©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sulfated Oligosaccharides Promote Hepatocyte Growth Factor Association and Govern Its Mitogenic Activity (*)

Thomas F. Zioncheck (§) , Louise Richardson , Jun Liu , Ling Chang , Kathleen L. King , Gregory L. Bennett , Pèter Fügedi (1), Steven M. Chamow , Ralph H. Schwall , Robert J. Stack (1)(¶)

From the (1)From Genentech, Inc., South San Francisco, California 94080-4990 and Glycomed Inc., Alameda, California 94501

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Hepatocyte growth factor (HGF) is a potent mitogen, motogen, and morphogen for various epithelial cell types. The pleiotropic effects of HGF are mediated by its binding to a specific high affinity receptor, c-Met. In addition, HGF binds to heparan sulfate proteoglycans on cell surfaces and within the extracellular matrix. Incubation of HGF with 0.1, 1.0, and 10 µg/ml of heparin, heparan sulfate, or dextran sulfate resulted in a concentration-dependent increase in mitogenic potency in a primary rat hepatocyte bioassay, whereas sodium sulfate or fucoidan did not. Although co-incubation of HGF with sulfated compounds that enhanced HGF-dependent mitogenesis did not alter the binding isotherm of HGF for the c-Met receptor in a solid phase assay, an increase in autophosphorylation of the c-Met receptor in intact A549 cells was observed upon their addition. A series of chemically sulfated malto-oligosaccharides varying in unit size and charge was tested in the bioassay in order to provide additional insights into the nature of the HGF-heparin interaction. While sulfated di-, tri-, tetra-, and pentasaccharides did not significantly potentiate HGF-dependent mitogenesis, larger oligosaccharides such as the sulfated hexa-, hepta-, or a sulfated oligosaccharide mixture containing decasaccharides resulted in an approximate 2-, 4-, and 7-fold enhancement, respectively. We observed a correlation between the sulfated oligosaccharide preparations that enhanced mitogenic potency and those that promoted HGF oligomerization in vitro, as measured by gel filtration and analytical ultracentrifugation. These findings indicate that heparin-like molecules can stabilize HGF oligomers, which may facilitate c-Met receptor dimerization and activation.


INTRODUCTION

Hepatocyte growth factor (HGF),()also referred to as scatter factor, is a pleiotropic polypeptide produced by stromal cells. In addition to being an important mitogen in liver growth and regeneration, HGF is thought to be a critical factor in embryogenesis, angiogenesis, and wound healing. Indeed, HGF has mitogenic, motogenic, and morphogenic activity on various epithelial and endothelial cell types in culture (for review, see Ref. 1). A relatively large (82-84 kDa) growth factor with sequence similarity to plasminogen(2) , HGF is secreted as a single-chain promitogen. It requires endoproteolytic processing at the Arg-Val bond by an extracellular serine protease to produce an active heterodimer(3, 4, 5) . Processing to the heterodimer results in an -chain (69 kDa) consisting of a N-terminal hairpin loop followed by four kringle domains. The -chain (32 kDa) bears sequence similarity to serine proteases, although the Ser and His residues critical for catalysis are absent(2) .

Structure-function studies indicate that a region within the N-terminal 175 amino acids of HGF is necessary and sufficient for receptor binding, although additional determinants within the -chain are required for full mitogenic activity(6) . The effect of HGF on DNA synthesis, motility, and cell morphology are thought to be mediated by binding to a single high affinity (K = 20-30 pM) tyrosine kinase receptor called c-Met(7, 8) . Emerging evidence suggests that autophosphorylation of these receptors results from ligand-induced receptor dimerization, with subsequent transphosphorylation of cytoplasmic domains(9) . Dimeric growth factors, such as platelet-derived growth factor, induce receptor dimerization when each molecule of the dimer binds independently to a separate receptor(10) . A bivalent receptor binding mechanism has also been demonstrated for non-kinase receptors such as the growth hormone receptor(11) . In this case, the ligand is a monomer but contains two receptor binding sites that can induce receptor dimerization by forming a 1:2 (ligand-receptor) complex(12, 13) .

Although the primary sequence of HGF is distinct from other known polypeptide mitogens, HGF is similar to a growing list of growth factors with respect to its affinity for heparin(14, 15) . HGF, basic and acidic fibroblast growth factor (bFGF and aFGF, respectively), vascular endothelial cell growth factor, and platelet-derived growth factor bind tightly to heparin affinity columns and require high concentrations of NaCl (0.8 M) for elution. Domain deletion mutants of HGF deficient in either the N-terminal hairpin loop or the second kringle domain do not bind to heparin affinity columns, suggesting that the ability of HGF to bind heparin may reside in one or both of these domains(16) . Examination of the primary sequence within these domains revealed a cluster of basic amino acids, presumed to be important for binding anionic heparin or heparin-like molecules.

