Enzymatic elimination of heparan sulfate (HS)
causes abnormal mesodermal and neural formation in Xenopus
embryos, and HS plays an indispensable role in establishing the
embryogenesis and tissue morphogenesis during early Xenopus
development (Furuya, S., Sera, M., Tohno-oka, R., Sugahara, K.,
Shiokawa, K., and Hirabayashi, Y. (1995) Dev. Growth
Differ. 37, 337-346). In this study, HS was purified from
Xenopus embryos to investigate its disaccharide composition
and binding ability to basic fibroblast growth factor (bFGF) and
follistatin (FS), the latter being provided in two isoforms with core
sequences of 315 and 288 amino acids (designated FS-315 and FS-288)
originating from alternative mRNA splicing. Disaccharide
composition analysis of the purified Xenopus HS showed the
preponderance of a disulfated disaccharide unit with uronic acid
2-O-sulfate and glucosamine 2-N-sulfate, which
has been implicated in the interactions with bFGF. Specific binding of
the HS to bFGF and FS-288, the COOH-terminal truncated form, was
observed in the filter binding assay, whereas HS did not bind to
FS-315, indicating that the acidic Glu-rich domain of FS-315 precluded
the binding. The binding of the HS to bFGF or FS-288 was markedly
inhibited by heparin (HP) and various HS preparations, but not by
chondroitin sulfate, supporting the binding specificity of HS. The
binding specificity was further investigated using FS-288 and bovine
intestinal [3H]HS. Competitive inhibition assays of the
HS binding to FS-288 using size-defined HP oligosaccharides revealed
that the minimum size required for significant inhibition was a
dodecasaccharide, which is larger than the pentasaccharide required for
bFGF binding. The binding affinity of FS to HS increased in the
presence of activin, a growth/differentiation factor, which could be
inactivated by direct binding to FS. These results, taken together,
indicate that the structural requirement for binding of HS to bFGF and FS is different. HS may undergo dynamic changes in its structure during
early Xenopus embryogenesis in response to the temporal and
spatial expression of various growth/differentiation factors.
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INTRODUCTION |
Heparan sulfate proteoglycans
(HS-PG)1 are ubiquitous
components of the extracellular matrix and cell surface of eukaryotic cells, where they exert a variety of biological functions (for a
review, see Ref. 1). In recent years, various effects of HS-PG on
growth factor-related cellular events have been observed. Basic
fibroblast growth factor (bFGF) is a typical growth factor and has been
detected as a complex with HS-PG in the extracellular matrix. Thus,
HS-PG is involved in protecting bFGF from protease digestion or
heat/acid-driven inactivation (2). More importantly, bFGF binds to the
cell surface HS-PG and the binding is essential for the interaction of
bFGF with its high affinity receptor molecule (3). HS-PG has also been
postulated to participate in mesoderm formation in early
Xenopus embryos (4-6). Immunohistochemistry using the
anti-HS mouse monoclonal antibody, HepSS-1, revealed that HS-PG occurs
mainly in the animal hemisphere in the early gastrulae, and then
appears predominantly on the sheath of the neural tube, the notochord
and the epithelium (6). Furthermore, elimination of HS-PG by
heparitinases induced abnormal mesodermal differentiation.
Embryogenesis is thought to be regulated by signaling factors, such as
FGFs (7), bone morphogenetic proteins (8, 9), activin (10, 11), midkine
(12), hepatocyte growth factor (13), Vg1 (14), Wnt (15, 16) and Sonic
Hedgehog (17), which are expressed in early stages in
Xenopus embryogenesis. Since most of these growth factors
have a heparin (HP)/HS binding property, at least some of these
signaling factors, probably exert their functions during embryogenesis
through interactions with HS-PG.
In early Xenopus development, the expression of bFGF is
turned on simultaneously from anterior and posterior regions at
mid-neurula stage and greatly increases during the late neurula and
tailbud stages (7). Disruption of the bFGF signaling pathway resulted in severe inhibition of invagination and neural tube closure in the
posterior region of embryos (18), suggesting that the bFGF signaling
pathway plays an important role in the formation of the posterior and
mesoderm in Xenopus embryogenesis. Follistatin (FS), which
was originally identified as an endogenous inhibitor for section of
follicle-stimulating hormone from pituitary cells (for a review, see
Ref. 19), is a potential neural inducer. FS occurs as two isoforms
(FS-315 and FS-288) originating from alternatively spliced mRNA.
