©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Analysis of Putative Heparin-binding Domains of Fibroblast Growth Factor-1
USING SITE-DIRECTED MUTAGENESIS AND PEPTIDE ANALOGUES (*)

(Received for publication, July 24, 1995)

Pauline Wong Brian Hampton Ewa Szylobryt Anne M. Gallagher Michael Jaye (1) Wilson H. Burgess (§)

From the Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855 and Rhône-Poulenc Rorer Central Research, Collegeville, Pennsylvania 19426

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The fibroblast growth factor (FGF) (^1)family consists of at least nine structurally related proteins(1, 2, 3) . 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 (4, 5, 6) and protect both FGF-1 and FGF-2 from proteolytic and heat inactivation (7, 8, 9) . In addition, heparin increases the apparent affinity of FGF-1 for high affinity FGF receptors(10, 11) . 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(12, 13, 14, 15, 16, 17) . It has also been reported that cell surface HSPGs are capable of binding and internalizing FGF-2(18) . 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(4) . 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(19) . This mutant is, however, an extremely poor mitogen for all cells tested(4) . 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(19) . In contrast, exogenous heparin is not required for FGF-1-induced mesoderm formation in Xenopus animal cap assays(20) .

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 I-fluorescein-heparin(21) . 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.(22) 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(23, 24) . 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(23) . 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(19, 25) . A basic residue in this position following the XBBBXXBX consensus is not common among known heparin-binding proteins(23, 24) . 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(7) , it does not appear to be consistent with the dramatic reduction in heparin affinity exhibited by the lysine 132 mutant(19) . Margalit et al.(26) 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 beta-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(27) .

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 (28) 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 PROCEDURES

Materials

Heparin-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 NaI were from Amersham Corp. Other chemicals were reagent grade.

Construction of Wild-type and Mutant FGF-1 Prokaryotic Expression Plasmids

The plasmids expressing wild-type or mutant human FGF-1 were constructed exactly as described previously(19) . 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(29) . An aliquot was used for the polymerase chain reaction. These reactions were performed using human FGF-1 sense and antisense primers as described previously(29) . 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(30) . This construct was used to transform BL21(DE3)pLysS Escherichia coli cells(30) . Production and purification of the recombinant proteins was performed as described previously(31) .

Heparin-binding Properties of Wild-type and Mutant FGF-1s

Cultures 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 beta 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 times 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 Analysis

Aliquots of the fractions eluted from the heparin cartridge were subjected to electrophoresis using the SDS-polyacrylamide gel electrophoresis system of Laemmli(32) . 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 I-protein A as described previously(19) .

Peptide Synthesis and Characterization

Peptides 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(33) . 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 Assays

Balb 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 times 10M 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(19) . 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 Assay

NIH 3T3 cells overexpressing FGF receptor-1 (flg) were prepared as described previously(11) . 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 I-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 counting.


RESULTS AND DISCUSSION

The 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 (23, 24) 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(23, 24) . 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(19) . 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(23, 24) , 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 1: Comparison of the amino acid sequences of human and bovine FGF-1. The complete (154-residue) amino acid sequence of human FGF-1 is shown. Those residues in the bovine sequence that differ from the human sequence are indicated. Ac refers to the acetylation of the amino-terminal alanine residue in both proteins. The lysine residues subjected to site-directed mutagenesis are boxed. Regions of the primary sequence studied using synthetic peptides are indicated by solid lines. Amino acid residues are identified using the single-letter code.




Figure 2: Heparin-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.




Figure 3: Ability of wild-type and mutant FGF-1 to compete with I-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 (box), unlabeled 132 mutant (circle), or unlabeled 127 mutant FGF-1 to displace labeled wild-type protein is shown.



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 4: UV 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.



