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Volume 270, Number 49, Issue of December 8, 1995 pp. 29586-29593
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Heparan Sulfate Oligosaccharides That Bind to Hepatocyte Growth Factor (*)

(Received for publication, August 17, 1995)

Satoko Ashikari ,  Hiroko Habuchi ,  Koji Kimata (§)

From the Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-11, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Proteoglycans from rat liver had the ability to bind hepatocyte growth factor (HGF). Digestion of the proteoglycans with heparitinase resulted in the complete loss of the activity, while the digestion with chondroitinase ABC had no effect. Heparan sulfate (HS)-conjugated gel also bound HGF, and the binding was competitively inhibited by heparin and bovine liver HS, but not by Engelbreth-Holm-Swarm sarcoma HS, pig aorta HS, or other glycosaminoglycans, suggesting the specific structural domain in HS for the binding of HGF.

Among limited digests with heparitinase I of bovine liver HS, octasaccharide is the minimal size to bind HGF. Comparison of the disaccharide unit compositions revealed a marked difference in IdoA(2SO(4))-GlcNSO(3)(6SO(4)) unit between the bound and unbound octasaccharides. The contents of this disaccharide unit were calculated to be 2 mol/mol for the bound octasaccharide but 1 mol/mol for the unbound one. Considering both the substrate specificity and properties of heparitinase I, the above results suggest that the bound octasaccharide should contain two units of IdoA(2SO(4))-GlcNSO(3)(6SO(4)) contiguously or alternately in the vicinity of the reducing end. The bound decasaccharide was more than 20 times as active as the unbound one with regard to the ability to release HGF bound to rat liver HS proteoglycan. The ability was comparable to the one-fourth of that of heparin.

INTRODUCTION

HS (^1)has been shown to have activities to bind to various molecules(1) . Of those, heparin-binding growth factors are particularly important, considering the physiological significance of potential ligands of HS(1) . bFGF is such a typical molecule and was detected as a complex with HSPG in the extracellular matrix such as basement membranes of the kidney glomerulus(2) . In addition, the low affinity receptor for bFGF on the cell surface was identified to be a cell-surface HSPG(3, 4) . Recent studies (5, 6, 7, 8) have shown that the binding of bFGF to the cell-surface and/or extracellular matrix HSPG is essential for the interaction of bFGF with its high affinity receptor. Heparin or HS may also be involved in protecting bFGF from protease digestion or heat/acid inactivation(9) . It is of note here that the binding of bFGF to HS requires the domain structure composed of a cluster of IdoA(2S)-GlcNS units(10, 11, 12, 13) .

HGF was identified initially as a mitogen for hepatocytes(14, 15) . Subsequently, HGF was found to be identical not only with a scatter factor (16) but also with a tumor cytotoxic factor(17) . Thus, HGF promotes the dissociation of epithelial cells and vascular endothelial cells in vitro and stimulates angiogenesis in vivo(18, 19) . In addition, HGF is considered to be a unique pleiotropic factor that acts as a mitogen, a tumor suppresser, a motogen, and a morphogen. Further, HGF may mediate epithelial and mesenchymal interactions during embryogenesis, organ repair, and neoplasia(20) .

HGF is known to have the ability to bind to heparin, and there are two classes of receptors for HGF with the different affinities(16, 21, 22, 23, 24) . The high affinity receptor (K 4.6 pM) (21) on rat hepatocytes was identified as the c-met proto-oncogene product, a transmembrane tyrosine kinase that is expressed predominantly on epithelial cells(16, 22, 25) . The low affinity receptor (K 275 pM) (21) was found to be a HSPG at the cell surface. Possible functional consequences after binding are as follows; stabilization of HGF(26, 27) , induction of conformational changes to fit HGF to the high affinity receptor(28, 29) , or, conversely, blocking of the biological activity due to ligand sequestering(30) . HSPGs in rat liver are identified as perlecan, syndecan, and fibroglycan(31, 32, 33, 34) . However, it remains to be determined which is likely for a low affinity receptor. A mutant HGF without the affinity for heparin showed neither the affinity for c-met protein nor the biological activity(35, 36, 37, 38, 39) . However, exogenous addition of heparin reduced the interaction of HGF with c-met protein (23, 28) and, consequently, reduced the mitogenic (40, 41) and motogenic (42) responses of cells to HGF. This was explained by the observation that a HGF-exogenous heparin complex could not be bound to c-met protein(28) , which suggests, interestingly, that exogenous heparin does not function as the cell-surface HSPG. Certain molecular structures and/or spatial localization of endogenous HSPG may be important in regulating the binding of HGF to c-met protein(28) . Therefore, the significance of interaction between cell-surface HSPG and HGF may be the same as that of bFGF, but the mechanism appears to be different and complex. To understand it, the precise analysis for the interaction between HSPG and HGF is needed.

In this study, we fractionated HS oligosaccharides prepared from the HS digested with heparitinase I, in accordance with the different affinities to HGF, and characterized a possible structure involved in the HGF binding. In addition, we showed that the addition of oligosaccharides with HGF binding activity to dishes coated with HSPG could release bound HGF from the HSPG.

