©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Heparin Binding by Fibronectin Module III-13 Involves Six Discontinuous Basic Residues Brought Together to Form a Cationic Cradle (*)

(Received for publication, May 15, 1995)

Thomas F. Busby W. Scott Argraves Shelesa A. Brew Igor Pechik (1) (2) Gary L. Gilliland (1) (2) Kenneth C. Ingham (§)

From the  (1)Holland Laboratory, American Red Cross, the Center for Advanced Research in Biotechnology of the Maryland Biotechnology Institute, and the (2)National Institute of Standards and Technology, Rockville, Maryland 20855

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The thirteenth type III domain of fibronectin binds heparin almost as well as fibronectin itself and contains a so-called heparin-binding consensus sequence, Arg^6-Arg^7-Ala^8-Arg^9 (residues 1697-1700 in plasma fibronectin). Barkalow and Schwarzbauer (Barkalow, F. J., and Schwarzbauer, J. E. (1991) J. Biol. Chem. 266, 7812-7818) showed that mutation of Arg^6-Arg^7 in domain III-13 of recombinant truncated fibronectins abolished their ability to bind heparin-Sepharose. However, synthetic peptides containing this sequence have negligible affinity for heparin (Ingham, K. C., Brew, S. A., Migliorini, M. M., and Busby, T. F.(1993) Biochemistry 32, 12548-12553). We generated a three-dimensional model of fibronectin type III-13 based on the structure of a homologous domain from tenascin. The model places Arg, Lys, and Arg parallel to and in close proximity to the Arg^6-Arg^7-Ala^8-Arg^9 motif, suggesting that these residues may also contribute to the heparin-binding site. Domain III-13 and six single-site mutants containing Ser in place of each of the above-mentioned basic residues were expressed in Escherichia coli. All of the purified mutant domains melted reversibly with a Tm near that of the wild type indicating that they were correctly folded. When fluorescein-labeled heparin was titrated at physiological ionic strength, the wild type domain increased the anisotropy in a hyperbolic fashion with a K of 5-7 µM, close to that of the natural domain obtained by proteolysis of fibronectin. The R54S mutant bound 3-fold weaker and the remaining mutants bound at least 10-fold weaker than wild type. The results point out that the Arg^6-Arg^7-Ala^8-Arg^9 consensus sequence by itself has little affinity for heparin under physiological conditions, even when presented in the context of a folded domain. Thus, the heparin-binding site in fibronectin is more complex than previously realized. It is formed by a cluster of 6 positively charged residues that are remote in the sequence but brought together on one side of domain III-13 to form a ``cationic cradle'' into which the anionic heparin molecule could fit.


INTRODUCTION

The interaction of heparin or heparan-sulfate glycosaminoglycans (GAGs) (^1)with proteins occurs in a variety of physiological processes including blood coagulation, lipoprotein metabolism, cell adhesion and migration, and regulation of growth factor activity(1, 2, 3, 4) . Efforts to understand the nature of such interactions are hampered by the fact that GAGs are generally heterogeneous in their size and charge density(5) . Furthermore, there is no example of a detailed structure of a GAGbulletprotein complex determined by x-ray or NMR methods, although some attempts at molecular modeling have been made(6, 7, 8, 9) . That electrostatic forces are involved is evident from the fact that the complexes can be disrupted by increasing ionic strength. It is clear that positively charged Arg and/or Lys residues on the protein play an important role because their modification by chemical (10, 11) or recombinant (12, 13, 14) means usually leads to a reduction in the affinity for heparin, the most widely studied GAG. This is supported by numerous studies with synthetic heparin-binding peptides. However, in cases where the affinities have been measured, the peptides rarely bind as tightly as the parent protein on which their sequence is based. This probably means that the tertiary structure of the protein holds the peptide in a conformation that is more complementary to the GAG or that other residues not contained in the peptide contribute to the binding site in the protein, or both.