HGF binding to heparin in vitro may signify a biologically important interaction with heparan sulfate (HS) in vivo. HS is similar in composition and structure to heparin (for review, see Refs. 17-19) and is a major component of proteoglycans expressed on cell surfaces and secreted into the extracellular matrix(20) . In vitro studies indicate that hepatocytes possess a large number of heparin elutable (K 260-400 pM) binding sites for HGF (21) and that as much as 85% of HGF bound on the cell surface may be released by washing with heparin(22) .

Although heparan sulfate proteoglycans (HSPGs) are found on all mammalian cell surfaces, their structures are cell type- and possibly differentiation-specific. Emerging evidence suggests that HSPGs serve as important regulators of cellular signaling by modulating the stability, diffusability, and biological activity of heparin binding growth factors(19) . For example, Yayon et al. demonstrated that the interaction of bFGF with cell surface HSPGs, or exogenous soluble heparin, was required for high affinity binding and receptor activation(23) . They proposed that HSPG or heparin binding may change the conformation of bFGF to allow for a higher affinity interaction between bFGF and its receptor. More recently, an alternative mechanism has been proposed for aFGF. In this case, soluble heparin induced oligomerization of aFGF, resulting in subsequent FGF receptor dimerization and activation(24) . Heparin-like molecules are also apparently required for the interaction of vascular endothelial cell growth factor with high affinity receptors on vascular endothelial cells(25, 26) .

In contrast to FGF, investigations of the physiological importance of glycosaminoglycan binding to HGF are limited. In the present study, we investigated the effect of heparin, heparan sulfate, dextran sulfate, and a series of sulfated oligosaccharides of defined unit size on HGF-dependent mitogenesis. We demonstrate that heparin (0.1-10 µg/ml) and other highly sulfated oligosaccharides are capable of enhancing the potency of HGF and of increasing c-Met receptor phosphorylation in the presence of HGF. Investigations into the biochemical mechanism(s) underlying this effect revealed a correlation between compounds that enhanced mitogenic activity and those that promoted HGF oligomerization. We postulate, therefore, that heparan sulfate and related molecules promote the stability of HGF oligomers, which subsequently promotes receptor dimerization and activation.


EXPERIMENTAL PROCEDURES

Materials

Low molecular weight heparin, heparan sulfate, dextran sulfate, malto-oligosaccharides (from maltose through maltoheptaose), and a malto-oligosaccharide mixture (maltotetraose-decaose mixture) containing 4-10 glucose units were purchased from Sigma (St. Louis, MO). Malto-oligosaccharides were chemically sulfated using pyridine-sulfur trioxide complex (27) and desalted by gel filtration on columns of Bio-Gel P-4 resin (Bio-Rad). Size exclusion chromatography and SDS-PAGE molecular weight standards were from Bio-Rad (Richmond, CA). Anti-HGF polyclonal antibodies were raised in guinea pigs. Several bleeds were pooled and partially purified by saturated ammonium sulfate precipitation followed by Protein A chromatography. Anti-c-Met-IgG antibodies were raised in rabbits and purified as described above. The biotin-conjugated antiphosphotyrosine antibodies and alkaline phosphatase-conjugated streptavidin used for immunoblotting were purchased from Zymed Laboratories, Inc. (South San Francisco, CA). Chemiluminescent substrate was purchased from Tropix (Bedford, MA), and [H]thymidine was obtained from Amersham Corp.

Preparation of Recombinant HGF

The heterodimeric form of recombinant human HGF (rhHGF) was produced in Chinese hamster ovary cells using a procedure modified from that of Naka et al. (4). rhHGF-transfected cells were grown in a 400-liter bioreactor in medium containing 2% fetal bovine serum for 8 days. Culture supernatant was clarified and concentrated and then conditioned by the addition of solid NaCl to 0.3 M. rhHGF was purified in a single step using cation-exchange chromatography as follows: conditioned, concentrated culture supernatant was loaded onto a column of S-Sepharose Fast Flow equilibrated in 20 mM Tris, pH 7.5, 0.3 M NaCl. After washing out unbound protein, rhHGF was eluted in a linear gradient from 20 mM Tris, pH 7.5, 0.3 M NaCl to 20 mM Tris, pH 7.5, 1.2 M NaCl. rhHGF-containing fractions were pooled based on SDS-PAGE analysis, concentrated, and exchanged into 20 mM Tris, pH 7.5, 0.5 M NaCl by gel filtration on Sephadex G-25. This material, at a final concentration of 3-5 mg/ml, was stored frozen, and aliquots were thawed for subsequent use. Protein was quantitated by absorbance at 280 nm (A = 2.2).