EXPERIMENTAL PROCEDURES

Materials

Heparin was purchased from Sigma. HSs from pig aorta, pig liver, bovine liver, and EHS sarcoma were gifts of K. Yoshida and T. Harada, Seikagaku Corp. Chondroitin 4-sulfate from whale cartilage, chondroitin sulfate E from squid cartilage, dermatan sulfate from pig skin, hyaluronic acid, heparitinase I (Flavobacterium heparinum, EC 4.2.2.8), heparitinase II (F. heparinum, no number assigned), heparinase (F. heparinum, EC 4.2.2.7), and chondroitinase ABC (Proteus vulgaris, EC 4.2.2.4) were obtained from Seikagaku Corp (Tokyo, Japan). [I]NaI (17.4 Ci/mg) and [^3H]NaBH(4) (21 Ci/mmol) were purchased from Amersham Japan (Tokyo, Japan). Recombinant human HGF was a gift of Mitsubishi Kasei Co. (Yokohama, Japan). Sephadex G-50, CNBr-activated Sepharose 4B, and epoxy-activated Sepharose 6B were purchased from Pharmacia (Uppsala, Sweden). Anti-digoxigenin-AP, Fab fragments, 5-bromo-4-chloro-3-indolyl phosphate, 4-toluidine salt, and nitro blue tetrazolium chloride were purchased from Boehringer Mannheim Biochemica (Mannheim, Germany).

Preparation of Proteoglycans from Rat Liver

Liver was quickly excised. Livers from five rats (total wet weight, approximately 65 g) were rinsed with PBS, cut into small pieces, and then homogenized in the 4 M guanidine HCl extraction solution containing 50 mM sodium acetate, 10 mM EDTA, 10 mMN-ethylmaleimide, 1 mM phenylmethanesulfonyl fluoride, 0.1 M 6-aminohexanoic acid, 20 mM benzamidine HCl, 2% (v/v) Triton X-100. The homogenate (approximately 360 ml) was stirred at 4 °C for 48 h. Insoluble residues were removed by centrifugation at 12,000 times g for 30 min at 4 °C. The supernatant was recovered. Twenty ml of the supernatant solution were diluted with 19 volumes of 7 M urea buffer (7 M urea, 20 mM Tris-HCl, pH 7.2, 10 mM EDTA, 5 mMN-ethylmaleimide, 0.5 mM phenylmethanesulfonyl fluoride, 2% (v/v) Triton X-100), and was applied to DEAE-Sephacel (2 ml) equilibrated with 7 M urea buffer at 4 °C. The column was washed with 10 ml of 0.2 M NaCl in 7 M urea buffer. Proteoglycans were eluted with 6 ml (3 volumes of the column) of 2 M NaCl in 7 M urea buffer. For the complete separation, the elute was diluted with 9 volumes of 7 M urea buffer, then applied to the second DEAE-Sephacel (1 ml). The column was washed twice with 5 ml of 0.2 M NaCl in 7 M urea buffer. A proteoglycan fraction was eluted with 3 ml of 2 M NaCl in 7 M urea buffer, precipitated with 3 volumes of 95% (v/v) ethanol containing 1.3% (w/v) potassium acetate. The precipitate was dissolved in 300 µl of H(2)O.

Preparation of Digoxigenin-conjugated HGF and [I]HGF

Digoxigenin-conjugated or I-labeled HGF was prepared according to the method recommended by the manufacturer. Briefly, 10 µg of HGF in 200 µl of 0.2 M phosphate buffer, pH 8.5. were added into N-acetylated heparan sulfate and then mixed with 8.75 nmol of digoxigenin in dimethyl sulfoxide followed by 2 h of incubation at room temperature. The HGF solution was applied to 0.5 ml of heparin-Sepharose gel equilibrated with phosphate-buffered saline (PBS; 0.1 M sodium phosphate, 1.37 M NaCl, 2.7 mM KCl, pH 7.2) containing 0.02% (v/v) Triton X-100 and 1 mg/ml BSA (solution A). Heparin-Sepharose gels were washed with 5 ml of solution A. Digoxigenin-conjugated HGF was then eluted with 2.5 ml of 2 M NaCl in solution A.

IODO-BEADS (Pierce) were kept in 100 µl of 0.1 M sodium phosphate containing 0.5 mCi of [I]NaI at room temperature for 5 min. Then 3 µg of HGF were added, and the suspension was kept for 10 min at room temperature. I-Labeled HGF was desalted using a Sephadex G-25 column (0.9 cm times 3.9 cm). Specific radioactivity of I-HGF was 2.5 5.7 times 10^4 dpm/ng.