In order to eliminate the complication of peptide dimerization mediated by the cysteine residue corresponding to position 131, we synthesized the peptide corresponding to residues 122-137 with position 131 occupied by a glycine. There was no detectable heparin binding activity associated with the peptide. We examined the structural requirements of position 131 to support heparin binding by synthesizing two mixed peptides, each containing 10 different amino acids at position 131 and testing their heparin binding. The results are shown in Fig. 5. The fractions corresponding to the peaks of heparin binding activity were subjected to amino acid sequence analysis. We were able to establish that peptides containing a histidine, tryptophan, methionine, cysteine, lysine, or arginine at position 131 were able to bind immobilized heparin. The remaining peptides with the exception of serine 131 were identified in the flow-through of the run. We synthesized a single peptide with serine at position 131 and observed binding similar to the Cys, Lys, and Arg-containing peptides. It is of interest that no peptide (even those containing Lys or Arg) exhibited higher apparent heparin affinity then the parent (Cys) peptide. We could not determine the identity of the amino acid at position 131 in the peak labeled X. This could represent an artifact of peptide synthesis or cleavage (i.e. failure to remove blocking group or other chemical modification). Together these results demonstrate an important role for nonbasic positions in mediating the heparin binding activity of this peptide. A role for other residues has not been established.


Figure 5: UV absorbance profiles of heparin affinity-based chromatography of two mixtures of synthetic peptides containing the 20 possible variants of residue 131 in FGF-1. The chromatography conditions were identical to those described in Fig. 2and Fig. 4. Panel A shows the elution profile of synthetic peptides corresponding to residues 122-137 of human FGF-1 with the exception that position 131 was occupied by either a Gly, Ala, Val, Leu, Pro, Met, Trp, His, Ser, or Asp residue. Panel B is the same with the exception that position 131 was occupied by a Lys, Arg, Cys, Glu, Phe, Ile, Asn, Gln, Thr, or Tyr residue. The peptides that bound heparin were identified by amino acid sequence analysis, and the elution positions are noted in the figure by the single-letter code for the amino acid identified at position 131.



As shown in Fig. 6, the apparent heparin affinity of intact bovine FGF-1 is slightly lower than that of the human protein. A peptide corresponding to residues 122-137 (peptide 1 in Fig. 1) was synthesized based on the bovine sequence, and this peptide exhibited a similar reduction in heparin affinity compared with the human peptide (Fig. 6). This reduction in apparent affinity of the bovine peptide is not due to a difference in the number of basic residues in the bovine sequence. This result indicates that the heparin binding properties of synthetic peptides may serve some predictive value in the design of mutants of the parent protein with altered heparin binding activity.


Figure 6: UV absorbance profiles of heparin affinity-based chromatography of recombinant human and bovine FGF-1 and synthetic peptides based on their respective sequences. The elution profiles of recombinant human (panel A), bovine (panel B), and synthetic peptides corresponding to residues 122-137 of the human (panel C) and bovine (panel D) sequences are shown.



Human FGF-1 is highly dependent on the presence of exogenous heparin for optimal biological activity(19) . We examined the ability of the peptide 122-137 to inhibit the mitogenic activity of FGF-1 using Balb MK cells. Assays were performed in the presence of 0.3 unit/ml heparin. As can be seen in Fig. 7, addition of the highest concentration of peptide to the standard growth media (epidermal growth factor and serum) had no effect on thymidine incorporation. In contrast, the peptide was able to inhibit quantitatively the FGF-1-induced DNA synthesis. This observation is consistent with the heparin-binding activity of the peptide and with the fact that the mitogenic activity of FGF-1 for these cells is absolutely dependent on the presence of exogenous heparin. In contrast, FGF-1 is able to induce DNA synthesis in NIH 3T3 cells in the absence of exogenous heparin. Heparin does, however, potentiate the activity as much as 10-fold. The effects of increasing concentrations of the synthetic peptide on FGF-induced DNA synthesis in these cells is shown in Fig. 8. It can be seen that there is no effect of the peptide on the activity of FGF-1 in the absence of exogenous heparin. In contrast, the peptide can inhibit the heparin potentiation of FGF-1 activity, although the inhibition can be overcome with increasing concentrations of heparin. Together these results indicate that the mechanism of inhibition of the peptide is simple competition with FGF-1 for the biologically competent heparin fraction. In addition, they demonstrate that there is no direct effect of the synthetic peptide on FGF-1 (i.e. altered stability, accelerated proteolysis, etc.).