Preparation of c-Met Receptor-IgG Fusion Protein

c-Met-IgG was constructed as described by Mark et al.(28) and produced in Chinese hamster ovary cells. The c-Met-IgG was purified in a single step using affinity chromatography on immobilized Protein A (Bioprocessing, Inc., Princeton, NJ), using an elution scheme modified from a previously described procedure(29) . This material, at a final concentration of 2-4 mg/ml, was stored frozen, and aliquots were thawed for subsequent use. Protein was quantitated by absorbance at 280 nm (A = 1.05).

HGF Bioassay

The biological activity of rhHGF was determined by measuring [H]thymidine incorporation into rat hepatocyte DNA in primary culture as described previously(3) . Briefly, hepatocytes were isolated from the livers of female Sprague-Dawley rats (180-220 g, obtained from Harlan) by perfusing a 0.02% collagenase solution through the portal vein. After washing in HEPES-buffered saline, pH 7.4, hepatocytes were washed and resuspended in serum-free medium (Williams Medium A, glutamine, penicillin-streptomycin, 10 µg/ml transferrin, 1 µg/ml insulin, gentamicin, and 1 mg/ml bovine serum albumin) to a final concentration of 5 10 cells/ml. Hepatocytes (100 µl/well) were added to 96-well microtiter plates containing 100 µl/well of assay medium, HGF, and, when indicated, HGF in the presence of sulfated oligosaccharides. After overnight incubation, at 37 °C in 5% CO, [H]thymidine (0.5-1.0 µCi/well) was added to each well, and the incubation was continued overnight. Cells were harvested using a Packard Filtermate Harvester, and radioactivity was measured using a Packard Microplate scintillation counter.

c-Met Autophosphorylation in A549 Cells

Human lung carcinoma cells (A549) were plated at 2 10 cells/150 cm culture dish in RPMI 1640 medium, containing 10% fetal bovine serum (Hyclone), 2 mM glutamine, and 100 units/ml penicillin streptomycin solution (JRH Biosciences) at 37 °C in a 5% CO humidified atmosphere. Cells were grown in monolayer to approximately 80% confluence before changing to serum-free medium (RPMI 1640 medium, containing 1 mg/ml bovine serum albumin). Cells were maintained in serum-free medium for 24-48 h; spent medium was aspirated and replaced with medium containing 5 ng/ml rhHGF in the presence or absence of sulfated maltohexaose. After 5 min at 37 °C, medium was removed and cells were washed in cold PBS 2 times prior to the addition of 1 ml of lysis buffer (150 mM NaCl, 1.5 mM MgCl, 1% Triton X-100, 4 µg/ml phenylmethylsulfonyl fluoride, 0.15 units/ml aprotinin, 1 mM sodium orthovanadate, 1 mMp-nitrophenylphosphate). Cell lysates were clarified by centrifugation for 5 min at 4 °C, and the supernatants were then immunoprecipitated by adding anti-c-Met-IgG polyclonal antibodies for 2 h followed by protein-G Sepharose for 0.5 h. Immune complexes were washed 3 times in PBS prior to boiling in 2 sample buffer. Samples were separated by SDS-PAGE and electroblotted to nitrocellulose. Phosphotyrosine-containing proteins were visualized using a biotinylated anti-phosphotyrosine antibody followed by streptavidin with chemiluminescence detection.

Binding of rhHGF to c-Met Receptor-IgG Fusion Protein

The effect of heparin on rhHGF binding to c-Met was tested using a microtiter plate assay. Affinity-purified F(ab`) rabbit anti-human IgG, Fc-specific, was adsorbed to each well (0.2 µg) by overnight incubation at 2-8 °C. The solution was discarded, and 150 µl of blocking buffer (PBS, 0.5% bovine serum albumin, 0.01% thimerosal, pH 7.4) was added to each well for 1.5 h. Wells were washed 6 with PBS, 0.05% Tween 20, pH 7.4 (wash buffer) prior to adding c-Met receptor-IgG fusion protein. The fusion protein was allowed to bind for 3 h, and the wells were washed again. rhHGF standards (0.27-200 ng/ml) were added to the appropriate wells in the presence or absence of 0.1, 1.0, or 10 µg/ml heparin or heparin-like compounds. Biotinylated polyclonal anti-HGF was added and incubated for 1.75 h. Plates were washed and horseradish peroxidase-streptavidin conjugate was added and allowed to incubate for an additional 1.75 h at ambient temperature with gentle agitation. Plates were washed 6 times with wash buffer, and 100 µl of substrate (o-phenylenediamine) solution was added. The colored reaction product was allowed to develop for 5 min before adding 50 µl of stopping solution (4.5 N HSO) to all wells. Absorbance at 490 nm was measured, and concentrations were determined from standard curves.