Binding Assay of Digoxigenin-HGF to PG from Rat Liver

15 µl of the PG fraction (equivalent to 0.2 g of rat liver) was subjected to 5% SDS-PAGE under nonreducing conditions, electrotransferred to a poly(vinylidene fluoride) membrane (ProBlott) (Applied Biosystems Japan) at 10 V and 4 °C overnight. Each membrane was blocked with a blocking solution (Boehringer Mannheim Biochemica) at room temperature for 30 min and then digested with a mixture of 10, 1, and 10 milliunits/ml heparitinase I, II, and heparinase (the HSase mixture) plus or minus chondroitinase ABC in 50 mM Tris-HCl, pH 7.2, 1 mM CaCl(2), 0.5 mg/ml BSA in the presence of protease inhibitors excepted no addition of EDTA as described previously(43) . Some membranes were digested only with 33 milliunits/ml chondroitinase ABC in 0.5 mM Tris-HCl, pH 8.0, 0.5 mg/ml BSA at 37 °C for 1 h. Membranes were washed three times with TBS (50 mM Tris HCl, pH 7.5, 0.15 M NaCl), and then subjected to HGF binding in the solution containing 0.2 µg/ml digoxigenin-HGF, 0.2 mg/ml chondroitin 4-sulfate, 0.9 mM CaCl(2). After 1 h at room temperature, unbound digoxigenin-HGF was removed by washes with TBS as described above. Membranes were then treated with anti-digoxigenin-AP, Fab fragments (1:500 dilution) for 1 h. Unbound antibodies were washed out as described above, and membranes were soaked in 5-bromo-4-chloro-3-indolyl phosphate, 4-toluidine salt (1:200 dilution) and nitro blue tetrazolium (1:260 dilution).

Preparation of HS-conjugated Sepharose Gel

HS-Sepharose gel was prepared by the method reported previously with a minor modification(44) . 3-Amino-2-hydroxypropyl-derivatized Sepharose gel was prepared from epoxy-activated Sepharose 6B gel. A portion (1 g) of amino-Sepharose gels thus obtained was suspended in 1 ml of 0.2 M phosphate buffer, pH 7.2, and conjugated with 30 mg of HS (pig liver) by adding 3 mg of NaBH(3)CN. The suspension was kept at room temperature for 48 h with a gentle shaking. The gel was washed several times with PBS. The amount of immobilized HS was 2.4 mg/ml of gel. The gels were, then, suspended in PBS(+) containing 20 mg/ml BSA, and gently stirred for 1 h at room temperature to block nonspecific binding sites. After an extensive wash with PBS(+), the gels were suspended in PBS(+) containing 0.02% NaN(3) to give a 25% (w/v) suspension and stored at 4 °C until use.

Competitive Inhibition Assay of [I]HGF Binding to Immobilized HS with GAGs

The binding reaction was performed in 100 µl of solution containing 1.25% (w/v) HS-conjugated gel, 1 times 10^4 dpm of I-HGF, 0.1100 µg/ml GAG, and 1 mg/ml BSA. After 1 h of incubation at 4 °C with gentle agitation, the mixture was diluted with 3 volumes of PBS(+) and centrifuged (630 times g, 3 min) in a microcentrifuge tube with a membrane filter (UFC30HV00; Millipore, Bedford, MA). The gel on the membrane was washed thoroughly with PBS(+), and the radioactivity bound to the gel was determined in a -radiation counter. Nonspecific binding was determined as the radioactivity bound to the gel in the presence of 100 µg/ml heparin.

Fractionation of HS

Bovine liver HS was fractionated by Dowex 1 column chromatography. The fraction eluted with 0.5 1.25 M NaCl was termed bovine liver HS fraction 1. The fraction eluted with 1.25 1.75 M NaCl was further fractionated by DEAE-Sephacel column chromatography. The subfractions eluted with 0.42 0.48 M and 0.48 0.62 M NaCl in 50 mM Tris-HCl, pH 7.2, were termed bovine liver HS fractions 2 and 3, respectively. bFGF-bound HS and unbound HS were prepared from EHS mouse sarcoma HS by the method reported previously (10) .

Preparation of Bovine Liver HS Oligosaccharides

25 mg of bovine liver HS fraction 2 was digested with 0.25 unit of heparitinase I at 37 °C for 1 h. To prepare ^3H-labeled oligosaccharides, a portion of the digest (2 mg) was dissolved in 200 µl of 0.1 M Tris-HCl, pH 8.8, and reduced with 2 mCi of [^3H]NaBH(4) (specific activity, 1.25 Ci/mmol) in 170 µl of 0.1 M NaOH and kept for 4 h at room temperature. The solution was adjusted to pH 4 with acetic acid to destroy excess [^3H]NaBH(4) and then to pH 7 with NaOH. [^3H]Heparan sulfate oligosaccharides were precipitated twice with 75% (v/v) ethanol to concentrate. ^3H-Labeled and nonlabeled HS oligosaccharides were fractionated, respectively, by chromatography on Sephadex G-50 column (1.2 cm times 124 cm) equilibrated with 0.2 M ammonium acetate. Fractions containing HS oligosaccharides were pooled as shown in Fig. 2. Each fraction was rechromatographed. Apparent molecular weights of those fractions (HS-I, HS-II, HS-III, HS-IV, HS-V, and HS-VI) were 800, 1300, 1700, 2100, 2600, and 3000, respectively. The specific activities of those ^3H-labeled fractions were 8.9 times 10^4, 4.3 times 10^4, 2.6 times 10^4, 2.5 times 10^4, 2.2 times 10^4, and 3.9 times 10^4 dpm/nmol, respectively.

Figure 2: Sephadex G-50 chromatography of [^3H]heparan sulfate oligosaccharides. 25 mg of bovine liver HS fraction 2 were subjected to partial digestion with 0.25 unit of heparitinase I at 37 °C for 1 h. A portion of the digest (2 mg) was then reduced with [^3H]NaBH(4). ^3H-Labeled oligosaccharides were subjected to the gel chromatography, and fractions (1 ml/tube) were collected. The fractions shown by solid horizontal bars were pooled and desalted for further analysis. These pooled fractions are as referred to in Fig. 3. V(0), void volume; V, total volume. Elution positions of molecular weight markers are indicate by arrows: a, [^3H]heparin octasaccharide; b, [^3H]chondroitin hexasaccharide; c, [^3H]chondroitin tetrasaccharide; d, DeltaDi-0S; e, [^3H]D-glucosamine.