FS-288 lacks the unique carboxyl-terminal extension with a glutamic
acid cluster present in FS-315 (20). Both FS-288 and FS-315 neutralize
the diverse actions of activin by forming a complex with activin (21,
22), a member of the transforming growth factor-
superfamily, which
induces dorso-anterior mesoderm. Although FS-288 shows
affinity for HS-PG, FS-315 does not, probably due to the presence of
the carboxyl-terminal extension (20). Thus, FS most likely plays a
significant role in the regulation of various actions of activin.
During the Xenopus embryogenesis, FS is expressed
predominantly in the Spemann organizer and the notochord, tissues known
to be potent neural inducers. Indeed, recent studies have suggested
that FS functions as a potential neural inducer through blocking of
activin actions (23, 24). Since HS-PG is also expressed in the
notochord (6), it may be involved in the blocking of activin by FS. FS
has been shown to associate with the cultured rat granulosa cell
surface was markedly inhibited by HP and HS, and also by treatment of
the cell surface with HP/HS-degrading enzymes (25), suggesting that FS
has a high affinity for the cell surface HS-PG.
In this study, the binding specificities of bFGF and FS to
Xenopus embryo HS were comparatively investigated as
representatives of HP/HS-binding growth/differentiation factors. The
relationship between the fine structure and the bFGF binding ability of
HP/HS has been investigated by several groups (26-29), and the minimum pentasaccharide sequence required for bFGF binding (28) has been
elucidated, although the biologically functional domain is larger than
the binding domain (30). On the other hand, the structural requirement
of HS for FS binding is not well understood. As mentioned above, the
spatial and temporal expression patterns of bFGF and FS, as well as
their roles in early Xenopus development, are different.
Hence, the precise structural requirement for the binding of bFGF and
FS within the HS chains may vary. In this study, HS was isolated from
early Xenopus embryos and its interactions with bFGF and FS
were investigated to compare structural requirements for the
binding.
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EXPERIMENTAL PROCEDURES |
Materials--
Heparan sulfate (HS) from Xenopus
embryos (stage 30) was prepared by exhaustive chondroitinase ABC
digestion of the sulfated GAG fraction that was purified from the early
tailbud embryos as reported (6). HS preparations from bovine kidney and
bovine intestinal mucosa were purchased from Sigma. Porcine intestinal HP was purchased from Nacalai Tesque (Kyoto, Japan). Chondroitin sulfate isoforms A, B, C, D and E, heparitinases I and II
(Flavobacterium heparinum), heparinase (Flavobacterium
heparinum), and chemically modified HP derivatives were obtained
from Seikagaku Corp. (Tokyo, Japan). A king crab cartilage chondroitin
sulfate K preparation was a gift from the late Dr. Nobuko Seno
(Ochanomizu University, Tokyo, Japan). Bovine liver glycosaminoglycans
(GAGs) were isolated basically as described previously (31), and
further purified by chromatography on a DEAE-cellulose column through
stepwise elution with 1.0 M LiCl (fraction 1) and 2.0 M LiCl (fraction 2). Fractions 1 and 2 contained HS and
chondroitin sulfate in a ratio of 1:1 and 7:3, respectively, as judged
by amino sugar analysis. Bovine lung HS was provided by Dr. Martin B. Mathews (University of Chicago, Chicago, IL). HS produced by the
Engelbreth-Holm-Swarm mouse tumor was isolated as described previously
(31). Human recombinant bFGF (rh-bFGF) was a gift from Dr. Koichi
Igarashi (Discovery Research Loboratories II, Takeda Chemical Ind.
Ltd., Tsukuba, Japan) (32). Human recombinant FS-288 (rhFS-288) and FS-315 (rhFS-315) were gifts from Dr. Shunichi Shimasaki (University of
California, San Diego, CA) and Dr. Yuzuru Eto (Ajinomoto Co., Kawasaki,
Japan), respectively. Porcine glycosylated follistatin isoforms
FS-288-1CHO and FS-303-1CHO, as well as activin isoforms activin A,
B, and AB, were purified from porcine ovaries. [3H]Acetic
anhydride (500 mCi/mmol) was purchased from Amersham Pharmacia Biotech
(Tokyo, Japan).