Determination of rhHGF Apparent Molecular Weight by Size Exclusion HPLC

The apparent molecular weight of rhHGF in the presence and absence of sulfated oligosaccharides in buffers of varying ionic strength was measured by size exclusion HPLC. Molecular weight standards were used for calibration. rhHGF was diluted into PBS, pH 7.2, to a final concentration of 0.26 mg/ml, and sulfated compounds were added at 10 µg/ml unless otherwise stated. Samples were analyzed using a TSK-Gel (TosoHaas) G3000SW XL column using a flow rate of 1 ml/min at ambient temperature. Chromatography was carried out using a PBS mobile phase, and rhHGF was detected in the eluant by monitoring absorbance at 280 nm.

Analytical Ultracentrifugation

Sedimentation equilibrium experiments were conducted in a Beckman XLA ultracentrifuge using charcoal-filled Epon six-channel Yphantis cells(30) . rhHGF was diluted with PBS, pH 7.2, to a final concentration of 0.2 mg/ml and then incubated with sulfated oligosaccharides at various concentrations. Experiments were performed at rotor speeds of 10,000 and 15,000 rpm at 20 °C. The attainment of equilibrium was verified by comparing successive scans. The partial specific volume of rhHGF (0.714 ml/g) was calculated from its amino acid and average carbohydrate composition. The sedimentation data were edited using a PC program, REEDIT (kindly provided by Dr. David Yphantis, University of Connecticut). Data points were truncated around the bottom of cell and meniscus regions to avoid nonlinear deviation or base-line noise. The average molecular weight was obtained by fitting the data as a single ideal species using a nonlinear least squares method(31) .


RESULTS

Heparin, Heparan Sulfate, and Dextran Sulfate Enhanced the Mitogenic Activity of rhHGF

Previous work has demonstrated that HGF is a potent mitogen for primary cultures of rat hepatocytes(32, 33) . Addition of increasing concentrations of rhHGF results in a bell-shaped dose-response curve, with mitogenic activity increasing from 1 to 100 ng/ml and declining at higher concentrations. We used this assay to study the effect of heparin and heparin-like molecules on the mitogenic activity of HGF. As expected, addition of rhHGF alone, at concentrations of 1-100 ng/ml resulted in a dose-dependent increase in DNA synthesis. Co-incubation of rhHGF with heparin (0.1-10 µg/ml) in this assay resulted in an increase in rhHGF potency (decrease in the EC). This effect was maximal at a heparin concentration of 10 µg/ml. Higher concentrations of heparin (100 µg/ml) were not as effective and in some cases inhibited HGF action. Fig. 1A shows a representative experiment in which HGF was co-incubated with heparin at three different concentrations (1, 10, and 100 µg/ml) prior to addition to primary rat hepatocyte cultures.


Figure 1: Sulfated polysaccharides enhance HGF-dependent mitogenesis. A, HGF (0.1-100 ng/ml) was added to primary cultures of rat hepatocytes in the absence () and presence of 1 (), 10 (), and 100 µg/ml heparin (). B, similarly, HGF (0.1-100 ng/ml) was added to primary cultures of rat hepatocytes in the absence () and presence of 10 µg/ml heparin (), heparan sulfate (), or dextran sulfate (). Data represent the average cpm/well (n = 3) ± S.D. for a given HGF concentration.



The specificity of this effect was examined by testing other sulfated compounds such as heparan sulfate, dextran sulfate, fucoidan, and NaSO at 10 µg/ml. Inclusion of heparin or heparan sulfate resulted in an approximate 3-5-fold enhancement in rhHGF potency (Fig. 1B). There was little or no significant difference in the maximal mitogenic response, which occurred at an rhHGF concentration of 100 ng/ml. Dextran sulfate showed a slightly greater (5-7-fold) enhancement of rhHGF-dependent mitogenesis than heparin, suggesting that the potentiation effect was not specific for glycosaminoglycans. The charge density of dextran sulfate varies, but it is generally higher than that of heparin. These sulfated polysaccharides did not increase [H]thymidine uptake in the absence of rhHGF over the time course of the experiment. Interestingly, neither fucoidan (a polysaccharide containing sulfated fucose) nor sodium sulfate (0.1-10 µg/ml) had any enhancing effect when tested in this assay (data not shown). Taken together, these data suggest that enhancement of HGF-dependent mitogenesis does not specifically require N-sulfation (as found in heparin or HS) but that compounds with high negative charge and only O-sulfation can increase rhHGF potency in a mitogenic assay. These results prompted us to examine the effects of a highly sulfated series of chemically sulfated malto-oligosaccharides.