Figure 3: Percent proportions of oligosaccharides with the binding activity to HGF affinity column. Oligosaccharide fractions (4 nmol) containing 1 times 10^5 dpm of ^3H-label which were prepared from bovine liver HS fraction 2 as shown in Fig. 2A and from heparin by degradation with nitrous acid at pH 1.5 (B) were subjected to a HGF affinity chromatography as described under ``Experimental Procedures.'' After incubated at 4 °C for 1 h, the column was washed with solution B and then eluted with 2 M NaCl, 10 mM Tris-HCl, pH 7.2. The elution was analyzed for radioactivity.


Preparation of Heparin Oligosaccharides

Degradation of heparin with nitrous acid at pH 1.5 and reduction of the products with [^3H]NaBH(4) were carried out as described by Shively and Conrad(45) . ^3H-Labeled heparin oligosaccharides were fractionated by gel chromatography as described in the last section using the same column. Fractions containing tetra- to dodecasaccharides were designated Hep-4, Hep-6, Hep-8, Hep-10, and Hep-12, in order of their molecular sizes, and had the specific activities of 3.9 times 10^5, 5.9 times 10^5, 3.0 times 10^5, 4.0 times 10^5, and 1.7 times 10^5 dpm/nmol, respectively.

Preparation of HGF-conjugated Sepharose Gel

HGF-conjugated Sepharose gel was prepared by the reported method(10) . HGF (1 mg) was coupled to 1.8 ml of CNBr-activated Sepharose 4B gel according to the method recommended by the manufacture. N-Acetylated heparin (10 mg) was added to the coupling reaction mixture to protect the heparan sulfate-binding sites in HGF.

HGF Affinity Chromatography of HS and Heparin Oligosaccharides

About 4 nmol of the HS or heparin oligosaccharide fractions containing 1 2 times 10^5 dpm of ^3H-label were dissolved in 1 ml of 10 mM Tris-HCl, pH 7.2, 0.15 M NaCl, 0.9 mM CaCl(2), 0.2 mg/ml chondroitin 4-sulfate (solution B), and applied to a syringe column of HGF-Sepharose (1 ml) equilibrated with solution B at 4 °C. Chondroitin 4-sulfate was included in solution B to prevent the nonspecific binding. The column was shaken gently for 1 h, then washed with 10 ml of solution B, and eluted with 3 ml of 2 M NaCl in10 mM Tris-HCl, pH 7.2. The radioactivity of the eluate was detected in a liquid scintillation counter.

Mono Q Column Chromatography

The eluate from HGF affinity column was subjected to gel chromatography on Sephadex G-50 (1.2 cm times 120 cm) to remove coexisting chondroitin 4-sulfate. The oligosaccharides were recovered from the retarded fractions and then desalted using a fast desalting column (Pharmacia). The fractions were applied to a mono Q column (Pharmacia). The chromatography was performed by a linear gradient elution from 0 to 2.0 M NaCl in 50 mM Tris-HCl, pH 7.2.

Composition Analysis of HS and Its Oligosaccharides

About 1 µg of HS or HS oligosaccharides was digested with a mixture of 1 milliunit of heparitinase I, 0.1 milliunit of heparitinase II, and 1 milliunit of heparinase in 50 µl of 50 mM Tris-HCl, pH 7.2, 1 mM CaCl(2), 5 µg of BSA at 37 °C for 1 h. Unsaturated disaccharide products were analyzed by HPLC using a polyamine-bound silica PAMN column (YMC). The elution was performed with a linear gradient from 40 to 550 mM KH(2)PO(4) and with a subsequent wash with 550 mM KH(2)PO(4) at a flow rate of 1.2 ml/min at 40 °C. The elution was monitored by uv absorption at 232 nm. Each peak was identified by its retention time which was standardized with authentic unsaturated disaccharides as described previously(46) .

Degradation of about 1 µg of HS oligosaccharides with nitrous acid at pH 1.5 and reduction of degradation products with [^3H]NaBH(4) were carried out as described by Shively and Conrad(45) . The products were desalted using Fast desalting columns. The fractions containing disaccharides were collected and analyzed by HPLC on a Partisil-10 SAX column (Whatman, Clifton, NJ) as described by Bienkowski and Conrad(47) . The elution was monitored by measuring the radioactivity in a liquid scintillation counter.