Disaccharide Composition Analysis of Xenopus Embryo
HS--
Xenopus embryo HS (1.0 µg) was digested with a
mixture of 1.3 mIU of heparinase, 1.3 mIU of heparitinase I, and 0.7 mIU of heparitinase II in a total volume of 75 µl of 20 mM sodium acetate buffer, pH 7.0, containing 2 mM Ca(OAc)2 at 37 °C for 3 h. Reactions were terminated by boiling for 1 min. Resultant disaccharides were analyzed according to recently developed methods (33), whereby
disaccharides were tagged with a fluorophore 2-aminobenzamide (2-AB)
and analyzed by HPLC. Briefly, after the sample was concentrated to
dryness in a vacuum concentrator, a 5-µl aliquot of a 0.25 M 2-AB solution in glacial acetic acid/dimethyl sulfoxide
(7/3, v/v) and 50 µl of a 1.0 M NaCNBH3
solution were added to the sample, and the mixture was incubated at
65 °C for 2 h, and concentrated to dryness. An aliquot of the
sample corresponding to 16 ng was analyzed by HPLC on an amine-bound
silica PA03 column basically as described (34), except that a
linear gradient of NaH2PO4 was made from 16 to
800 mM over 60 min. Eluates were monitored using a
fluorometric detector RF-535 (Shimadzu Co., Kyoto, Japan) with
excitation and emission wavelengths of 330 and 420 nm,
respectively.
Preparation of [3H]Acetyl-labeled HS
Polysaccharides--
N-[3H]Acetyl labeling of
HS was carried out basically according to the procedure of Shaklee and
Conrad (35). Xenopus embryo HS or bovine intestinal HS (180 µg each) was mixed with 100 µl of hydrazine monohydrate (Nacalai
Tesque) containing 1.0 mg of hydrazine sulfate in a test tube, which
was then sealed and heated at 96 °C for 6 h. The mixture was
concentrated to dryness, reconstituted in 100 µl of water, and
evaporated to dryness. This process was repeated once more to remove
hydrazine. The polysaccharides were isolated by gel filtration on a
column (0.8 × 56 cm) of Sephadex G-25 eluted with 0.25 M NH4HCO3, 7% propanol. The
polysaccharide fraction was desalted by repeated evaporation with water
and dissolved in 200 µl of 10% methanol containing 0.05 M Na2CO3. The solution was mixed
with 2.5 mCi of [3H]acetic anhydride on ice in a fume
hood. The pH of the reaction mixture was kept at 7.0 by addition of
10% methanol containing 0.05 M
Na2CO3. The reaction was continued for a total
of 2 h, with repeated addition of 2.5 mCi of
[3H]acetic anhydride every 20 min. During a 1-h period, 1 µl of unlabeled acetic anhydride was added three times. The pH was
maintained at 7.0 by adding 10% methanol containing 0.05 M
Na2CO3. The reaction mixture was applied to a
column (1 × 46 cm) of Sephadex G-50, which was eluted with 0.25 M NH4HCO3, 7% propanol. Labeled
materials excluded from the gel were pooled, concentrated to dryness,
and desalted by repeated evaporation with water.
Filter Binding Assay--
Various amounts of rh-bFGF were
incubated with Xenopus embryo [3H]HS in 50 µl of 50 mM Tris-HCl, pH 7.4, containing 130 mM NaCl and 0.5 mg/ml bovine serum albumin at room
temperature for 3 h. The growth factor, along with any bound
[3H]HS, was recovered by quick passage of the samples
through nitrocellulose filters (Sartorius, pore size 0.45 µm; 25 mm
diameter), which had been placed onto a 12-well vacuum-assisted
manifold filtration apparatus. The filters were prewashed with 10 ml of
50 mM Tris-HCl, 130 mM NaCl, pH 7.4, before
application of the samples, which was immediately followed by washing
five more times with 2 ml of the same buffer. Protein-bound
radioactivity was determined after submersion of the filters in 1 ml of
1 M NaCl, 0.05 M diethylamine, pH 11.5, for 30 min; radioactivity in the eluate was determined in a liquid
scintillation counter (Aloka LSC-700) using a scintillation fluid
containing 1.2% (w/v) 2,5-diphenyloxazole and 33% (w/v) Triton
X-100.
FS (rhFS-288 or rhFS-315) was incubated with Xenopus embryo
[3H]HS in 50 µl of 20 mM HEPES-NaOH, pH
7.3, containing 150 mM NaCl and 0.5 mg/ml bovine serum
albumin at room temperature for 3 h. In competition experiments,
various unlabeled GAGs were included as inhibitors in the incubation
mixture. Binding of [3H]HS to FS was determined as for
bFGF.
