From the
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.
Hepatocyte growth factor (HGF),
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
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
(
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
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.
A second sulfated
hexasaccharide-like compound, bis-(
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.
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.
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
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 HGF
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)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) .
-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) .
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.
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) .
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
H
SO
) 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) .
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.
-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.
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.
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.
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.
(
)
c-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
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.