The Effect of Sulfated Oligosaccharide Unit Size on rhHGF Activity

A series of chemically sulfated malto-oligosaccharides varying in size from 2 to 7 glucose units was prepared in an attempt to define minimal size requirements for modulating rhHGF activity. We observed that rhHGF potency could be enhanced by the addition of such compounds, but the enhancement was dependent on oligosaccharide unit size. Sulfated malto-oligosaccharide 6 glucose units or larger significantly stimulated HGF-dependent mitogenesis (), whereas the sulfated di-, tri-, and tetrasaccharides showed no significant enhancement. While the effect of sulfated maltopentaose was variable, the sulfated maltohexaose or maltoheptaose at 10 µg/ml increased the potency of rhHGF (approximately 2- and 4-fold, respectively). Importantly, malto-oligosaccharides did not enhance HGF-dependent mitogenesis if they were not chemically sulfated (data not shown), suggesting the observed effect is related to both oligosaccharide size and charge.

A second sulfated hexasaccharide-like compound, bis-(-maltotriosyl)ethylene glycol sulfate, was prepared by chemically linking two maltotriose sulfate units with an ethylene glycol bridge(34) . The bis-(-maltotriosyl)ethylene glycol sulfate also significantly enhanced rhHGF activity, whereas the untethered maltotriose sulfate did not. The observed enhancement was comparable with that of maltohexaose sulfate (). These results further demonstrate that sulfated hexasaccharides, but not trisaccharides, are capable of potentiating rhHGF action.

An additional experiment was performed with a sulfated mixture of malto-oligosaccharides containing 4-10 glucose units, as pure malto-oligosaccharides containing 8 or more glucose units are not commercially available. Interestingly, this mixture of sulfated oligosaccharides enhanced HGF potency to the same degree (5-7-fold) as high molecular weight polymers such as dextran sulfate. The relative effects of sulfated maltotetraose, maltoheptaose, and the maltotetra-decaose mixture are shown graphically in Fig. 2.


Figure 2: The effect of oligosaccharide unit size on HGF dependent mitogenesis. HGF (0.1-100 ng/ml) was added to primary cultures of rat hepatocytes in the presence and absence (control) of 10 µg/ml of sulfated maltotetraose (Tetrasaccharide), sulfated maltoheptaose (Heptasaccharide), or sulfated maltotetraose-decaose mixture (Decasaccharide). Data represent the average cpm/well (n = 3) ± S.D. for a given HGF concentration.



Incubation of rhHGF with Maltohexaose Sulfate Enhances c-Met Phosphorylation

Autophosphorylation is the earliest detectable event in the mitogenic signaling cascade. Addition of rhHGF results in an increase in the phosphotyrosine content of c-Met expressed by A549 cells(3) . To investigate one possible mechanism by which sulfated compounds might enhance rhHGF potency, we compared the phosphorylation state of the c-Met receptor in A549 cells after the addition of rhHGF (5 ng/ml), rhHGF, and sulfated maltohexaose (10 µg/ml) or sulfated maltohexaose alone (Fig. 3). The addition of the sulfated hexasaccharide alone (in the absence of HGF) did not change the phosphotyrosine content of the c-Met receptor subunit (145 kDa) above basal levels (compare lanes1 and 2). However, co-incubation of rhHGF (5 ng/ml) and the sulfated hexasaccharide resulted in a distinct and reproducible increase in phosphorylation of the c-Met subunit, as compared with the above, or as compared with cells treated with rhHGF alone. The phosphotyrosine content of the c-Met receptor after treatment with 5 ng/ml rhHGF plus sulfated hexasaccharide was similar to the level of phosphorylation typically observed after treatment with a 10-fold higher concentration (50 ng/ml) of rhHGF alone (compare lanes4 and 6). Interestingly, the sulfated compound had no effect on the phosphorylation state of c-Met receptor when rhHGF was present at relatively high concentrations (50 ng/ml). In addition, we also observed an increase in tyrosine-phosphorylation of a 60-kDa protein in cells treated with rhHGF in the presence of the sulfated compound. These results suggest that activation of the c-Met receptor is enhanced by lower levels of rhHGF when highly sulfated hexasaccharides (and presumably larger oligosaccharides) are present.


Figure 3: The effect of sulfated maltohexaose on c-Met receptor autophosphorylation. Human lung epithelial cells (A549) were maintained in serum free medium for 24-48 h; spent medium was aspirated and replaced with fresh medium alone (lane1), with fresh medium containing hexasaccharide (lane2), fresh medium containing 5 ng/ml HGF in the absence (lane3) and presence (lane4) of hexasaccharide, and 50 ng/ml HGF in the presence (lane5) and absence (lane6) of hexasaccharide. After 5 min at 37 °C, cells were lysed, and c-Met receptor was immunoprecipitated. Immune complexes were separated by SDS-PAGE and transferred to nitrocellulose by electroblotting. Phosphotyrosine-containing proteins were detected using anti-phosphotyrosine antibodies and visualized by chemiluminescence.



