Fibroblast growth factors (FGFs) are polypeptides involved in the control of cell growth, migration, and differentiation and thereby in the regulation of processes such as embryonic development and tumor angiogenesis (Friesel and Maciag, 1995). To date, more than 15 distinct FGF species have been identified. These growth factors bind strongly to heparin, a polysaccharide found in the intracellular granules of connective tissue mast cells. In vivo, the major polysaccharide ligand for FGFs is probably heparan sulfate (HS), that occurs as HS proteoglycans at cell surfaces and in the extracellular matrix throughout the body. Interactions of FGF-1 and FGF-2 (acidic and basic FGF, respectively) with HS have been shown to be essential for the biological activity of the growth factors (Rapraeger et al., 1991; Guimond et al., 1993; Ishihara, 1994). These, and perhaps all, FGFs fail to bind and activate cell surface FGF receptors in cells that lack endogenous HS, whereas the FGF responsiveness can be restored by addition of HS or heparin. Furthermore, HS has been implicated in the regulation of tissue localization (Friedl et al., 1997) and proteolytic degradation of FGFs (Saksela et al., 1988).
HS is a structurally complex sulfated glycosaminoglycan consisting of hexuronic acid-glucosamine disaccharide units (Kjellén and Lindahl, 1991; Salmivirta et al., 1996). Some regions of the initial glucuronic acid-N-acetylglucosamine (GlcA-GlcNAc)n polymer are modified during the HS biosynthesis by N-deacetylation and N-sulfation of GlcNAc units. These N-sulfated regions (NS-domains) are further modified by C5 epimerization of GlcA residues into iduronic acid (IdoA) residues and O-sulfate substitution, mainly at the C2 position of IdoA and C6 position of GlcN. NS-domains alternate with sequences of N-acetylated disaccharide units (NA-domains), largely devoid of O-sulfate groups. In addition, the HS polymers contain domains composed of alternating N-sulfated and N-acetylated disaccharide units (NS/NA-domains), which generally contain few 2-O-sulfate groups but may carry >50% of all 6-O-sulfate groups of the polysaccharide (Maccarana et al., 1996). The type and extent of HS polymer modification reactions vary between different cell types (Turnbull and Gallagher, 1990; Sanderson et al., 1994) and tissues (Maccarana et al., 1996) and are subject to modulation during development (Brickman et al., 1998), aging (Feyzi et al., 1998), and clinical (Lindahl and Lindahl, 1997) and experimental (Kjellén et al., 1983) disease conditions. Heparin, frequently used as a substitute for HS in experimental work, consists almost exclusively of highly modified NS-domains and lacks most of the regioselective modifications characteristic of HS (Kjellén and Lindahl, 1991).
The interactions of HS with FGFs (Turnbull et al., 1992; Maccarana and Lindahl, 1993; Mach et al., 1993; Fromm et al., 1997) and other peptide growth factors (Lyon et al., 1994; Ashikari et al., 1995; Feyzi et al., 1997) involve predominantly the NS-domains of the polysaccharide. Biochemical and crystallographic analyses of FGF-2-heparin oligomer interactions show that the minimal saccharide domain binding to FGF-2 encompasses a pentamer containing N-sulfate groups and an essential IdoA(2-OSO3) residue (Maccarana et al., 1993; Faham et al., 1996). The shortest heparin fragments binding to an FGF-1 affinity matrix with high affinity have been reported to be octasaccharides (Ishihara, 1994), and HS fragments of similar length are protected against HS-lyase cleavage in the presence of FGF-1 (Fromm et al., 1997). However, only 4-5 sugar units seem to make contact with the protein (Mach et al., 1993; DiGabriele et al., 1998), suggesting that the minimal sequences binding to FGF-1 and FGF-2 are similar in length. While studies with partially desulfated heparin preparations (Ishihara, 1994) and the structure of FGF-1-heparin co-crystals (DiGabriele et al., 1998) suggest a role for 6-O-sulfate groups in the heparin-FGF-1 interaction, no detailed information regarding the sugar structures mediating the interaction in the more physiological HS ligand has been available. The minimal 4-5-mer domains sufficient for FGF binding fail to promote the mitogenic activity of the growth factors. In the case of FGF-2, the mitogenic response requires a dodecasaccharide or longer sequence containing both IdoA(2-OSO3) and GlcNSO3(6-OSO3) residues (Guimond et al., 1993; Ishihara, 1994). The dodecasaccharide domain has been proposed to mediate complexing between FGF-2 and FGF receptor by concomitant interactions with both components (Guimond et al., 1993), alternatively dimerization of FGF-2 (11). Shorter (8-10-mer) heparin fragments appear sufficient to promote FGF-1 induced FGF receptor phosphorylation (Ornitz et al., 1992), cell proliferation (Ishihara, 1994) and formation of saccharide-linked dimers of FGF-1 (Spivak-Kroizman et al., 1994). This study was undertaken in order to characterize the FGF-1 binding HS domain and to study its structural relationship to the previously characterized domain interacting with FGF-2.
