(Received for publication, August 17, 1995)
From the
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)-GlcNSO
(6SO
) 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
)-GlcNSO
(6SO
)
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.
HS ()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.
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
3.9 cm). Specific radioactivity of
I-HGF was 2.5
5.7
10
dpm/ng.
Figure 2:
Sephadex G-50 chromatography of
[H]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
[
H]NaBH
.
H-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
, void volume; V
,
total volume. Elution positions of molecular weight markers are
indicate by arrows: a,
[
H]heparin octasaccharide; b,
[
H]chondroitin hexasaccharide; c,
[
H]chondroitin tetrasaccharide; d,
Di-0S; e,
[
H]D-glucosamine.
Figure 3:
Percent proportions of oligosaccharides
with the binding activity to HGF affinity column. Oligosaccharide
fractions (4 nmol) containing 1 10
dpm of
H-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.
Degradation of about 1 µg of HS
oligosaccharides with nitrous acid at pH 1.5 and reduction of
degradation products with [H]NaBH
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.
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.''
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)-GlcNSO
-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.
Each H-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
, 0.2 mg/ml
chondroitin 4-sulfate). After a wash with solution B, the bound
H-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
H-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.
Figure 4:
Mono Q FPLC of heparan sulfate
oligosaccharides fractions. A, HGF column-unbound () and
-bound (
) fractions of [
H]HS-IV (2
10
and 4
10
dpm, respectively) were
desalted, freed from chondroitin 4-sulfate, concentrated, and applied
to mono Q column. B, HGF column-unbound (
) and -bound
(
) fraction of [
H]HS-V (7.5
10
and 2
10
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
H-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 Di-(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
)-GlcNSO
(6SO
) 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
)-GlcNSO
(6SO
) 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)-GlcNSO
(6SO
), 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
[H]NaBH
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
)AMan
(6SO
), and only 2%
were GlcA(2SO
)AMan
(6SO
). Therefore,
HexA(2SO
)-GlcNSO
(6SO
), 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
)-GlcNSO
(6SO
) 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
and GlcA-GlcNSO
(6SO
)
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
)-GlcNSO
(6SO
) units
and one N-sulfated component (HexA-GlcNSO
or
HexA-GlcNSO
(6SO
)) may form the binding site for
HGF.
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 (), V-UB (
), and
heparin (
) 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.
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)-GlcNSO
(6SO
) 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
)-GlcNSO
(6SO
) 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)-GlcNSO
(6SO
)
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(6SO
) residues. However,
according to their results, no contiguous sequence of two or more
IdoA(2SO
)-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
) residue still retained a
HGF affinity.
It is in question in our present study whether
HexA(2SO)-GlcNSO
units are involved in the
binding of HGF to HS directly, since these
HexA(2SO
)-GlcNSO
units comprised only 3.2% of
the starting material, bovine liver HS fraction 2. However,
HexA(2SO
)-GlcNSO
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
)-GlcNSO
units (10) , showed no significant inhibition in the HGF binding to
HSPG (Table 1). Furthermore, the content of the
IdoA(2SO
)-GlcNSO
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
)-GlcNSO
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)-GlcNSO
(±6SO
)
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.