HGF-releasing Activity of HS Oligosaccharides and Heparin

The releasing activity was measured by ELISA by the method recommended by the manufacture with a minor modification. A 96-well Nunc-Immuno Plate MaxiSorp (A/S Nunc, Roskilde, Denmark) was coated with 0.1 nmol (as hexuronic acid) of rat liver proteoglycans overnight at 4 °C. Wells were washed three times with 200 µl of PBS and then blocked with 200 µl of PBS containing 10 mg/ml BSA (solution C) for 1 h at 37 °C with a gentle shaking. Wells were washed three times with 200 µl of PBS. Then 100 µl of the solution C containing 0.2 µg/ml digoxigenin-HGF, 0.2 mg/ml chondroitin 4-sulfate, 0.9 mM CaCl(2) was added into each well. After 1 h at room temperature, unbound digoxigenin-HGF was removed by washes as described above. Then, 100 µl of PBS containing 1 ng to 10 µg of heparin or 1 pmol to 1 nmol as hexuronic acid of HS oligosaccharides were added into wells. After 1 h at room temperature, wells were washed as above, and then alkaline phosphatase-conjugated Fab fragments of anti-digoxigenin antibody (1:500 dilution) were added. After 1 h at room temperature, unbound Fab fragments were removed by washing, and the alkaline phosphatase substrate (1 mg/ml of pNPP in 1 mol/liter diethanolamine, pH 9.8, containing 0.5 mmol/liter) was added into each well. The enzyme activity in each well was measured by a MTP-100 microplate reader (Corona Electric Co., Ibaragi, Japan).

RESULTS

Binding of HGF to Rat Liver Proteoglycans

PG preparations from whole rat liver were subjected to SDS-PAGE. PGs separated on the gel were transferred to a membrane for the blot analysis of HGF binding using digoxigenin-conjugated HGF. At least three species of PGs showed the affinity for HGF, of which molecular masses were 220, 180, and 120 kDa (Fig. 1, lane 1). When these PGs on the membrane were digested with a mixture of heparitinases I and II and heparinase (the HSase mixture) before exposing to HGF, none of them could bind HGF (Fig. 1, lane 2). However, the digestion of the PGs with chondroitinase ABC had no effect on the HGF binding (Fig. 1, lane 3). The results, therefore, suggested that HGF appeared to bind to proteoglycans only with HS chains, but not with chondroitin sulfate or dermatan sulfate chains.

Figure 1: Analysis for digoxigenin-HGF binding activity of PG fraction from rat liver. 15 µl of the PG fraction from rat liver which was prepared as described under ``Experimental Procedures'' were subjected to SDS-PAGE, and subsequently transferred to a poly(vinylidene fluoride) membrane. The membrane was treated with PBS (lane 1), with a mixture of heparitinases I and II and heparinase (lane 2), with chondroitinase ABC alone (lane 3), and with a mixture of heparitinases I and II, heparinase, and chondroitinase ABC (lane 4) at 37 °C for 1 h. After being washed, the membrane was subjected to analysis for digoxigenin-HGF binding as described under ``Experimental Procedures.''


HGF Binding Activities of Various Glycosaminoglycans

The activities of various GAGs were assessed by their capacities to inhibit I-HGF binding to pig liver HS-conjugated Sepharose gel as described under ``Experimental Procedures.'' Table 1shows IC values of various GAGs which were their concentrations to inhibit 50% the total radioactivity of I-HGF bound to the HS-conjugated gels. Heparin exhibited the highest inhibition activity (IC = 0.15 µg/ml). Bovine liver HS fraction 3 (IC = 0.75 µg/ml) was approximate in the inhibition activity to heparin. Bovine liver HS fraction 2 exhibited inhibition activity less than that of heparin (IC = 5.4 µg/ml). When bovine liver HS fractions 2 and 3 were digested with the HSase mixture (see ``Experimental Procedures'') prior to the addition, the inhibition activity completely disappeared (data not shown). This result also supported the fact that HS chains bound HGF. However, bovine liver HS fraction 1 and pig liver HS exhibited weak inhibition activity (IC = 45 and 38 µg/ml, respectively), and neither pig aorta HS nor EHS tumor HS showed inhibition activity. The results suggest that HSs vary depending on their differences in species and tissue origins with respect to their affinity for HGF. None of other GAGs tested exhibited inhibition activity.

Heparin and bovine liver HS fraction 3 that showed the high inhibition activity are higher in the sulfation degree (2.59/disaccharide and 2.12/disaccharide, respectively) than other GAGs, suggesting the involvement of the negative charge in the activity. However, bovine liver HS fraction 2 with the significant inhibition activity is apparently lower in the sulfation degree than chondroitin sulfate E or chemically sulfated dermatan sulfate that showed no inhibition activity (1.21/disaccharide for bovine liver HS fraction 2, compared with 1.43/disaccharide for chondroitin sulfate E or 1.31/disaccharide for chemically sulfated dermatan sulfate). Taken together, it is likely that binding of HGF to HS/heparin is not simply due to an electrostatic interaction, but may depend on some unique structural units in HS. Indeed, because HS from EHS tumor, which had such units for bFGF-binding (IdoA(2SO(4))-GlcNSO(3)-rich domain)(10) , had no inhibition activity, binding of HGF to HS may require structural units of HS distinct from the ones for bFGF binding.

Fractionation of HGF-bound HS Oligosaccharides

To determine HGF binding structures in HS, we first prepared HS oligosaccharide with various HGF binding activities from bovine liver HS fraction 2. Limited digestion of the fraction with heparitinase I was performed, which attacks preferentially glucosaminic linkages to nonsulfated hexuronic acid residues in HS. Oligosaccharide products were reduced with [^3H]NaBH(4), and ^3H-labeled HS oligosaccharides thus obtained were subjected to a molecular size fractionation by Sephadex G-50 column chromatography (Fig. 2). Fractions of HS oligosaccharides with different sizes were rechromatographed on the same column for further purification and designated as shown in Fig. 2. The apparent molecular weights of HS oligosaccharide fractions calculated from their relative elution positions to those of standard oligosaccharides were as follows; HS-I, 800; HS-II, 1300; HS-III, 1700; HS-IV, 2100; HS-V, 2600; and HS-VI, 3000.