The present study concerns fibronectin, a large modular glycoprotein that interacts with a variety of macromolecules in the extracellular matrix and with cell-surface molecules such as integrins and heparan-sulfate proteoglycans. Such interactions are important in regulating cell behavior including growth, adhesion, spreading, migration, and differentiation(15) . Recent studies have shown that certain cell types require both the GAG binding and cell binding regions of fibronectin for efficient spreading and formation of focal contacts(16, 17, 18) . The interaction of fibronectin with heparin is dominated by the COOH-terminal hep-2 region which can be isolated as a 30-kDa proteolytic fragment consisting of fibronectin type III domains 12 through 14. Previous work has shown that domain III-13, when isolated by further proteolysis or expressed in Escherichia coli as an independent domain, binds heparin in solution with almost the same affinity as the parent fragment(19) . A cationic cluster, Arg^6-Arg^7-Ala^8-Arg^9, located near the NH(2) terminus of III-13, matches one of two patterns that are commonly found in heparin-binding proteins, namely BBXB, where B represents a basic residue, Arg, Lys, or His, and X represents any residue(7) . Arg^6 and Arg^7 were shown to be critical in that their simultaneous mutation abolished the ability of truncated recombinant fibronectins (``deminectins'') to bind heparin-Sepharose under physiological conditions(12) . However, synthetic peptides containing this cluster have negligible affinity for heparin under physiological conditions, indicating that tertiary structure and/or other residues are important (19) .

Fibronectin type III domains are among the most ubiquitous of protein modules, occurring in about 2% of animal proteins(20) . The three-dimensional structures of several type III domains have been elucidated, and they all show a similar beta-sandwich fold which resembles that of the immunoglobulin C domain(21, 22, 23, 24) . To gain insight into the three-dimensional arrangement of cationic residues in the folded structure of III-13, a molecular model was generated based on the known three-dimensional structure of a homologous type III domain in tenascin(21) . The model predicted that in addition to the above-mentioned arginines 6, 7, and 9, which would be expected to fall close to each other, 3 additional cationic residues should be located nearby in which case they might also contribute to the heparin-binding site. Based on the model, all 6 residues were individually mutated, and the resulting expression products were tested for the presence of compact structure and their ability to bind heparin in the fluid and solid phases. The results are embodied in the title of this paper.


MATERIALS AND METHODS

Molecular Modeling

The initial model of fibronectin domain III-13 was generated using as a template the three-dimensional structure of a homologous domain III-3 from tenascin(21) . The amino acid sequences of III-13 that differed from tenascin III-3 were replaced and adjusted manually to avoid stearic overlap using the program FRODO(25) . This initial model was then subjected to energy minimization and molecular dynamics using the X-PLOR 3.1 program package(26) . In the first step of this process, the model was subjected to 150 cycles of conjugate gradient energy minimization to further reduce stearic contacts. During this initial minimization, coordinates of side chain atoms of conservative residues and all C atoms were held near their initial positions by applying additional harmonic potential restraints. The coordinates were modified by subjecting the model to a two-stage molecular dynamics simulation. In the first stage the model constrained as above was heated from 0 to 300 K in 5 ps and then equilibrated for 50 ps. To increase the mobility of the side chains in this simulation, the partial atomic charges were reduced to 0.5 of their initial values, and hydrogen bond energy terms were turned off. In the second stage of the simulation, the model was equilibrated for another 50 ps with the harmonic restraints of the model removed, the partial atomic charges restored to their original values, and the hydrogen bond energy terms turned on. The resulting model was then subjected to a final 200 cycles of conjugate gradient energy minimization to optimize the geometry and stereochemistry of the final structure.

Expression of Recombinant Domains

Expression of recombinant III-13 and its mutants as fusion proteins with maltose-binding protein was accomplished in E. coli using a modified pMAL-p2 expression vector (New England Biolabs) in a manner similar to that previously described(27) . cDNA fragments encoding residues 1-89 of domain III-13, i.e. Asn-Thr of plasma fibronectin(28) , were prepared by PCR using 21-base synthetic primers flanking the desired regions and cDNA encoding human fibronectin kindly provided by Dufour et al.(29) . The primers also contained the sequence for the BamHI and HindIII restriction sites for ligation into the pMAL-p2 expression vector. Approximately 20 ng of the ligated DNA was used to transform TB1 E. coli cells by electroporation. The electroporated cell/DNA mixture was grown in LB media at 37 °C for 1 h. Serial dilutions were spread onto plates covered with LB media containing ampicillin and incubated at 37 °C overnight. After screening for the presence of insert by restriction analysis and/or PCR analysis, individual colonies were grown overnight at 37 °C with shaking at 225 revolutions/min, and diluted 100-fold with fresh ampicillin-containing media. After growing at 37 °C for about 2.5 h or until the absorbance at 600 nm reached about 0.6, the cells were induced with isopropyl-1-thio-beta-D-galactopyranoside at a final concentration of 0.3 mM and grown for an additional 3 h. Although the fusion proteins were preceded by a signal peptide sequence, the bulk of the product was found in the cytoplasm. The cells were harvested by centrifugation, lysed by sonication in 0.005% Triton X-100, and the fusion protein was purified immediately by affinity chromatography on immobilized amylose and/or heparin. The yield of fusion protein varied between 5 and 15 mg/liter. For unknown reasons, storage of the lysate at 4 °C resulted in progressively lower yield of material binding to the amylose resin. Similarly, the efficiency of rebinding of maltose-binding protein and fusion protein to the amylose resin, even after exhaustive dialysis, was too low to be useful in further steps of purification. Therefore, after digestion of the fusion proteins with factor Xa(30) , the liberated intact III-13 domains were purified to homogeneity by rechromatography on heparin-Sepharose and/or QMA anion-exchange media (Waters) and size-exclusion chromatography as needed. The amino terminus of the final products contained 2 additional residues, Ile and Leu, that are not part of the III-13 domain.