Heparin Does Not Alter the Binding Affinity of rhHGF for the c-Met Receptor Extracellular Domain

To determine whether heparin binding to rhHGF alters its ability to interact with the extracellular domain of c-Met, we measured the affinity of rhHGF binding to a c-Met-IgG fusion protein. This fusion construct, first reported by Mark et al.(28) , includes only the extracellular domain of c-Met; rhHGF binds this c-Met-IgG fusion protein with an affinity comparable with that reported for c-Met expressed on cell surfaces. We found that heparin did not alter, and was not required for high affinity binding of rhHGF to the c-Met extracellular domain (Fig. 4). Similarly, co-incubation of HGF with 10 µg/ml dextran sulfate, maltohexaose sulfate, or maltoheptaose sulfate did not change the binding isotherm of HGF for its receptor (data not shown). These data suggest that the enhancement of HGF mitogenic activity by sulfated compounds was not a result of these compounds increasing the affinity of HGF for the c-Met receptor.


Figure 4: Heparin has no effect on HGF binding to a c-Met receptor-IgG fusion protein. rhHGF standards were added (0.27-200 ng/ml) in the presence of 0, 0.1, 1.0, or 10 µg/ml of heparin to plates containing immobilized c-Met receptor-IgG fusion protein. A biotinylated anti-HGF polyclonal Ab, followed by a horseradish peroxidase-streptavidin conjugate, was added to all wells and allowed to incubate at ambient temperature with gentle agitation. Plates were washed prior to adding substrate, and the colored reaction product was allowed to develop for 5 min. Absorbance at 490 nm was measured, and concentrations were determined from standard curves.



Incubation of rhHGF with Sulfated Oligosaccharides Promotes HGF Association

Size exclusion chromatography experiments were performed to determine the effect of sulfated malto-oligosaccharides on the oligomerization state of rhHGF in solution. We obtained poor recoveries of rhHGF from the column using a PBS mobile phase (<10%, data not shown), perhaps due to ionic interactions between rhHGF and the column matrix (the recovery of rhHGF could be significantly improved if high salt was included in the elution buffer, data not shown). The apparent molecular mass of rhHGF under these conditions was 73-76 kDa, consistent with the molecular mass predicted from amino acid composition data for a monomeric HGF. We noted that recovery could also be improved if rhHGF was co-incubated with the sulfated malto-oligosaccharide preparations instead of NaCl prior to chromatography (Fig. 5A). Interestingly, the percent recovery of rhHGF was greatest when sulfated oligosaccharides 6 glucose units and larger were tested, although chromatography in the presence of sulfated maltotri-, tetra-, and pentaose also improved recovery relative to rhHGF alone. Optimal recovery of rhHGF required at least an equimolar concentration of sulfated oligosaccharide.


Figure 5: Size exclusion chromatography of HGF in the presence of sulfated maltooligosaccharides. A, rhHGF was incubated with sulfated maltose (2), maltotriose (3), maltotetraose (4), maltopentaose (5), maltohexaose (6), maltoheptaose (7), and a sulfated maltotetraose-decaose mixture (4-10) for 15 min prior to chromatography. rhHGF was eluted using a PBS, pH 7.4, mobile phase at a flow rate of 1 ml/min. Representative chromatograms show the relative retention time and recovery of rhHGF. B, molecular weight standards were run under the same conditions, and the apparent molecular weights of rhHGF in the presence of each of the sulfated oligosaccharides were calculated and plotted as a function of sulfated oligosaccharide unit size.



Perhaps more significantly, we also observed that the sulfated oligosaccharides differentially affected the apparent molecular weight of rhHGF (Fig. 5A). Chromatography of rhHGF in the presence of the sulfated hexa- and heptasaccharide compounds yielded retention times of 7.6-7.7 min compared with 8.6-8.9 min for the di-, tri-, tetra-, and pentasaccharides. Comparison of these retention times to those of molecular weight standards allowed for estimation of apparent molecular weights. rhHGF ranged from 238-261 kDa in the presence of the hexa- and heptasaccharide, compared with 75-101 kDa in the presence of the di-, tri-, tetra-, or pentasaccharide. Subsequent testing of the sulfated oligosaccharide mixture containing tetra- to decasaccharides gave results similar to those seen for 6 and 7 saccharide units.

The multimeric state of rhHGF was plotted as a function of oligosaccharide unit size in Fig. 5B. While rhHGF appeares to exist as a monomer in the presence of sulfated oligosaccharides containing 2-5 glucose units, it appears to associate into trimers (or elongated dimers) in the presence of sulfated malto-oligosaccharides containing 6 or more glucose units. Size exclusion chromatography fractionates molecules on the basis of their hydrodynamic properties. Thus, two globular proteins cross-linked by a sulfated oligosaccharide may form an elongated dimer, resulting in an artificially high molecular weight. These data suggest that a discontinuity in the oligomerization state of rhHGF occurs upon binding oligosaccharides of a unit size between 5 and 6. Importantly, only these larger sulfated oligosaccharides are capable of significantly enhancing the mitogenic activity of rhHGF () or promoting the phosphorylation of c-Met in the presence of HGF (Fig. 3).

