(Received for publication, June 20, 1995; and in revised form, October 23, 1995)
From the
Ryudocan, a heparan sulfate proteoglycan, was isolated from human endothelium-like EAhy926 cells by a combination of ion-exchange and immunoaffinity chromatography. Purified human ryudocan has biochemical properties similar to those of rat ryudocan isolated from microvascular endothelial cells. Human ryudocan contains only heparan sulfate (HS) glycosaminoglycan chains along with a core protein with an apparent molecular mass of 30 kDa. We evaluated the interactions between purified human ryudocan and several extracellular ligands by using a solid-phase binding assay. We found that basic fibroblast growth factor (bFGF), midkine (MK), and tissue factor pathway inhibitor (TFPI) exhibit significant ryudocan binding. Heparitinase (but not chondroitin ABC lyase) treatment destroyed the ability of ryudocan binding to bFGF, MK, and TFPI. Heparin and HS, but not chondroitin sulfate, inhibited such ryudocan binding. Thus, the HS chains of ryudocan appear to be responsible for its binding to bFGF, MK, and TFPI. The apparent dissociation constants for purified ryudocan were as follows: bFGF, 0.50 nM; MK, 0.30 nM; and TFPI, 0.74 nM. Immunohistochemical analysis revealed that ryudocan was expressed in fibrous connective tissues, peripheral nerve tissues, and placental trophoblasts. These findings suggest that ryudocan may possess multiple biological functions, such as bFGF modulation, neurite growth promotion, and anticoagulation, via HS-binding effectors in the cellular microenvironment.
Ryudocan is an integral membrane heparan sulfate proteoglycan
(HSPG) ()that was originally isolated from endothelial cells
as an anticoagulant molecule(1, 2) . This compound is
a member of the syndecan family composed of four cell membrane
intercalated HSPGs: syndecan (syndecan-1), fibroglycan (syndecan-2), N-syndecan (syndecan-3), and ryudocan (syndecan-4), which have
homologous transmembrane and intracellular domains, but very distinct
extracellular regions(3) . The best characterized molecule of
this family is syndecan, the prototypical member that was isolated from
mouse mammary epithelial cells(4) . Syndecan selectively binds
via its heparan sulfate (HS) chains to a variety of matrix components,
including fibrillar collagens(5) , fibronectin(6) ,
thrombospondin(7) , tenasin(8) , and
amphoterin(9) , which suggests that syndecan may be an
extracellular matrix receptor.
Syndecan also binds to the heparin-binding growth factors, such as basic fibroblast growth factor (bFGF)(10, 11, 12, 13, 14) . The potential importance of this interaction is underscored by recent reports demonstrating the involvement of cell-surface HSPGs in bFGF signaling mechanisms(15) . N-Syndecan is another member of the syndecan family. This molecule was recently identified and cloned in rat Schwann cells and chicken embryos(16, 17) . N-Syndecan possesses a high degree of specificity for bFGF through its glycosaminoglycan (GAG) chains(18) . N-Syndecan was also isolated as a heparin binding growth-associated molecule (pleiotrophin) receptor and was anticipated to mediate the neurite outgrowth-promoting signal from a heparin binding growth-associated molecule to the cytoskeleton of growing neurites(19) .
Several syndecan-4 cDNAs have been characterized for the rat (ryudocan(2) ), human (ryudocan (20) and amphiglycan(21) ), and chicken (22) forms. Little is known, however, about the biological function of this molecule. Thus, we purified human ryudocan from endothelium-like cells (EAhy926 cells) and examined its interactions with various biological ligands by using a solid-phase binding assay. The ligands tested for ryudocan binding were bFGF, midkine (MK), and tissue factor pathway inhibitor (TFPI). Among them, MK is a heparin-binding growth factor unrelated to fibroblast growth factor(23, 24) , promotes neurite outgrowth (25) and neuronal cell survival(26) , and enhances plasminogen activator activity in aortic endothelial cells(27) . TFPI is an important regulator of the extrinsic pathway of blood coagulation through its ability to inhibit factor Xa- and factor VIIa-tissue factor activity(28, 29) . We also investigated the expression of human ryudocan by using an immunohistochemical method utilizing a specific anti-human ryudocan antibody.