Each ^3H-labeled HS oligosaccharide fraction (4 nmol) was applied to a column of HGF-conjugated Sepharose equilibrated with solution B (10 mM Tris-HCl, pH 7.2, 0.15 M NaCl, 0.9 mM CaCl(2), 0.2 mg/ml chondroitin 4-sulfate). After a wash with solution B, the bound ^3H-labeled oligosaccharides were eluted with 2 M NaCl in 10 mM Tris-HCl, pH 7.2. The percent proportion of the bound radioactivity to the applied radioactivity for each fraction is shown in Fig. 3A. The proportion increased as the molecular size increased. However, a sharp increase in the proportion was observed between HS-III and HS-IV (4 and 17%, respectively). The results suggest that HS-IV is the smallest size of the structures required for HGF binding, which was estimated to be HS octasaccharide judging from its molecular weight and disaccharide composition as described below (see Table 2). The chain size dependence of the heparin-binding to HGF was also determined using ^3H-labeled heparin oligosaccharides (Fig. 3B). The octasaccharide (Hep-8) was also the smallest fraction to show a sharp increase in the binding proportion, although the proportions tended to increase as the size of oligosaccharides increased.

The results suggest that the sizes of HS/heparin saccharides are one of the structural factors required for the binding of HS/heparin to HGF and the octasaccharides are the minimal.

Characterization of HGF-bound and -unbound Oligosaccharides

Bound and unbound oligosaccharides of HS-IV were prepared as described under ``Experimental Procedures.'' Rechromatography of the HS-IV-unbound fraction showed that more than 95% of the radioactivity passed through the HGF column reproducibly (data not shown), indicating no significant contamination of HGF-bound species. Both bound and unbound fractions of HS-IV were further fractionated in accordance with their negative charges by ion-exchange chromatography on a Mono-Q column (Fig. 4). Most of HS-IV-bound fraction was eluted at the NaCl concentration of above 0.88 M (fractions 43-50; designated IV-B in Fig. 4A). On the other hand, the HS-IV-unbound fraction was eluted with a broad distribution pattern. But 16% of the HS-IV-unbound fraction was recovered in the subfraction similar in the elution positions to HS-IV-bound fraction (fractions 44-50; designated IV-UB in Fig. 4A). Therefore, the difference in HGF affinity between IV-B and IV-UB may be due to structural factors other than their net negative charges.

Figure 4: Mono Q FPLC of heparan sulfate oligosaccharides fractions. A, HGF column-unbound (bullet) and -bound (circle) fractions of [^3H]HS-IV (2 times 10^5 and 4 times 10^4 dpm, respectively) were desalted, freed from chondroitin 4-sulfate, concentrated, and applied to mono Q column. B, HGF column-unbound (bullet) and -bound (circle) fraction of [^3H]HS-V (7.5 times 10^4 and 2 times 10^4 dpm, respectively) were treated as described above. The elution was performed with the indicated NaCl gradient in 50 mM Tris-HCl, pH 7.2, and fractions (1 ml) were served for the measurement of the radioactivity. Fractions were pooled as shown by closed and open bars and designated as indicated above. In a separate experiment for the compositional analysis, nonlabeled fractions corresponding to ^3H-labeled fractions as described above (unbound HS-IV, 10 nmol; bound HS-IV, 2 nmol; unbound HS-V, 4.4 nmol; bound HS-V, 2.6 nmol) were also applied to the same mono Q column as above. Fractions corresponding to labeled fractions shown by closed and open bars were pooled and desalted for analysis.


Both nonlabeled IV-B and IV-UB, after the extensive digestion with the HSase mixture, were subjected to the compositional analysis by HPLC on a polyamine silica column as described under ``Experimental Procedures'' (Table 2). Comparison of the unsaturated disaccharide compositions between them showed a marked difference: 47% of the disaccharides obtained from IV-B were DeltaDi-(N,6,U)triS, whereas only 26% were in those obtained from IV-UB. Considering the molecular weights of IV-B and IV-UB, these composition data suggested that IV-B and IV-UB corresponded to the octasaccharide (4 disaccharide units) containing at least 2 HexA(2SO(4))-GlcNSO(3)(6SO(4)) units and a mixture of the octa- and decasaccharides containing only 1 above unit, respectively. Moreover, considering both the substrate specificities and catalytic properties of enzymes used for the preparation of these HS oligosaccharides, nonreducing ends of the HS oligosaccharides are supposed to have nonsulfated unsaturated HexA. Hence, 2 HexA(2SO(4))-GlcNSO(3)(6SO(4)) units in HGF-bound octasaccharides should be localized contiguously or alternately at or near the reducing ends.