Figure 7: Effect of the heparin-binding peptide on DNA synthesis in BALB-MK cells. Cells were serum starved and then treated with growth media (GM) in the presence of 100 µg of peptide, 0.3 ng/ml FGF-1 with the indicated amounts of synthetic peptide or left untreated. [^3H]Thymidine incorporation into DNA was measured as described in the text. The values are the mean of triplicate assays and the standard derivations are indicated by the error bars (not detectable for three of the treatments).




Figure 8: Effect of the heparin-binding peptide on DNA synthesis in NIH 3T3 cells. Cells were serum starved and then treated with the indicated concentrations of FGF-1, heparin, and synthetic peptides. [^3H]Thymidine incorporation into DNA was measured (as described in the text). The values are the mean of triplicate assays.



The subtle (i.e. synthetic peptides based on bovine versus human sequence) differences and the drastic (i.e. synthetic peptides with cysteine 131 replaced by a glycine or other residues) difference in the heparin-binding activity of these peptides provides further evidence that heparin-binding domains cannot be identified based simply on the distribution of clusters of basic amino acids. Overall, our results are similar to those obtained from site-directed mutagenesis of the heparin-binding protein lipoprotein lipase(34) . In those studies, basic residues in three homologous ``consensus'' sequences for heparin-binding were altered by site-directed mutagenesis to alanine residues. Alterations in two of the three consensus sequences were shown to affect heparin-binding. In addition, only certain basic residues within the two heparin-binding domains appear important to heparin-binding. That is, a change of certain basic residues that affect the consensus had little effect on the actual heparin-binding. The studies presented here demonstrate that point mutations of basic residues in one of the regions of FGF-1 that fit the consensus sequence are without affect on heparin affinity, those in another consensus sequence have minimal effect on heparin affinity, and those in a third consensus sequence have varying effects on the heparin binding properties of the protein.

Examination of the three-dimensional structure of FGF-1 (28) reveals that the amino-terminal residues that fit the consensus sequence are not well oriented and extend from the core structure. The fact that mutation of these basic residues did not affect heparin binding is consistent with their lack of ``structure'' and with the findings of Imamura et al.(35) , who demonstrated that these residues could be deleted from FGF-1 without altering significantly the apparent heparin affinity of the truncated protein. More importantly, our results are, for the most part, consistent with the studies of Zhu et al.(36) , who reported the solution of a crystal structure of bovine FGF-1 and sucrose octasulfate in a 1:1 complex. Sucrose octasulfate is thought to bind to similar sites on FGFs as heparin and other proteoglycans(17, 37, 38) . They demonstrated that sucrose octasulfate binds to the region of FGF-1 composed of residues 126-141 and that the primary contacts were with lysine 126, arginine 130, lysine 132, and arginine 136. Our data support the notion that residues 126 and 132 are important for heparin binding. Residue 130 is a serine in the human sequence, and we have not examined directly the role of arginine 136. This residue is included in the heparin-binding peptide described above.

The majority of basic residues in FGF-1 are banded like an equator around the spherical FGF-1 structure. Our data are consistent with a model whereby residues in the region of amino acids 122-137 form a primary recognition site for heparin, which allows the heparin molecule to subsequently interact with additional basic residues that contribute affinity. Such a model would also explain the ability of heparin to protect FGF-1 from trypsin digestion in that the extended heparin molecule could establish contact with all or most of the basic residues found in the protein. However, formal proof of this model will require solution of the three-dimensional structure of the FGF-1bulletheparin complex.