Minimal length of FGF-1 binding heparin/HS domain
The minimal size of the FGF-1 binding heparin domain was studied by testing the binding of even-numbered, 3H-labeled heparin oligomers to FGF-1 using a well established filter-trapping assay (see Materials and methods). Saccharide-protein complexes, formed in free solution under physiological pH and ionic strength, were trapped onto nitrocellulose filters and the bound saccharide was quantified by scintillation counting. The shortest heparin oligomers showing affinity toward FGF-1 were hexasaccharides, which displayed a low level of binding clearly discernible from that of tetrasaccharides (Figure
Figure 1. Minimal size of heparin oligomers and NS-domains from bovine intestinal HS required for binding to FGF-1. FGF-1 (0.5 µg) was incubated with 20,000 d.p.m. of 3H-labeled even-numbered heparin oligomers or HS-derived NS-domains as described in Materials and methods. Following a 2 h incubation, the samples were passed through nitrocellulose filters and the protein-bound saccharide was quantified in a liquid scintillation counter. Results are expressed as mean values of two incubations. Composition of the FGF-1 binding HS domain
To analyze the disaccharide composition of FGF-1 binding HS domains, 3H-labeled decameric NS-domains from bovine intestinal HS were fractionated into FGF-1 bound (representing ~4% of the added saccharide) and unbound pools using the filter-trapping procedure in preparative mode (see Materials and methods). Decasaccharides were employed as the saccharide ligand because the lower apparent affinity of hexa- and octasaccharides, that more closely corresponded to the minimal binding domain, did not allow recovery of sufficient amounts of FGF-1 bound material for compositional disaccharide analysis. The decameric NS-domains showed a dose-dependent and saturable binding to FGF-1 (see Figure
Figure 2. Compositional disaccharide analysis of FGF-1 bound and unbound NS-domains. Decameric NS-domains were fractionated according to binding to FGF-1 using the nitrocellulose filter procedure in a preparative fashion (see Materials and methods). The bound and unbound oligosaccharides were cleaved by HNO2 at pH 1.5. The resultant disaccharides were radiolabeled by reduction with NaB3H4 and analyzed on a Partisil-10 HPLC SAX column eluted with a step-wise gradient of KH2PO4 (broken line). The peaks representing different disaccharide species were identified by comparing their elution positions with those of standard heparin disaccharides and are numbered as follows: (1) GlcA-aManR(6-OSO3); (2) IdoA-aManR(6-OSO3); (3) IdoA(2-OSO3)-aManR; (4) IdoA(2-OSO3)-aManR(6-OSO3). The peaks marked with (*) represent tetrasaccharides, partly due to 'anomalous" ring contraction (Shively and Conrad, 1976), which were not included in the quantification of disaccharide species shown in Figure 3.
Figure 3. Disaccharide composition of FGF bound and unbound oligosaccharides. 3H-Labeled, decameric NS-domains from bovine intestinal HS (A) and preferentially 6-O-desulfated heparin hexasaccharides (B) were fractionated according to FGF binding and the disaccharide compositions of the bound and unbound oligomers were determined by anion-exchange HPLC (Figure 2) and high voltage paper electrophoresis (see Materials and methods). The proportions of each disaccharide species were quantified from the chromatography data by peak area measurement.