Mutagenesis

Arg Ser and Lys Ser mutations were prepared by the method of Ho et al.(31) . Synthetic complementary 21-base primers containing the desired mutation and spanning the region to be mutated, as well as two primers exterior to the type III-13 insert, were annealed to the wild type III-13 cDNA template. PCR extension of these primers resulted in two cDNAs that were then annealed to each other through their 21-base overlapping region (containing the mutation) and, using the primers exterior to the III-13 insert, extended again to produce the complete mutated inserts. The 6-fold degeneracy in the codon for serine was exploited, when possible, to introduce unique restriction sites in the mutated insert. In those cases, digestion with the unique restriction enzyme followed by electrophoresis could be used as an initial test for the presence of the mutation before the insert was ligated into the plasmid. The R6S mutation (TCG for AGA) introduced a second TaqI site (T-CGA), the R7S mutation (TCG for AGG) added a third MboI site (-GATC), the R9S mutation (TCT for CGT) inserted a unique XmnI site (GAANN-NNTTC), the R23S mutation (TCG for AGA) produced a unique SalI site (G-TCGAC), the K25S mutation (TCG for AAG) introduced a second TaqI site (T-CGA), the R47S mutation added a unique SalI site (G-TCGAC), and the R54S mutation inserted a unique XhoI site (C-TCGAG). After initial confirmation of the mutation by digestion of the PCR products at these unique sites, the mutated inserts were ligated into the pMAL-p2 plasmid, transformed, expressed, and purified as outlined above. Repeated attempts to generate the K25S mutation using an AGT codon for the serine (unique ScaI site) were unsuccessful; only about 25% of the plasmid was cleavable, and induced cells produced no fusion protein. This was not a problem when the TCG codon was used.

With the exception of the R54S mutant, all fusion proteins were soluble and monomeric. All cleaved and purified III-13 domains were homogeneous by analytical size-exclusion chromatography on Superdex-75 (Pharmacia) and/or SDS-polyacrylamide gel electrophoresis in 8-25% gradient polyacrylamide gels (Pharmacia Phast system). All mutations were confirmed by sequencing at both the DNA and the protein level. Protein sequencing was done with a Hewlett Packard G1000S protein sequencing system. With the Arg^6, Arg^7, Arg^9, Arg, and Lys mutants, this was done on the intact domains because the mutations were close enough to the NH(2) terminus of the domain. With the Arg and Arg mutants, it was necessary to first cleave chemically or enzymatically, sequence the resulting mixture of peptides, and compare the results with the known sequence of domain III-13 using custom software developed by G. Argraves (Shelton, CT).

Thermal Stability

The structural integrity of the recombinant III-13 domains was assessed by heating a solution of the domain at a concentration of 0.1 mg/ml at 1 °C/min in the SLM 8000C spectrofluorometer while monitoring the ratio of fluorescence at 350 nm to that at 320 nm with excitation at 280 nm(32) . The fluorescence ratio provides a convenient and sensitive means of detecting the spectral shift that accompanies denaturation. Changes in the fluorescence ratio permitted detection of melting transitions and assessment of relative stability and the degree of reversibility of the denaturation upon cooling.