In summary, the results presented here support the conclusions of Hata et al.(34) , that whereas the concept of consensus sequences for heparin-binding may serve some predictive value, the predictions require experimental verification. They do not support the spatial distribution analysis of Margalit et al.(26) . It should be noted that this study was limited to computer modeling and did not provide experimental verification. In addition, our findings demonstrate that within a verified heparin-binding domain that fits one of the consensus sequences, the contributions of the specific basic residues to heparin affinity are not equal and that the nature of the nonbasic residues can be extremely important. Finally, we have shown that a short sequence from FGF-1 retains a relatively high apparent affinity for immobilized heparin (when compared with heparin-binding domains of other proteins) and that this peptide is able to inhibit the heparin-dependent mitogenic activity of FGF-1. The correlations established between the relative apparent affinities of different heparin-binding peptides and the relative apparent affinities of the intact proteins containing these sequences indicate that synthetic peptide analogues may be useful in the design of FGF-1 mutants with altered and predictable heparin affinities.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL35762. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0654; Fax: 301-738-0465.

(^1)
The abbreviations used are: FGF, fibroblast growth factor; HSPG, heparan sulfate proteoglycans; HPLC, high performance liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl.


ACKNOWLEDGEMENTS

We thank Thomas Maciag and Jeffrey Winkles for critical comments, Cathryn Wawzinski for help in the preparation of the manuscript, and Mark Ravera for help in the generation of FGF-1 mutants.