Figure 4. Discrete NS-domains are involved in binding to FGF-1 and FGF-2. (A) Re-binding of FGF-1 bound and unbound decameric NS-domains from bovine intestinal HS to FGF-1. 3H-Labeled NS-domains were fractionated (using the preparative filter trapping procedure) into FGF-1 bound and unbound pools and aliquots of these (10,000 d.p.m.) were tested for re-binding to FGF-1 (0.5 µg/incubation). Binding of an equal amount of the unfractionated NS-domains is shown for comparison. The data are the mean values of two incubations. (B) Dose-dependent binding to FGF-2 of NS-domains from the FGF-1 unbound fraction. The FGF-1 unbound fraction of 3H-NS-domains from the experiment shown in (A) was subjected to an additional affinity fractionation step and increasing amounts of the unretained (depleted) fraction were tested for binding to FGF-1 and FGF-2 (0.5 µg) using the filter-trapping assay. The dose-dependent binding of the unfractionated NS-domains to FGF-1 and FGF-2 is also shown. Each data point is the mean of duplicate incubations.
Further, we analyzed the sulfation pattern of FGF-1 bound, partially O-desulfated heparin hexasaccharides. The hexasaccharide structure spans the minimal FGF-1 binding domain (Mach et al., 1993; DiGabriele et al., 1998; Figure
Collectively, the structures of FGF-1 bound HS and heparin-derived oligosaccharides suggest a role for closely positioned IdoA(2-OSO3) and GlcNSO3(6-OSO3) residues in the interaction. Regardless of the length of the saccharide ligand, the trisulfated IdoA(2-OSO3)-GlcNSO3(6-OSO3) units amounted to at least one per oligosaccharide chain in the FGF-1 bound fractions and were more abundant in the bound than in the unbound fractions (Figure HS decamers depleted of FGF-1 binding domains retain their ability to bind FGF-2
To study the binding specificity of the FGF-1 bound NS-domains isolated by affinity fractionation, a series of re-binding experiments were performed with the FGF-1 bound and unbound oligosaccharide pools recovered from preparative experiments. First, FGF-1 bound (4% of total) and unbound [3H]decamers were retested for binding to FGF-1. The once bound pool showed clearly enhanced binding whereas the unbound pool was found to bind less efficiently than the unfractionated decamer preparation (Figure
Table I. Differential binding of intact HS chains to FGF-1 and FGF-2
Unlike the isolated, size-defined NS-domains studied above, native HS chains contain multiple NS-domains of varying length and degree of O-sulfation as well as additional sulfate groups outside the NS-domains (Maccarana et al., 1996). We therefore asked whether differential O-sulfation of NS domains regulates FGF-1 and FGF-2 binding also at the level of native, full-length HS polymers. To this end we tested the FGF binding properties of human aorta HS from a young and an old subject using the filter-trapping assay. We recently found (Feyzi et al., 1998) that human aging is associated with increased 6-O-sulfation and increased proportions of IdoA(2-OSO3)-GlcNSO3(6-OSO3) units in the NS-domains of aorta HS, whereas the degree of N-sulfation, IdoA 2-O-sulfation, and O-sulfation outside the NS-domains were not affected by subject age. This structural change resulted in an enhanced binding of HS to platelet-derived growth factor (PDGF) isoforms containing polybasic cell retention sequences (Feyzi et al., 1998) but did not affect the binding to FGF-2. HS from an old subject (76 years) was found to bind FGF-1 ~6-fold more efficiently than HS from a young subject (22 years) (Figure
Figure 5. Age-related difference in binding of human aorta HS to FGF-1. (a) FGF-1 and FGF-2 were tested for binding to 3H-labeled human aorta HS isolated from a young (22 years) and an old (76 years) individual. Increasing amounts of 3H-HS (up to 17 pmol) were incubated with recombinant FGF-1 or FGF-2 (31 pmol and 29 pmol/incubation, respectively) as described in Materials and methods. Binding was assessed using the filter trapping assay. The results are the mean values of duplicate incubations.