The high molecular weight complexes that formed were stable over the time course of chromatography and did not require the inclusion of oligosaccharide in the elution buffer. Incubation of rhHGF with a molar excess of these oligosaccharides did not appreciably affect the average oligomer molecular weight, while there was evidence of peak broadening (data not shown). The high molecular weight rhHGF complexes could be dissociated in the presence of high NaCl (0.5-1.0 M), and co-incubation of rhHGF with sulfated oligosaccharides did not change rhHGF mobility following SDS-PAGE under reducing or nonreducing conditions (data not shown); thus, the complexes are presumed to be a result of noncovalent interactions.

Analytical ultracentrifugation experiments confirmed the association of rhHGF observed in the size exclusion experiments. These experiments were performed under near physiological (0.15 M NaCl) and high (0.5 M NaCl) salt conditions. The molecular mass of rhHGF determined was 94 kDa under near physiological salt conditions (0.2 mg/ml HGF in PBS, pH 7.4) and 85 kDa under high salt conditions (Fig. 6). When rhHGF was then incubated with sulfated maltoheptaose prior to centrifugation, an increase in the average molecular weight of rhHGF consistent with dimerization was observed. The average molecular weight increased from 94 to approximately 200 kDa in the presence of the sulfated maltoheptaose, and this maximum was obtained at a 2-fold molar excess of oligosaccharide to protein.


Figure 6: Analytical centrifugation of HGF in the presence of sulfated maltoheptaose. HGF was diluted with PBS (0.15 M) or PBS containing high NaCl (0.5 M), pH 7.2, to a final concentration of 0.2 mg/ml and subjected to centrifugation. HGF (in PBS) was also incubated with sulfated maltoheptaose at various concentrations to produce molar ratios from 2:1, HGF/maltoheptaose, to 1:64, HGF/maltoheptaose. Experiments were performed at rotor speeds of 10,000 and 15,000 rpm at 20 °C. The average molecular weight was obtained by fitting the data as a single ideal species using a nonlinear least squares method.




DISCUSSION

The study of glycosaminoglycans and their role in regulating the biological activities of various heparin binding growth factors has become an area of intense research(19) . We have demonstrated that sulfated polysaccharides such as heparin, heparan sulfate, and dextran sulfate can enhance the potency of rhHGF in a primary rat hepatocyte assay. The concentration of soluble heparin used in the assay was important to the biological outcome. For example, low concentrations (0.1-10 µg/ml) of heparin enhanced rhHGF-dependent mitogenesis in primary rat hepatocytes, whereas high concentrations (greater than or equal to 100 µg/ml) were not as effective and in some experiments were inhibitory.

Other reports have described the inhibitory effects of high concentrations of heparin on mitogenic (35, 36) and scattering (37) responses, as well as inhibition of the cross-linking of HGF to c-Met receptors on intact cells(22) . In addition, evidence for a direct interaction between HGF and HS, and the structural requirements within HS necessary for HGF binding, were recently reported by Lyon et al.(38) . These authors reported that 6-O-sulfation of HS was a critical determinant for high affinity interaction with HGF. They also reported that oligosaccharides as small as hexasaccharides bound to HGF affinity columns, although larger oligosaccharides (containing 10-12 saccharide units) bound with higher affinity.

The structure of rat liver HS was recently shown to have a particularly high charge density compared with HS from other sources, containing an average of 1.34 sulfates/disaccharide(39) . It was characterized as an extreme member of the HS family with a considerable portion of heparin-like structure asymmetrically concentrated to the distal part of the chain. Thus, the sulfation profile of HS in liver appears to be well suited for binding and regulating HGF biological activity.

In the present study, we report a direct correlation between the oligomerization state of HGF, induced by sulfated saccharides (greater than or equal to 6 glucose units), and the potentiation of rhHGF biological activities. rhHGF bound the extracellular domain of c-Met with affinity comparable with that observed for cell-associated c-Met receptor in the absence of heparin. Furthermore, inclusion of heparin at 0.1-10 µg/ml had no effect on the rhHGF binding isotherm over the concentration range of rhHGF tested. These data would suggest that heparin or heparin-like molecules are not required and do not significantly alter the binding affinity of rhHGF for its receptor. However, we demonstrated that a sulfated compound of sufficient length (6 glucose units) to enhance HGF biological activity could also enhance receptor phosphorylation when coincubated with low levels of rhHGF, suggesting a potential role for endogenous HSPG in the regulation of receptor activation. From these data we cannot rule out the possibility that the sulfated maltooligosaccharides, in addition to inducing HGF dimerization, prevent binding of HGF to HSPG on cell surfaces. This could make more HGF available for high affinity c-Met receptor binding.