REFERENCES

  1. Burgess, W. H., and Maciag, T. (1989) Annu. Rev. Biochem. 58, 575-606 [CrossRef][Medline] [Order article via Infotrieve]
  2. Burgess, W. H., and Winkles, J. A. (1995) in Regulation of the Proliferation of Neoplastic Cells (Pusztai, L., Lewis, C. E., and Yap, E. eds) pp. 155-218, Oxford University Press, Oxford, United Kingdom
  3. Miyamoto, M., Naruo, K.-I., Seko, C., Matsumoto, S., Kondo, T., and Kurokawa, T. (1993) Mol. Cell. Biol. 13, 4251-4259 [Abstract]
  4. Burgess, W. H., Shaheen, A. M., Ravera, M., Jaye, M., Donohue, P. J., and Winkles, J. A. (1990) J. Cell Biol. 111, 2129-2138 [Abstract]
  5. Mueller, S. N., Thomas, K. A., Di Salvo, J., and Levine, E. M. (1989) J. Cell. Physiol. 140, 439-448 [Medline] [Order article via Infotrieve]
  6. Damon, D. H., Lobb, R. R., Damore, P. A., and Wagner, J. A. (1989) J. Cell. Physiol. 138, 221-226 [Medline] [Order article via Infotrieve]
  7. Rosengart, T. K., Johnson, W. V., Friesel, R., Clark, R., and Maciag, T. (1988) Biochem. Biophys. Res. Commun. 152, 432-440 [Medline] [Order article via Infotrieve]
  8. Lobb, R. R. (1988) Biochemistry 27, 2572-2578 [Medline] [Order article via Infotrieve]
  9. Gospodarowicz, D., and Cheng, J. (1986) J. Cell. Physiol. 128, 475-484 [Medline] [Order article via Infotrieve]
  10. Schreiber, A. B., Kenney, J., Kowalski, W. J., Friesel, R., Mehlman, T., and Maciag, T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6138-6142 [Abstract]
  11. Kaplow, J. M., Bellot, F., Crumley, G., Dionne, C. A., and Jaye, M. (1990) Biochem. Biophys. Res. Commun. 172, 107-112 [Medline] [Order article via Infotrieve]
  12. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991) Cell 64, 841-848 [Medline] [Order article via Infotrieve]
  13. Rapraeger, A. C., Krufka, A., and Olwin, B. B. (1991) Science 252, 1705-1708 [Medline] [Order article via Infotrieve]
  14. Klagsbrun, M., and Baird, A. (1991) Cell 67, 229-231 [Medline] [Order article via Infotrieve]
  15. Partanen, J., Vainikka, S., Korhonen, J., Armstrong, E., and Alitalo, K. (1992) Prog. Growth Factor. Res. 4, 69-83 [Medline] [Order article via Infotrieve]
  16. Ornitz, D. M., Yayon, A., Flanagan, J. G., Svahn, C. M., Levi, E., and Leder, P. (1992) Mol. Cell. Biol. 12, 240-247 [Abstract]
  17. Spivak-Kroizman, T., Lemmon, M. A., Dikic, I., Ladbury, J. E., Pinchasi, D., Huang, J., Jaye, M., Crumley, G., Schlessinger, J., and Lax, I. (1994) Cell 79, 1015-1024 [Medline] [Order article via Infotrieve]
  18. Moscatelli, D., Flaumenhaft, R., and Saksela, O. (1991) Ann. N. Y. Acad. Sci. 638, 177-181 [Medline] [Order article via Infotrieve]
  19. Burgess, W. H., Shaheen, A. M., Hampton, B., Donohue, P. J., and Winkles, J. A. (1991) J. Cell. Biochem. 45, 131-138 [Medline] [Order article via Infotrieve]
  20. Slack, J. M. W., Isaacs, H. V., and Darlington, B. G. (1988) Development 103, 581-590 [Abstract]
  21. Mehlman, T., and Burgess, W. H. (1990) Anal. Biochem. 188, 159-163 [Medline] [Order article via Infotrieve]
  22. Baird, A., Schubert, D., Ling, N., and Guillemin, R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2324-2328 [Abstract]
  23. Cardin, A. D., and Weintraub, H. J. R. (1989) Atherosclerosis 9, 21-32
  24. Jackson, R. L., Busch, S. J., and Cardin, A. D. (1991) Physiol. Rev. 71, 481-539 [Free Full Text]
  25. Harper, J. W., and Lobb, R. R. (1988) Biochemistry 27, 671-678 [Medline] [Order article via Infotrieve]
  26. Margalit, H., Fischer, N., and Ben-Sasson, S. A. (1993) J. Biol. Chem. 268, 19228-19231 [Abstract/Free Full Text]
  27. Burgess, W. H., Friesel, R., and Winkles, J. A. (1994) Mol. Reprod. Dev. 39, 56-61 [Medline] [Order article via Infotrieve]
  28. Zhu, X., Komiya, H., Chirino, A., Faham, S., Fox, G. M., Arakawa, T., Hsu, B. T., and Rees, D. C. (1991) Science 251, 90-93 [Medline] [Order article via Infotrieve]
  29. Winkles, J. A., and Gay, C. G. (1991) Cell Growth & Differ. 2, 531-540
  30. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89 [Medline] [Order article via Infotrieve]
  31. Jaye, M., Burgess, W. H., Shaw, A. B., and Drohan, W. N. (1987) J. Biol. Chem. 262, 16612-16617 [Abstract/Free Full Text]
  32. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  33. Hampton, B. S., Marshak, D. R., and Burgess, W. H. (1992) Mol. Biol. Cell 3, 85-93 [Abstract]
  34. Hata, A., Ridinger, D. N., Sutherland, S., Emi, M., Shuhua, Z., Myers, R. L., Ren, K., Cheng, T., Inoue, I., Wilson, D. E., Iverius, P.-H., and Lalouel, J.-M. (1993) J. Biol. Chem. 268, 8447-8457 [Abstract/Free Full Text]
  35. Imamura, T., Engleka, K., Zhan, X., Tokita, Y., Forough, R., Roeder, D., Jackson, A., Maier, J. A. M., Hla, T., and Maciag, T. (1990) Science 249, 1567-1570 [Medline] [Order article via Infotrieve]
  36. Zhu, X., Hsu, B. T., and Rees, D. C. (1993) Structure 1, 27-34 [Medline] [Order article via Infotrieve]
  37. Folkman, J., Szabo, S., Stovroff, M., McNeil, P., Li, W., and Shing, Y. (1991) Surgery 214, 414-427
  38. Prestrelski, S. J., Fox, G. M., and Arakawa, T. (1992) Arch. Biochem. Biophys. 293, 314-319 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.