We performed similar binding experiments using metabolically 3H-labeled HS from chlorate-treated MDCK cells. Chlorate selectively impedes cellular sulfation reactions and results in a dose-dependent inhibition of 2-O- and 6-O-sulfation but does not significantly affect the degree of N-sulfation (F.Safaiyan, K.Prydz, S.O.Kolset, and M.Salmivirta, unpublished observations). Whereas HS from untreated cells bound well to both FGF species, HS from cells treated with 50 mM chlorate showed a selective loss of binding to FGF-1 (Figure
Figure 6. Selective effect of chlorate treatment on binding of HS from MDCK cells to FGF-1. 3H-HS was isolated from MDCK cells cultured in the absence or presence of sodium chlorate (5, 20, and 50 mM). Aliquots of 3H-HS (20,000 dpm) were tested for binding to FGF-1 and FGF-2 (1 µg). The FGF-binding of HS from cells without chlorate treatment is set as 100%. The binding was assessed by the nitrocellulose filter trapping method. The data are means of duplicate incubations.
The present study provides evidence that FGF-1 and FGF-2 are recognized by different HS structures, expressed in discrete NS domains of the HS polymers. We show that (1) the FGF-1 and FGF-2 bound NS-domains clearly differed with regard to disaccharide composition, (2) NS-domains depleted of FGF-1 binding sequences retained their ability to bind FGF-2 and (3) changes in HS sulfation occurring in vivo, or induced by chlorate treatment in vitro, selectively affected binding of the polysaccharide to FGF-1. The key determinant of the FGF-2 binding HS site is an IdoA(2-OSO3) residue (Maccarana et al., 1993; Faham et al., 1996). 6-O-Sulfated GlcN residues, on the other hand, do not make contacts with the protein in FGF-2/heparin hexasaccharide co-crystals (Faham et al., 1996) and appear redundant for HS-FGF-2 interaction in binding assays (Maccarana et al., 1993; Ishihara, 1994). By contrast, 6-O-sulfate groups were found to promote the FGF-1-HS interaction in the present study, as shown by an enrichment of the FGF-1 bound NS-domains with regard to 6-O-sulfated disaccharide species. Interestingly, this enrichment was restricted to the trisulfated IdoA(2-OSO3)-GlcNSO3(6-OSO3) units, which amounted to at least one per FGF-1 bound oligosaccharide and thus appeared to hallmark the FGF-1 binding HS domain.
Crystal structures of heparin oligomers complexed with FGF-1 (DiGabriele et al., 1998) and FGF-2 (Faham et al., 1996) provide interesting clues as to why different saccharide sequences are required for binding to these FGF species with highly conserved structures. Analysis of the crystal structure of dimeric FGF-1, bridged by a heparin decasaccharide, revealed as many as eight different modes of interaction, distinguished by variable saccharide chain orientation and amino acid contacts (DiGabriele et al., 1998). Considering the sulfate groups contacting FGF-1, the crystallography data largely agree with the conclusions from our study by showing that N-, 2-O-, and 6-O-sulfate groups all make contact with one of the FGF-1 molecules. The other FGF-1 molecule, however, seemed to bind heparin without contribution of the 6-O-sulfate groups. Notably, in the complex involving each of the three sulfate substituents, the O-sulfate groups were assembled in the trisulfated IdoA(2-OSO3)-GlcNSO3(6-OSO3) unit. The heparin-protein contact unique for FGF-1 appears to involve N114 and the 6-O-sulfate group in a GlcNSO3(6-OSO3) unit, whereas the neighboring IdoA(2-OSO3) residue and a N-sulfate group were found to bind N18, Q127, and K118 in a fashion resembling the interaction described for FGF-2. In the FGF-2-heparin complex (Faham et al., 1996), sulfate groups in consecutive GlcNSO3 and IdoA(2-OSO3) residues have been proposed to mediate high affinity binding to N28/K126 and K126/Q135, respectively.