After dialysis against 0.15 M NaCl, 1 mM EDTA, and 50 mM Tris-HCl, pH 7.4, CHAPS was added to a concentration of 0.6%, and the proteoglycans eluted from the DEAE-Sephacel column were charged to a monoclonal antibody-Sepharose affinity column (CNBr-activated Sepharose conjugated with anti-human ryudocan monoclonal antibody). The column was washed with 5 column volumes of 0.15 M NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 7.4, and 0.6% CHAPS, and the proteoglycans were eluted with 3 column volumes of 0.15 M NaCl and 50 mM glycine, pH 2.5, followed by immediate neutralization with 0.2 volume of 1 M Tris-HCl, pH 7.8. The eluant from the monoclonal antibody affinity column was then loaded onto a second DEAE-Sephacel column (0.5 ml). This column was washed with 5 column volumes of 0.4 M NaCl and 50 mM sodium acetate, pH 5.0, followed by 5 column volumes of 0.3 M NaCl, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4. Proteoglycans were eluted with 1 M NaCl, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4. Purified human ryudocan was divided into aliquots and stored at -70 °C for further use.
To assure that the applied proteins stuck to the wells, we
performed an immunodetection assay as follows. After coating with the
proteins (0.5 µg/well) and blocking nonspecific binding sites as
described above, the wells were sequentially incubated with a specific
rabbit antibody against each ligand and a peroxidase goat anti-rabbit
IgG antibody (Dako Japan Co. Ltd., Kyoto, Japan) in 0.1% BSA/PBS
containing 0.05% Tween 20. After washing five times with distilled
water, the wells were incubated with 6.2 mMo-phenylenediamine dihydrate and 0.01% (v/v)
HO
in 75 µl of 0.1 M citrate/phosphate buffer, pH 5.0. The reaction was stopped by
adding 75 µl of 3 M H
SO
, and the
absorbance at A
of the sample in each well was
measured. In addition, we measured the amount of protein remaining in
the well after removal of ligand solution using a
I-labeled tracer for each protein. We labeled each ligand
protein with
I as described above and verified the purity
of each
I-labeled protein by SDS-PAGE and
autoradiography. The wells, which were coated with each ligand
containing
I-labeled tracer as described above, were
washed, and the amount of bound radioactivity was quantified.
To
verify that unlabeled ryudocan exhibits similar binding properties, we
also tested the competitive ability of unlabeled ryudocan against I-ryudocan binding. Thus, a solid-phase binding assay was
performed in the presence of serial amounts of unlabeled ryudocan.
After coating with 0.5 µg/well bFGF, MK, or TFPI, the microtiter
plate wells were incubated with 0.1 ng (1
10
cpm)/well
I-ryudocan containing unlabeled ryudocan
(0.1, 1, or 10 ng/well). The wells were washed, and the amount of bound
radiolabeled ryudocan in each well was quantified as described above.
To determine the purity of the
proteoglycan preparation and to identify the attached glycosaminoglycan
chains, this material was subjected to enzymatic degradation. After
radiolabeling with I, the human ryudocan preparation was
treated with purified Flavobacterium heparitinase or
chondroitin ABC lyase and analyzed by SDS-PAGE through a 4-15%
gradient (Fig. 1). Autoradiography of the resulting material
showed a diffuse band with an apparent molecular mass of 160 kDa.
Treatment of the sample with purified Flavobacterium heparitinase resulted in a single distinct band with an apparent
molecular mass of 30 kDa. This corresponded to the previously described
core protein size of rat ryudocan from microvascular endothelial
cells(1) . In contrast, chondroitin ABC lyase treatment did not
affect the migration of the iodinated 160-kDa molecule. These data
indicate that the purification procedure yielded pure human ryudocan,
which was predominantly composed of heparan sulfate GAG chains.
Figure 1:
Characterization of human ryudocan from
EAhy926 cells. After radiolabeling with I, purified human
ryudocan was subjected to heparitinase or chondroitin ABC lyase
treatment as indicated, analyzed by electrophoresis through a
4-15% SDS-polyacrylamide gradient gel, and
autoradiographed.