Fluorescence Anisotropy

Measurements were made with the SLM-8000C spectrofluorometer in the T format with excitation and emission wavelengths of 493 and 524 nm, respectively. All experiments described here were performed with Sephadex G-100 fraction no. 4 of fluoresceinamine-labeled heparin (FA-heparin) having an average molecular mass of 15,000 daltons(33) . Titrations of 0.1 µM FA-heparin with recombinant III-13 domains were performed in 0.02 M Tris buffer, pH 7.4, containing 0.02% NaN(3) and no NaCl (TB) or 0.15 M NaCl (TBS). Small amounts of a stock solution of the recombinant peptides were added continuously with a motorized syringe controlled by the same computer that controls the fluorometer. The change in anisotropy, DeltaA, as a function of titrant concentration was fitted to a single class of equivalent binding sites on the FA-heparin by using the following equation:

where [titrant] is the free concentration of fragment (or peptide), DeltaA(max) is the maximum anisotropy change that would be produced at saturating concentrations of titrant, and K is the apparent dissociation constant of the heparin-fragment complex(19) . Since in all cases the concentration of FA-heparin was low compared to the range of concentration of fragment, the free fragment concentration was taken as the total. The concentrations of the fragments were determined from the absorbance at 280 nm, using a molar extinction coefficient, = 10,800 M cm.

Analytical Affinity Chromatography

Heparin-Sepharose was prepared as described(33) . The recombinant peptides or fusion proteins were applied at 1 ml/min to a 1.7-ml column of heparin-Sepharose in either TB or TBS using a Pharmacia fast protein liquid chromatography system. A linear gradient to 0.6 M NaCl was used for elution, which was monitored by fluorescence at 340 nm with excitation at 280 nm.


RESULTS

Molecular Modeling of Domain III-13

The fibronectin type III domains form a beta sandwich with a folding topology similar to that of the immunoglobulin C domain(21, 22, 23, 24) . Domain III-13 of fibronectin was modeled after domain III-3 from tenascin(21) . The results are illustrated in Fig. 1where the charged side chains are highlighted. Arginines 6, 7, and 9 conform to one of the sequence patterns identified in a number of heparin-binding proteins(1, 7) . Arginines 6 and 7 are the ones whose mutation by Barkalow and Schwarzbauer (12) abolished binding of truncated fibronectins to heparin-Sepharose at physiological ionic strength. The model suggests that additional positively charged residues Arg, Lys, and Arg might also contribute to heparin binding since they lie in close proximity to arginines 6, 7, and 9. Although the positions of the side chains are not precisely determined by the model, it is clear that 6 of the 10 positive charges in this domain lie within a few nanometers of each other on one side of the structure, in a region devoid of negative charge. A similar structure has been identified as the GAG-binding site in human lactoferrin and has been termed a ``cationic cradle''(8) . In an effort to evaluate the relative importance of all 6 of these residues, each of them was mutated separately to a serine residue. The resulting constructs were expressed in E. coli and evaluated for folding integrity and heparin binding. Arginine 47, which in this model lies on the opposite side of the domain and is not predicted to be part of the binding site, was also mutated as a control for the possibility that simply reducing the net positive charge might affect heparin binding.


Figure 1: Three-dimensional structure of the heparin-binding domain III-13 modeled after the known structure of a homologous type III domain from tenascin. Negatively charged side chains are red, and positively charged Arg and Lys side chains are green; there are no His residues.



Reversible Unfolding of Recombinant III-13 and Its Mutants

The thermal stability of the recombinant III-13 domains was assessed as a measure of their structural integrity. The various recombinant peptides were heated at 1 °C/min in the fluorometer while monitoring the ratio of fluorescence at 350 nm to that at 320 nm as a measure of the spectral shift that accompanies unfolding(32) . As shown in Fig. 2, all of the products underwent a cooperative sigmoidal unfolding transition similar to that observed previously with natural fragments derived from the parent protein by proteolysis(32) . The transitions were highly reversible in that the fluorescence ratio returned to a value close to the original upon cooling. The T values varied between 60 and 71 °C (Table 1). These results show that all of the recombinant III-13 domains, as isolated, were folded into compact structures with stabilities similar to one another and to that of the natural domain.


Figure 2: Melting of recombinant fibronectin domain III-13 and its mutants. Samples were equilibrated in TBS at 0.1 mg/ml and heated at 1 °C/min while monitoring the ratio of fluorescence intensity at 350 nm to that at 320 nm with excitation at 280 nm. The dashed curves indicate reversibility on cooling.