HS-V fraction was also fractionated into HGF-bound and -unbound fractions by HGF affinity chromatography. Both V-B and V-UB were fractionated on a Mono-Q column (Fig. 4B), and the resulting fractions (V-B and V-UB) were subjected to the compositional analysis. V-B that was estimated to be a decasaccharide contained more than 50% HexA(2SO(4))-GlcNSO(3)(6SO(4)), but V-UB contained only 12% (Table 2). Thus, the composition analysis gave similar results to those obtained with IV-B and IV-UB fractions.

To identify the hexuronic acid residues participating in HGF binding, IV-B was treated with nitrous acid at pH 1.5 and then reduced with [^3H]NaBH(4) according to the method of Shively and Conrad(45) . 85% of the total labeled saccharides were recovered in the disaccharide fraction (data not shown). The disaccharides were identified by HPLC on a SAX column. Of these disaccharides, 52% were IdoA(2SO(4))AMan(R)(6SO(4)), and only 2% were GlcA(2SO(4))AMan(R)(6SO(4)). Therefore, HexA(2SO(4))-GlcNSO(3)(6SO(4)), which was a major disaccharide component of IV-B, was an IdoA-type. The identification of hexuronic acid residues was also performed with the other HGF-bound fraction, V-B. Molar ratios of disaccharides per mol of IV-B or V-B estimated from both the results of Table 2and the above identification of hexuronic acid residues are shown in Table 3. In both IV-B and V-B, IdoA(2SO(4))-GlcNSO(3)(6SO(4)) was the only component with the content close to or exceeding 2 mol/mol, suggesting an essential involvement of this disaccharide unit in the HGF binding. Other disaccharide components were present in less than 1 mol/mol. However, contents of N-sulfated disaccharides such as IdoA-GlcNSO(3) and GlcA-GlcNSO(3)(6SO(4)) were relatively high, compared to those of N-acetylated disaccharides, and the sum of these N-sulfated disaccharide contents was more than 1 mol/mol. The results suggest that clustering of 2 IdoA(2SO(4))-GlcNSO(3)(6SO(4)) units and one N-sulfated component (HexA-GlcNSO(3) or HexA-GlcNSO(3)(6SO(4))) may form the binding site for HGF.

HGF Releasing Activities of HS Oligosaccharides and Heparin from the Complex of HGF and HSPGs

Affinities to HGF of HS-bound and -unbound oligosaccharides and heparin were assessed by their releasing activities of HGF from the complex of HGF and HSPGs. The HSPG preparation from rat liver were used to coat ELISA plates. Digoxigenin-HGF was bound on the plate via coated HSPGs. After 1 h of incubation with oligosaccharides at various concentrations on the plate, digoxigenin-HGF yet bound on the plate was determined using anti-digoxigenin Fab fragment as described under ``Experimental Procedures.'' The HGF-releasing activity was compared among HGF-bound HS oligosaccharide (V-B), HGF-unbound HS oligosaccharide (V-UB), and heparin (Fig. 5). The concentrations to give a 50% release of bound HGF were 1.3, 5, and 110 ng/ml for heparin, V-B, and V-UB, respectively. The releasing activity of V-B was 20 times more active than V-UB and only one fourth less than heparin.

Figure 5: HGF releasing activity of V-B, V-UB, and heparin from the complex with HSPG. Releasing activity was detected by ELISA as described under ``Experimental Procedures.'' Digoxigenin-HGF was added into wells coated with rat liver proteoglycans (0.1 nmol as hexuronate). After 1 h, unbound digoxigenin-HGF was removed, and then V-B (circle), V-UB (bullet), and heparin (up triangle) at various concentrations were added. After 1 h, the wells were washed, then anti-digoxigenin-AP, Fab fragments were added to yield color. Nonspecific binding was determined using 100 ng/ml heparin.


DISCUSSION

Our present study has shown that HGF bound only to heparin and some species of HS, suggesting possible involvements of some unique structures on the chains in the binding (Table 1). HGF affinity gel chromatography of HS oligosaccharides prepared by a limited digestion of bovine liver heparan sulfate with heparitinase I has shown that minimal sizes of the chains for HGF binding are octasaccharide (Fig. 3). Bound and unbound octasaccharides thus obtained were subjected to structural analyses. HS-bound octasaccharides (IV-B) characteristically comprised 2 mol of IdoA(2SO(4))-GlcNSO(3)(6SO(4)) per molecule (Table 3). These results, considering the fact that their nonreducing ends were nonsulfated, unsaturated hexuronic acid, suggest that at least two IdoA(2SO(4))-GlcNSO(3)(6SO(4)) units are present contiguously or alternately each other at or near the reducing ends (see Fig. 6). The presence of this structural unit was also detected in the HS-bound decasaccharide fraction (V-B) (Table 3).

Figure 6: Minimal structures on heparan sulfate for HGF binding. Nonreducing ends of these HS octasaccharide were nonsulfated, unsaturated hexuronic acid. Structural variants in the HGF binding region are indicated by R. One R is the 6-O-sulfate group, the other R is hydrogen. N-Sulfate groups are not less than three groups in the molecule. Two IdoA(2SO(4))-GlcNSO(3)(6SO(4)) units (within the shaded boxes) are present contiguously (A and B) or alternately (C) at the reducing side or at the internal side.