Figure 2:
Binding of I-labeled human
ryudocan to potential ligands. Microtiter wells were coated with 1
µg/well fibronectin (FN), type I collagen (Col
I), bFGF, epidermal growth factor (EGF), insulin-like
growth factor (IGF), MK, granulocyte CSF (G-CSF),
monocyte CSF (M-CSF), granulomonocyte CSF (GM-CSF),
interleukin-3 (IL-3), ATIII, TFPI, or BSA and incubated with
6.9
10
cpm/well
I-ryudocan. After
washing, the amount of bound radioactivity was counted. Data from
triplicate samples (mean ± S.D.) are presented, and similar data
were obtained in two additional
experiments.
To assure that the applied proteins
stuck to the wells, we performed an immunodetection assay as described
under ``Experimental Procedures.'' The significant signals of
absorbance at A were obtained from all ligands
tested, compared with those from the BSA control (data not shown). In
addition, we measured the amount of protein remaining in the wells
after removal of ligand solution by using a
I-labeled
tracer for each protein. The amounts of the remaining ligands in the
wells ranged from 6.0 to 20.7%. These data suggest that a lack of
ryudocan binding could not result from a failure of the applied
proteins to stick to the wells.
We also performed a competition
assay to verify that unlabeled ryudocan exhibits similar binding
properties as described under ``Experimental Procedures.''
Unlabeled ryudocan inhibited dose-dependently the binding of I-ryudocan to bFGF, MK, and TFPI (Fig. 3).
Presumably, due to excess of coated ligands, a relatively large amount
of unlabeled ryudocan would be needed to block the binding of
I-ryudocan. Thus, the observed
I-ryudocan
binding might not be a consequence of the iodination reaction.
Figure 3:
Inhibition of I-ryudocan
binding by unlabeled ryudocan. Microtiter wells were coated with bFGF,
MK, or TFPI (0.5 µg/well) and then incubated with 0.1 ng (1.1
10
cpm)/well
I-ryudocan containing
unlabeled ryudocan (0.1 (open columns), 1 (hatched
columns), or 10 (closed columns) ng/well) as indicated.
After washing, the amount of bound radioactivity was counted. Data from
duplicate samples (mean ± S.D.) are presented, and similar data
were obtained in an additional experiment. The absolute counts
corresponding to 100% binding were 696 cpm for bFGF, 692 cpm for MK,
and 216 cpm for TFPI.
To
investigate the involvement of ryudocan GAG chains in ligand binding,
we digested the GAG chains of I-ryudocan with purified Flavobacterium heparitinase or chondroitin ABC lyase prior to
the solid-phase binding assay (Fig. 4). Heparitinase treatment
destroyed the ability of
I-ryudocan binding to bFGF, MK,
and TFPI. In contrast, chondroitin ABC lyase treatment did not affect
I-ryudocan binding to these potential ligands. These data
indicate that the HS chains, not the core protein of ryudocan, are
responsible for binding bFGF, MK, and TFPI.
Figure 4:
Glycosaminoglycan chains govern ryudocan
binding to bFGF, MK, and TFPI. Microtiter wells were coated with bFGF
(0.5 µg/well), MK (0.5 µg/well), or TFPI (0.5 µg/well) and
subsequently incubated with 3.4 10
cpm/well intact
I-ryudocan preparation (open columns) or that
which was pretreated with purified Flavobacterium heparitinase (closed columns) or chondroitin ABC lyase (hatched
columns) as indicated. After washing, the amount of bound
radioactivity was counted. Data from duplicate samples (mean ±
S.D.) are presented, and similar data were obtained in two additional
experiments. The absolute counts corresponding to 100% binding were
1690 cpm for bFGF, 1241 cpm for MK, and 162 cpm for
TFPI.
We also tested the
inhibition of ryudocan binding by heparin, HS, and CS (Fig. 5).
We was found that heparin distinctly inhibited the binding of I-ryudocan to bFGF, MK, and TFPI. HS also moderately
inhibited
I-ryudocan binding to these molecules, but at
high concentrations. In contrast, CS had no inhibitory effect on
I-ryudocan binding to these ligands.