Size exclusion HPLC and analytical ultracentrifugation both indicated that rhHGF will form stable oligomers in solutions containing sulfated maltooligosaccharides of unit length 6. Those compounds that promoted the oligomerization of rhHGF were again those that enhanced either [H]thymidine incorporation into rat hepatocyte DNA or c-Met phosphorylation. The rhHGF-oligosaccharide complexes so formed were stabilized by noncovalent interactions, as indicated by the observations that they could be disrupted by either boiling in SDS or including high salt in buffers prior to chromatography.

Prediction of the rhHGF unit size within the oligomeric complex by size exclusion chromatography was complicated due to the fact that this technique fractionates on the basis of a molecules hydrodynamic properties. Although Stokes radius is proportional to molecular weight for globular proteins, nonideal behavior may occur if two globular proteins were attached via a sulfated oligosaccharide. An elongated dimer would elute sooner and thus give an over estimation of molecular weight. Thus we feel that the size exclusion chromatography and sedimentation equilibrium experiments are consistent and suggest that HGF forms stable dimers in the presence of sulfated oligosaccharides of sufficient length. Another relevant observation related to rhHGF dimerization was recently made in x-ray crystallographic studies on the receptor binding domain, NK1, of HGF. The molecule crystallized as a dimer in the presence of sulfate-containing buffers such as HEPES sulfate.()

It is probable that the interaction between HGF and HSPG plays a critical role in regulating HGF activity both in vitro and in vivo, as is the case for the FGF family of proteins. Recently, heparin was reported to induce aFGF oligomerization and, as a consequence, subsequent FGF receptor dimerization and activation(26) . Complexes between FGF and HS are resistant to both heat denaturation and proteolytic degradation(40) , and HSPG may act to sequester and stabilize FGF and other heparin-binding growth factors in the extracellular matrix. Following cell or tissue injury, growth factors may be released by proteolytic or heparinase enzymes to provide rapid local proliferative responses. Consistent with this notion, HGF has a high affinity for HS in vitro(38) and binds to the surface of hepatocytes and to the extracellular matrix of liver through interactions with heparin-like molecules(41) , and HGF-like activity with affinity for heparin was shown to be sequestered in the hepatic subendothelial space(42) . HGF associated with heparan sulfate in the extracellular matrix has been shown to significantly enhance mitogenic activity and cell spreading(43) . Local secretion of endogenous HGF and sequestration by the extracellular matrix may allow HGF to function in a paracrine fashion mediating mesenchymal-epithelial cell interactions. HGF resident in the extracellular matrix may participate in the growth regulation of hepatocytes during liver regeneration, epithelial cells during wound healing, and endothelial cells during angiogenesis.

In summary, we have shown that the interaction of rhHGF with sulfated malto-oligosaccharides 6 glucose units and larger leads to greater receptor phosphorylation and enhanced DNA synthesis in rat hepatocytes. There was no apparent change in the HGFc-Met receptor binding isotherm in the presence of heparin, suggesting that HGF binding affinity to c-Met was not significantly altered by heparin. However, we clearly demonstrated a correlation between those oligosaccharide preparations that potentiated rhHGF activity and receptor phosphorylation with those that caused rhHGF to form oligomers in solution. These data lead to the hypothesis that the presentation of multivalent HGF, induced by the binding of HGF to sulfated epitopes 6 sugar units or larger on HSPG chains, may facilitate subsequent c-Met receptor dimerization, phosphorylation, and subsequent cellular proliferation.

  
Table: The effect of oligosaccharide unit size on the potentiation of HGF-dependent mitogenesis



FOOTNOTES

*
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: Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080-4990. Tel.: 415-225-3269; Fax: 415-225-6452.

Present address: P. O. Box 1709, Murphys, CA 95247.

The abbreviations used are: HGF, hepatocyte growth factor; HS, heparan sulfate; FGF, fibroblast growth factor; aFGF, acidic FGF; bFGF, basic FGF; HSPG, heparan sulfate proteoglycan; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; rhHGF, recombinant human hepatocyte growth factor; HPLC, high performance liquid chromatography.

M. Ultsch and A. de Vos, personal communication.


ACKNOWLEDGEMENTS

We thank David Peers for assistance in purification of rhHGF and c-Met-IgG, Reed Harris for amino acid analysis of rhHGF and c-Met-IgG, and Ed Cox and Hardat Prashad for performing the cell culture. We also thank Steve Shire for advice in the design and interpretation of the analytical ultracentrifuge experiments.


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