Analytical Affinity Chromatography on Heparin-Sepharose

Affinity chromatography was used as a qualitative test of the ability of the recombinant III-13 domains to bind heparin(19) . All of the products bound in high yield to the heparin-Sepharose column at room temperature, whether applied in the presence (TBS) or absence (TB) of 0.15 M NaCl. Natural III-13, derived from plasma fibronectin by proteolysis(19) , and wild type rIII-13 eluted similarly between 0.43 and 0.45 M in a gradient of NaCl (Table 1) indicating that the recombinant and natural domains are functionally similar. The remaining mutants all eluted earlier, between 0.25 and 0.38 M NaCl. The final concentration of NaCl required for elution was similar whether starting from 0.0 M (TB, Table 1) or 0.15 M (TBS, not shown). The R47S control mutant also bound and was eluted at the same salt concentration as the natural and recombinant wild type fragments.

Titration of Fluorescent-labeled Heparin

A fluorescence polarization anisotropy assay was used to obtain a more quantitative estimate of the effect of the various mutations on the affinity for heparin in the fluid phase. The results are presented in Fig. 3where solid curves represent best fits to . In TB, all of the products caused a dose-dependent increase in fluorescence anisotropy of FA-heparin. Dissociation constants ranged from 0.5 to 1.4 µM for the natural, wild type and R47S control domains and all of the mutants fell within this same range (Table 1). At physiological ionic strength, in TBS, the K values of the wild type and R47S control domains were close to each other and to that of the natural domain although the values for all three were approximately 10-fold higher than at low ionic strength. In contrast, the binding of the R6S, R7S, R9S, and R23S mutants was too weak to cause a significant increase in the anisotropy at the concentrations of the domains that were achieved (Fig. 3). The K for these mutants is conservatively estimated at geq100 µM, based on the reasonable assumption that the change in anisotropy caused by saturation with these mutants would be similar to that caused by the wild type. The same assumption was used in fitting the data for the K25S mutant, which caused a slight increase in anisotropy but still failed to achieve a substantial fractional saturation of the response at the highest concentration employed. The R54S mutant had the highest affinity of all the mutants with a K of 19 µM, still significantly weaker than the wild type or R47S control. The results indicate that the affinity of domain III-13 for heparin, as measured in the fluid phase at physiological ionic strength, is reduced at least 10-fold by elimination of a positive charge at positions 6, 7, 9, or 23, slightly less at position 25, and approximately 3-fold at position 54. By contrast, the affinity is essentially unaffected by neutralizing the positive charge at position 47 and, when measured at low ionic strength, is not sensitive to any of these mutations.


Figure 3: Titration of fluorescein-labeled heparin (0.1 µM) with recombinant fibronectin domain III-13 and its mutants at room temperature in 0.02 M Tris-HCl, pH 7.4, 0.02% NaN(3), in the absence (TB) and presence (TBS) of 0.15 M NaCl. Solid lines represent best fits of the data to . The corresponding values of Kare given in Table 1.




DISCUSSION

The results presented here indicate that binding of fibronectin to heparin at physiological ionic strength is more complex than previously appreciated. It involves at least 6 basic residues within type III domain 13. These include Arg^6, Arg^7, Arg^9, Arg, Lys, and Arg. Mutation of any 1 of these residues to a serine decreased the affinity of the isolated domain for fluorescent heparin in the fluid phase in TBS by at least an order of magnitude in four cases and to a lesser but still significant extent in the other cases. The mutant domains also exhibited decreases in the concentration of NaCl required for their elution from heparin-Sepharose. By contrast, in 0.02 M Tris without added salt, where the fluid-phase interaction between domain III-13 and FA-heparin is about 10-fold tighter than in TBS, the mutant domains bind as well as the wild type domain. This latter observation should serve as a cautionary note to those who would attempt to interpret the physiological significance of GAG binding measurements conducted at subphysiological ionic strength.