Figure 5:
Inhibition of ryudocan binding by
glycosaminoglycans. Microtiter wells were coated with bFGF (0.25
µg/well), MK (0.25 µg/well), or TFPI (2.5 µg/well) and then
incubated with 6.6 10
cpm/well
I-ryudocan in the presence of heparin (Hp), HS,
or CS at the indicated concentration. After washing, the amount of
bound radioactivity was counted. Data from duplicate samples (mean
± S.D.) are presented, and similar data were obtained in two
additional experiments. The absolute counts corresponding to 100%
binding were 1615 cpm for bFGF, 1040 cpm for MK, and 1006 cpm for
TFPI.
We examined the
concentration-dependent binding of ryudocan to bFGF, MK, and TFPI by a
solid-phase assay as described under ``Experimental
Procedures.'' We found that the binding of ryudocan to bFGF, MK,
and TFPI was saturable (Fig. 6). Scatchard analyses showed that
the apparent K values were 0.50 nM for
bFGF, 0.30 nM for MK, and 0.74 nM for TFPI (Fig. 6, inset).
Figure 6:
Concentration-dependent binding of
ryudocan to bFGF, MK, and TFPI. Microtiter wells were coated with bFGF (open circles; 0.25 µg/well), MK (closed circles;
0.25 µg/well), or TFPI (open squares; 2.5 µg/well) and
then incubated with various concentrations of I-ryudocan
(specific activity of 4.2
10
cpm/µg). After
washing, the amount of bound radioactivity was counted. The insets show Scatchard analyses of
I-ryudocan binding to
immobilized bFGF (open circles), MK (closed circles),
and TFPI (open squares).
Figure 7:
Immunostaining of human ryudocan in
various human tissues. The tissue sections were immunostained with
anti-human ryudocan as described under ``Experimental
Procedures.'' Cytotrophoblasts (A; magnification
400), peripheral nerve tissues (B;
200), fetal lung
capillaries (C;
200), fetal endocardium (D;
200), fibrous regions of Bowman's capsules (E;
200), and fibrous regions of coronary atheromatous plaques (F;
200) were distinctly stained. To assure the
specificity of the signals, the tissue sections were also immunostained
with anti-human ryudocan antibody pretreated (+) or
nontreated(-) with an excess amount of Ryu2 peptide as described
under ``Experimental Procedures.'' Cytotrophoblasts (G;
100) and fetal lung capillaries (H;
100) were distinctly stained with nontreated antibody(-),
whereas the specific signals faded away when anti-human ryudocan IgG
was preincubated with an excess amount of Ryu2 peptide
(+).
To demonstrate the specificity of the signals, we used Ryu2 peptide as a competitor with the native ryudocan molecule as described under ``Experimental Procedures.'' The signals of cytotrophoblasts as well as the fetal pulmonary endothelium faded away when rabbit anti-human ryudocan IgG was incubated with an excess amount of Ryu2 peptide prior to immunostaining (Fig. 7, G and H). These results thus suggest that the stained signals would be specific for the human ryudocan molecule.
Ryudocan is one of the syndecan family members that are type I integral membrane HSPGs. HS binds a variety of proteins, including peptide growth factors, extracellular matrix components, cell adhesion molecules, lipolytic enzymes, protease inhibitors, and circulating lipoproteins. Syndecan, the best characterized molecule in its family, has been shown to selectively bind a variety of extracellular matrix molecules, suggesting that syndecan may be an extracellular matrix receptor(5, 6, 7, 8, 9) .
Recently, it was shown that cell-surface HS appears to be required
for the binding of bFGF to its high affinity receptor(15) .
