 26 was without affect on the heparin affinity of FGF-1. Furthermore, mutation of lysines 114 or 115 was shown to have a slight affect on the affinity of FGF-1 heparin, and extension of peptide 1 to include these residues (peptide 3, Fig. 1) resulted in a small increase in the apparent affinity of the synthetic peptide for heparin (Fig. 4). The peak of absorbance eluting at ˜0.7 M NaCl is likely to represent a disulfide-linked dimer of peptide 3 as judged by the fact that we were not able to derivatize its cysteine residue without prior reduction. In contrast, extension of peptide 1 by the same amount in the C-terminal direction did not increase the apparent affinity of the peptide for heparin (data not shown). Together, these data demonstrate that a relatively short linear sequence within the primary structure of human FGF-1 that has a relatively high affinity for heparin can be identified. Furthermore, the studies with synthetic peptides overall correlate well with site-directed mutagenesis experiments.Figure 4UV absorbance profiles of heparin affinity-based chromatography of various synthetic peptides corresponding to regions of FGF-1 sequences. The chromatography conditions were identical to those described in Fig. 2. Panel A shows the elution profile of a synthetic peptide corresponding to residues 122-137 of human FGF-1. Panel B shows the elution profile of a synthetic peptide corresponding to residues 114-137 of human FGF-1.View Large Image