Recent reports indicate 6-O-sulfation of GlcN units as a biosynthesis step subject to major regulation in various biological and pathological contexts, including cell differentiation (Brickman et al., 1998; Salmivirta et al., 1998) and malignant transformation (Jayson et al., 1998; Safaiyan et al., 1998) in vitro and senescence (Feyzi et al., 1998) in vivo. The alterations in 6-O-sulfation frequently affect the proportions of IdoA(2-OSO3)-GlcNSO3(6-OSO3) units (Feyzi et al., 1998; Safaiyan et al., 1998; Salmivirta et al., 1998) and might therefore have functional impact in terms of FGF-1 binding capacity of a given HS species. The binding studies reported here, involving human aorta HS preparations with different contents of IdoA(2-OSO3)-GlcNSO3(6-OSO3) units (Feyzi et al., 1998), indeed demonstrated that the HS preparation from an old individual, enriched in such units, showed drastically enhanced binding to FGF-1. Notably, the contents of these units in HS from the old subject were ~1.5-fold higher whereas the FGF-1 binding capacity was ~6-fold increased as compared to HS from the young subject. This finding suggests that only a minor proportion of the IdoA(2-OSO3)-GlcNSO3(6-OSO3) units present in HS from the young subject is localized in sequences comprising the FGF-1 binding domain and that the age-associated increase in the 6-O-sulfate substitution of aorta HS occurs in a manner that efficiently accomplishes the FGF-1 binding site. We have previously demonstrated a similar relationship between aorta HS structure and binding to PDGF isoforms carrying polybasic cell retention sequences (Feyzi et al., 1998). The PDGF-binding HS domain is also characterized by the presence of trisulfated IdoA(2-OSO3)-GlcNSO3(6-OSO3) units (Feyzi et al., 1997) and may thus show similarity to the FGF-1 binding domain characterized in the present study.
In summary, the present study provides the first structural characterization of the HS domain involved in binding to FGF-1. In contrast to the widely expressed FGF-2 binding HS sequence, the binding of HS to FGF-1 depends on a more rarely occurring combination of sulfate groups, hallmarked by heparin-like IdoA(2-OSO3)-GlcNSO3(6-OSO3) units. The specificity of the FGF-1 binding HS domain raises exciting perspectives for finely tuned regulation of FGF activities. Such regulation has previously been shown to involve differential expression of FGF receptor species and their splice variants. Cellular regulation of HS domain structure is likely to represent an additional way of modulating cell sensitivity toward different FGF species. Glycosaminoglycan preparations
Heparin from pig intestinal mucosa (stage 14, Inolex Pharmaceutical Division, Park Forest South, IL) was purified as described previously (Lindahl et al., 1965) and used either unlabeled or radiolabeled by [3H]acetylation of free amino groups (specific activity, ~0.34 × 105 d.p.m./nmol disaccharide) as described previously (Höök et al., 1982). The preparations of partially O-desulfated heparin and heparin hexasaccharides and fully sulfated, 3H-labeled heparin oligosaccharides were prepared as described earlier (Feyzi et al., 1997a,b). HS from bovine intestinal mucosa was prepared from heparin by-products (a gift from KABI AB, Stockholm) by precipitation with cetylpyridium chloride in 0.5 M NaCl. The precipitated HS was eluted at 0.6-0.8 M NaCl on a column of DEAE-Sephacel. HSs from human aorta were isolated as described previously (Feyzi et al., 1998). For preparation of NS-domains, HS was N-deacetylated by hydrazinolysis (Guo and Conrad, 1989) as described previously (Safaiyan et al., 1998) and subjected to cleavage with HNO2 at pH 3.9 (Shively and Conrad, 1976). The resulting oligosaccharides were radiolabeled by reduction of the formed anhydromannose residues with NaB3H4 (Amersham Pharmacia Biotech) resulting in labeled 2,5-anhydromannitol (aManR) residues, and separated from the unincorporated radioactivity by chromatography on a column of Sephadex G-15 (Amersham Pharmacia Biotech) in 0.2 M NH4HCO3. The tetra- and larger oligosaccharide fractions were pooled, evaporated to dryness, and subjected to chromatography on a column of Bio-Gel P-10 (Bio-Rad) run in 0.5 M NH4HCO3 at a flow rate of 2 ml/h. Fractions corresponding to hexa-, octa-, deca-, and dodecasaccharides were pooled, desalted by repeated evaporation to dryness and stored at -20°C until further use. The specific radioactivities of the saccharides were determined by scintillation counting and the carbazole reaction for hexuronic acid (Bitter and Muir, 1962). HS from metabolically [3H]GlcN labeled Madin-Darby canine kidney (MDCK) cells, grown in the absence or presence of sodium chlorate, was isolated according to the protocol described earlier for purification of HS from mammary epithelial cells (Safaiyan et al., 1998). The culture conditions for MDCK cells have been described elsewhere (Svennevig et al., 1995). Binding experiments