Syndecan from mammary epithelial cells bound bFGF via its HS chains and
was considered a good candidate for a cell-surface HSPG low affinity
receptor(11) . However, overexpression of syndecan at the
surface of NIH 3T3 cells has been shown to increase fibronectin binding
and yet inhibit bFGF-induced cell proliferation(37) . It was
also reported that syndecan as well as glypican and fibroglycan block
heparin-dependent bFGF receptor binding due to competitive
inhibition(38) . In addition, perlecan, a large basal lamina
proteoglycan, has been identified as a major candidate for a bFGF low
affinity accessory receptor as well as an angiogenic modulator by
virtue of its differential HS structure(39) . Nevertheless, it
is still possible that bFGF high affinity interactions in vivo require cooperative effects between these cellular HSPGs. In this
study, we showed that ryudocan has a specific binding affinity for bFGF
via its HS chains, with an apparent K of 0.50
nM. We also demonstrated that ryudocan is distinctly expressed
in several fibrous tissues as well as in fetal lung capillaries and
endocardium. These data suggest that ryudocan may participate in
fibroblast growth and fetal angiogenesis through the bFGF interaction
either as a low affinity receptor or as an inhibitor of pathogenesis or
cellular development.
MK showed the highest affinity (apparent K = 0.30 nM) for ryudocan among
the three ligands tested. MK promotes both neurite outgrowth and the
survival of various embryonic neurons, and the neurite promoting
activity is in the COOH-terminal half of the MK molecule, which has
heparin binding activity(30) . MK has
50% sequence
identity to pleiotrophin(40) , also called heparin binding
growth-associated molecule(41) . MK and pleiotrophin have
similar functions and constitute a new family of heparin-binding
proteins involved in regulation of cellular growth and differentiation (42) . The neurite promoting activity was strongly inhibited by
heparin and only weakly by HS. (
)This mode of inhibition is
quite similar to that of ryudocan binding to MK described in this
paper. We also found that ryudocan is expressed abundantly in
peripheral nerve bundles. These findings imply that the interaction of
HS chains on ryudocan with MK may participate in the formation of a
neural network in peripheral nerve tissues. In this context, it is
noteworthy that N-syndecan was reported to be the receptor of
pleiotrophin in the promotion of neurite outgrowth of embryonic
neurons(19) . N-Syndecan is also considered to be a
potential co-receptor for bFGF during nerve tissue
development(18) .
Several investigators have shown that HSPG
is present on the endothelial cell surface and functions as an
anticoagulant(43) . Interestingly, ryudocan was originally
isolated from rat microvascular endothelial cells as an anticoagulant
HSPG(1, 2) . Immunohistochemical analysis revealed
that ryudocan is distinctly expressed in placental cytotrophoblasts as
well as in fetal lung capillaries. Placental tissues are known to be
resources for tissue factor involved in blood coagulation, and
placental dysfunction may cause disseminated intravascular
coagulopathy. It has been reported that TFPI is detected specifically
in macrophages in the villi of term placenta (44) and that
heparin enhances the rate of factor Xa inhibition by recombinant TFPI
in the presence of Ca(45) . In this study, we
demonstrated that purified ryudocan from EAhy926 cells possesses
significant affinity for TFPI, but not for ATIII. These data suggest
that human ryudocan may have an anticoagulant activity through its TFPI
interaction. This may be one physiological function of ryudocan in the
placental villus cytotrophoblasts.
The variation in structure and binding affinity of cell-surface HS on syndecan is a differentiated characteristic of each cell type(46) . This enables cells to respond to the specific HS-binding effectors in the cellular microenvironment. The attachment of different numbers of HS and CS chains to syndecan family members may alter interactions with specific proteins and hence modify the biological function of these components. It has been reported that ryudocan possesses three functional GAG attachment sites, that the sites are always occupied with GAG chains, and that each site is capable of bearing either HS or CS(47) . In addition, it was noted that the distribution of ryudocan isoforms and the length of ryudocan HS chains are altered when cells shift from the exponentially growing to the post-confluent state(47, 48) . These variations in structure of GAG chains may serve to expand the functional versatility of ryudocan and to allow it to participate in many different biological processes. We demonstrated in this report that purified human ryudocan, obtained from endothelium-like cells (EAhy926 cells), has specific affinities for such ligands as bFGF, MK, and TFPI. Ryudocan is distinctly expressed in peripheral nerve tissues, fibrous tissues, and placental trophoblasts. These results thus suggest that ryudocan may function as a bFGF modulator, as a neurite growth promoter, and as an anticoagulant via the HS-binding effectors present in the cellular microenvironment.