Lyon et al.(48) have also suggested that heparan sulfate with a high affinity to HGF apparently has a sequence rich in IdoA and GlcNSO(3)(6SO(4)) residues. However, according to their results, no contiguous sequence of two or more IdoA(2SO(4))-containing disaccharides appeared to be absolutely necessary for the interaction with HGF, because most of fragments prepared from fetal skin fibroblast HS by digestion with heparinase I which specifically attacks N-sulfated disaccharides containing IdoA(2SO(4)) residue still retained a HGF affinity.

It is in question in our present study whether HexA(2SO(4))-GlcNSO(3) units are involved in the binding of HGF to HS directly, since these HexA(2SO(4))-GlcNSO(3) units comprised only 3.2% of the starting material, bovine liver HS fraction 2. However, HexA(2SO(4))-GlcNSO(3) units were not condensed into the HGF-bound fractions such as IV-B (Table 2). In addition, bFGF-bound HS from EHS tumor, which has been shown to be composed of some clusters of IdoA(2SO(4))-GlcNSO(3) units (10) , showed no significant inhibition in the HGF binding to HSPG (Table 1). Furthermore, the content of the IdoA(2SO(4))-GlcNSO(3) unit was somewhat higher in HGF-unbound HS octasaccharides (IV-UB) than in the corresponding HGF-bound HS octasaccharides (IV-B). These results suggest that HGF-binding structures of HS are apparently distinct from the bFGF-binding structures, and IdoA(2SO(4))-GlcNSO(3) units may not be important for binding of HGF to HS.

There are some different types of HSs with respect to their affinity to HGF. As shown in Table 1, bovine liver HS had a notably high affinity, which was almost comparable to that of heparin, and pig liver HS also showed a significant affinity. However, it is of note that pig aorta HS and EHS sarcoma HS showed no detectable affinity. In relation to this difference, rat liver has been found to contain at least three species of PGs with HGF affinity. Indeed, it has been shown that some clusters of IdoA(2SO(4))-GlcNSO(3)(±6SO(4)) units were present in rat liver HS(49) . We have also characterized the presence of highly sulfated HS in lung with a HGF affinity (data not shown). It is now known that lung acts as an endocrine organ with respect to HGF production, and HGF is active in the organogenesis and development of lung(50) . The results that HSs derived from some organs have some activities to bind HGF may suggest that HS may be important in regulating functional HGF activity.

Exogenous heparin reduced the HGF/c-met protein interaction (23, 28) and mitogenic (40, 41) and motogenic (42) responses, and does not simply function as a soluble form of the cell-surface HSPG. It is possible that certain molecular arrangements of the active units in endogenous HSPGs may be important in regulating the cell growth. Whether this particular structure of HSPG is required to promote HGF/c-met receptor interaction remains to be elucidated.

HS sequences required to bind to both acidic FGF and K-FGF (FGF-1 and FGF-4, respectively) and promote their signaling might be different from that for basic FGF (FGF-2)(12, 13) . In this study, HGF-binding structures have been shown to be different from the bFGF-binding structure. In addition, HGF-bound HS oligosaccharides had HGF-releasing activity that was 20 times higher than that of HGF-unbound HS oligosaccharides. There may be some difference in HS structures required for binding of the growth factors and their activation, depending on the difference in growth factors. Such differences of the polysaccharide structure could regulate cellular responses to different heparin-binding growth factors.

HGF in plasma after intravenous administration disappeared rapidly with an early phase half-life of 4 min(51) . On the other hand, heparin-HGF complex exhibits much lower clearances for hepatic uptake and plasma disappearance than HGF itself(27) . Heparin has anticoagulative activity, and it has been shown that the presence of 3-O-sulfate of glucosamine residues is crucial for the binding of heparin and HS to antithrombin III(1) . Since we could not detect the presence of 3-O-sulfate of glucosamine residue in HGF-bound octasaccharides, the complex of HGF with HGF-bound HS octasaccharides such as IV-B may be promising as a novel drug delivery system for HGF.

FOOTNOTES

*
This work was supported in part by grants-in-aid from the Ministry of Education, Culture and Science, Japan, Special Coordination Founds of the Science and Technology Agency of the Japanese Government, and a special research fund from Seikagaku Corporation. 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. Tel.: 81-52-264-4811 (ext. 2088); Fax: 81-561-63-3532.

(^1)
The abbreviations used are: HS, heparan sulfate; HGF, hepatocyte growth factor; bFGF, basic fibroblast growth factor; PG, proteoglycan; GAG, glycosaminoglycan; EHS, Engelbreth-Holm-Swarm; GlcNSO(3), N-sulfoglucosamine; IdoA, L-iduronic acid; HexA, hexuronic acid; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; DeltaDi-0S, 2-acetamide-2-deoxy-4-O-(4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid)-D-glucose; DeltaDi-(N,6,U)triS, 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo - alpha - L-threo - hex - 4 - enepyranosyluronic acid)-6-O-sulfo-D-glucose; AMan, 2,5-anhydro-D-mannose (the subscript R following this abbreviation refers to the corresponding alditol formed by reduction of the compound with NaBH(4)); PBS, phosphate-buffered saline; PBS(+), PBS containing Ca and Mg; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay.

ACKNOWLEDGEMENTS

We thank Dr. T. Ishi, Mitsubushi Kasei Co., for providing us with recombinant HGF, Dr. N. Koide for allowing us to use the HGF for this experiment, and Drs. K. Yoshida and T. Harada for preparing heparan sulfates from various animal species.

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