Recombinant human FGF-1 and FGF-2 (PeproTech) produced in a bacterial expression system were used in all binding assays. In analytical experiments (Maccarana and Lindahl, 1993), 0.5 µg of FGF was incubated with radiolabeled saccharide in 200 µl of phosphate-buffered saline (PBS, pH 7.4) containing 0.1 mg/ml bovine serum albumin (Sigma) at room temperature for 2 h. The mixture was rapidly passed through a nitrocellulose filter (Sartorius, diameter 25 mm, pore size 0.45 µm) which was previously washed with PBS using a vacuum-assisted filtering apparatus, followed by two washes with 5 ml of PBS. Proteins and protein-bound saccharides bind to the filter whereas free saccharides pass through the filter. Radiolabeled saccharides were released from the filter with 2 M NaCl, and the radioactivity was measured in a scintillation spectrometer. In preparative experiments, 30 µg of FGF-1 and 20 µg FGF-2 were used with a larger filter (38 mm in diameter). The proteins were incubated together with 3H-labeled NS-domains or partially de-O-sulfated heparin hexasaccharides in 2 ml of PBS under conditions described for the analytical experiments. The bound and unbound oligosaccharides were recovered from the filter-bound and flow-through fractions, respectively, and desalted by passage through PD-10 or Hi-Trap columns (Amersham Pharmacia Biotech) in water. Finally, the desalted oligosaccharide fractions were dried in a centrifugal evaporator and subjected to compositional disaccharide analysis or to rebinding experiments. Compositional disaccharide analysis
NS-domains and heparin oligosaccharides were subjected to cleavage by HNO2 at pH 1.5 (Shively and Conrad, 1976). At this pH, the reagent cleaves glucosaminidic linkages at the GlcNSO3 residues and results in a near-quantitative degradation of the N-sulfated oligosaccharides into disaccharides. The disaccharide derivatives were radiolabeled by reduction with 0.25 mCi of NaB3H4 and recovered by passage through a column of Sephadex G-15 (1 × 190 cm) in 0.2 M NH4HCO3. Appropriate fractions were pooled, desalted by repeated evaporation to dryness, and analyzed by anion-exchange HPLC (Partisil-10 SAX, 4.6 × 250 mm; Whatman Inc.). The column was eluted by a step gradient of KH2PO4 at a flow rate of 1 ml/min, and radioactivity in the effluent was monitored by a flow radioactivity detector. The different HexA-aManR disaccharide derivatives were identified by comparing their elution positions with those of standard heparin disaccharides. The proportions of the non-O-sulfated HexA-aManR residues in the total disaccharide preparations were determined by high voltage paper electrophoresis on Whatman 3MM paper in 83 mM pyridine/50 mM acetic acid (pH 5.3; 80 V/cm). The paper strips were dried, cut into 1 cm pieces, and analyzed for radioactivity. The elution positions of the sample disaccharides were compared with those of 3H-labeled nonsulfated, monosulfated, and disulfated disaccharide standards.
We thank Dr. Dorothe Spillmann for the generous gift of heparin oligosaccharides and Dr. Emadoldin Feyzi for useful discussions. This study has been supported by the Swedish Medical Research Council (Grant K96-03P to M.S. and 2309 to U.L.), Swedish Cancer Society (Grant 3919-B97 to M.S.), Polysackaridforskning AB, Uppsala, and The Medical Faculty of Uppsala University.
aManR, 2,5-anhydromannitol; FGF, fibroblast growth factor; GlcA, d-glucuronic acid; GlcN, glucosamine; HPLC, high performance liquid chromatography; HS, heparan sulfate, IdoA, l-iduronic acid; NA, N-acetylated; NS, N-sulfated; PDGF, platelet-derived growth factor.
Preparation
Disaccharide composition (% of total disaccharides)
HexA-aManR
GlcA(2-OSO3)-aManR
GlcA-aManR (6-OSO3)
IdoA-aManR (6-OSO3)
IdoA(2-OSO3)-aManR
IdoA(2-OSO3)-aManR(6-OSO3)
Aorta HS
Old subject (76 yr.)
NAa
1
4
2
82
12
Young subject (22 yr.)
NA
1
3
2
86
8
Intestinal HS 10-mer
11
NDb
9
3
58
20
Pref. 6-O-desulfated 6-mer
14
ND
3
9
61
14
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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