All of the single site mutants exhibited cooperative reversible unfolding transitions as detected by sigmoidal changes in fluorescence upon heating. This proves that the recombinant domains were folded into a compact structure similar to that of the wild type and decreases the likelihood that the observed effects of the mutations on binding are due to improper folding. The choice of Ser as the substitute for Arg or Lys in the mutants has the effect of replacing the cationic side chain with a hydroxylated methlyene group which preserves a measure of hydrophillic character. Furthermore, the new group is similar to those which are abundant in the GAG thus minimizing the possibility that the mutation would actually interfere with heparin binding. The variation of T between the different mutants was outside the range of experimental error suggesting some subtle changes in stability. This could arise from the loss of attractive or repulsive electrostatic interactions involving the mutated residues or of hydrophobic interactions involving the methylene groups in their side chains. Note also the 9 °C lower T of wild type III-13 relative to its natural counterpart which was derived from fibronectin by proteolysis. The natural fragment is significantly longer, containing additional residues at the COOH-terminal end that are derived from domain III-14 and might participate in a stabilizing interaction with III-13(32) .

A three-dimensional model of domain III-13 suggests that these 6 cationic residues, all of which are conserved in human, bovine, rat, chick, and xenopus fibronectin(15, 34, 35) , are clustered on one side of the domain in a region devoid of negative charge. This situation resembles that recently described for lactoferrin, which contains two BBXB sequences that are separated by 24 residues and form a cationic groove or ``cradle'' into which the anionic polysaccharide was proposed to fit(8) . In fibronectin III-13 only a single BBXB sequence is involved, perhaps accounting for its 50-fold lower affinity for FA-heparin relative to that of lactoferrin. The other 3 basic residues that contribute to the GAG-binding site in III-13 are recruited from positions that are 14, 16, and 45 residues distant from Arg^9 in the primary structure. Mutation of Arg, which according to the model is located on the opposite side of the domain, had no effect on binding, indicating that mere reduction of the net positive charge from +2 to +1 cannot account for the observed effects of the other mutations. Other examples of proteins in which positively charged groups that are remote in sequence are brought together in the folded protein to form a GAG-binding site include antithrombin III(36) , lipoprotein lipase(37) , fibroblast growth factors(38) , and platelet factor 4(9, 39) . In the latter case, the clustering of positive charge is enhanced through self-association of the protein to form dimers and tetramers with progressively higher affinity for heparin(39) .

It is worth mentioning that domain III-14 of fibronectin has little affinity for heparin in spite of the fact that it has 13 basic and only 7 acidic residues for a net positive charge of +6, compared to +2 for domain III-13. Yet, the affinity of III-14 for heparin is at least 10-fold lower in comparison to III-13(19) . A model of domain III-14 (not shown) suggests that the cationic residues are dispersed more or less randomly over its surface with no obvious clusters that resemble anything like the cradle in III-13. The 3 contiguous basic residues Arg-Lys-Lys (residues 87-89 in the model) fall in close proximity to Glu which would diminish their cationic potential. Similarly, although basic residues 26, 28, and 76 are predicted to be in close proximity to each other, their cationic potential would be diminished by proximity to Asp^3. Thus, the relative affinities of these two homologous domains for heparin are consistent with the distribution of charge predicted by their respective models.

The RRAR sequence, when present in a synthetic peptide, has weak affinity for heparin in comparison to larger fragments containing this sequence(19, 40) . Furthermore, a proteolytic fragment of fibronectin that contains type III domain 12 plus a 17-residue COOH-terminal extension that includes the RRAR sequence of III-13 also has negligible affinity (fragment 10K12 in (19) ). It was argued that the low affinity might be due to the failure of the peptide, when isolated or appended to domain 12, to assume a tertiary structure consistent with its configuration in the folded domain. The present results show, however, that this sequence by itself does not promote high affinity for heparin, even when presented in the context of a properly folded domain, as in the R23S and K25S mutants, both of which bind substantially more weakly than the wild type domain. This same RRAR sequence also exists in the carboxyl-terminal lobe of lactoferrin where it is not functional as a GAG-binding site(8) . These observations help to explain why the list of actual heparin-binding proteins is much shorter than the list of proteins containing a putative heparin-binding ``consensus sequence''(7) . Many of the latter probably lack the additional strategically positioned cationic residues that are necessary for tight binding to heparin.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL21791 and HL44336. 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: American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0731; Fax: 301-738-0794.

^1
The abbreviations used are: GAGs, glycosaminoglycans; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We are indebted to Dr. David Mann for helpful suggestions throughout the course of this work and for valuable discussions during the preparation of the manuscript. Thanks also to Mary Migliorini for confirming the presence of mutations in all of the expressed recombinant domains by amino acid sequencing and to Gary Argraves for development of the sequence analysis and titration software.


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