Examination of effects of activin on the interaction between FS and HS
was performed as follows. Activin (0.6 µg of activin A, B, or AB
isoform) was preincubated with FS (0.3 µg of rhFS-288 or rhFS-315) in
25 µl of 20 mM HEPES-NaOH, pH 7.3, containing 150 mM NaCl and 0.5 mg/ml bovine serum albumin at room
temperature for 1 h. The reaction mixture was then incubated with
bovine intestinal [3H]HS (50 ng, 10000 cpm) in a total
volume of 50 µl of the same buffer at room temperature for 2 h.
Binding of [3H]HS to the activin-FS complex was
determined as described above.
Preparation of HP Oligosaccharides--
Even numbered HP
oligosaccharides were generated by enzymatic degradation. HP (15 mg)
was digested with 20 mIU heparinase in a total volume of 1 ml of 30 mM acetate-NaOH buffer, pH 7.0, containing 3 mM
Ca(OAc)2 and 1% bovine serum albumin. When the reaction
reached a plateau after 1 h as monitored by absorption at 232 nm,
it was terminated by heating at 100 °C for 1 min. The digest was
adjusted to 1.0 M NaCl and fractionated into even-numbered species on a column (1.6 × 95 cm) of Bio-Gel P-10, which was
equilibrated and eluted with 1.0 M NaCl, 10% ethanol.
Elution was performed with the same solution. Fractions (2 ml) were
collected and monitored by absorption at 232 nm. The separated
fractions were pooled, concentrated, and desalted by gel filtration on
a column (1.5 × 46.5 cm) of Sephadex G-25, and lyophilized.
Analytical Method--
Uronic acid was determined by the
carbazole method (36).
 |
RESULTS |
Our previous results suggested that HS played an indispensable
role in establishing the fundamental body plan during the early Xenopus development (6). In this study, HS was purified from stage 30 embryos and characterized for its structure, as well as for
the ability to interact with bFGF and FS as representatives of typical
HS-binding growth/differentiation factors.
Disaccharide Composition Analysis of Xenopus Embryo
HS--
Xenopus embryo HS was subjected to a disaccharide
composition analysis after digestion with a mixture of heparinase and
heparitinases I and II. The resulting disaccharides were labeled with a
fluorophore 2-AB and analyzed by anion-exchange HPLC according to the
recently developed fluorophore-tagging method (33). Nearly 55% of the disaccharides were N-sulfated, 24% contained a hexuronate
2-O-sulfate residue, and 17% were 6-O-sulfated
and relatively low as compared with N- or
2-O-sulfate (Table I). These
findings were in good agreement with the results obtained by the
disaccharide composition analysis after deamination with
HNO2 followed by reduction with NaB3H4 (data not shown). The levels of the
N-sulfation and 2-O-sulfation were higher,
whereas that of 6-O-sulfation was lower, if compared with a
commercial reference compound bovine kidney HS (37). Compared with HS
from porcine and bovine organs, the Xenopus HS was
characterized by a higher content of the HexA(2S)-GlcN(NS) disaccharide
unit representing 17.9% of the total disaccharides (Table I), where
HexA, 2S, and NS represent hexuronic acid, 2-O-sulfate, and
2-N-sulfate, respectively. These results may be relevant to the importance of N- and 2-O-sulfations for the
binding of the Xenopus embryo HS to FS and/or bFGF as
described below.
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Table I
Disaccharide composition analysis of Xenopus embryo HS
After Xenopus embryo HS was incubated with a mixture of
heparinase, heparitinases I and II at 37 °C for 3 h, the digest
was labeled with a fluorophore 2-AB and analyzed by HPLC as described under "Experimental Procedures." Eluates were monitored using a
fluorometric detector with excitation and emission wavelengths of 330 and 420 nm, respectively.
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Binding of Xenopus Embryo HS to rh-bFGF--
3H
labeling of the HS preparation was conducted by
N-deacetylation with hydrazine followed by
N-reacetylation with
[3H](CH3CO)2O. The
3H-labeled Xenopus embryo HS preparation was
incubated with rh-bFGF, and the binding ability was evaluated using the
nitrocellulose filter binding assay (see "Experimental
Procedures"). bFGF, along with any bound carbohydrate, was recovered
through nitrocellulose filters. The filters were examined in a
scintillation spectrometer. Specific binding of the Xenopus
embryo HS to rh-bFGF was concentration-dependent as shown
in Fig. 1. The direct binding of the
Xenopus embryo HS to rh-bFGF suggests that the HS contains
the pentasaccharide sequence -HexA-GlcN(NS)-HexA-GlcN(NS)-IdceA(2S)- required for specific binding to bFGF (28). To further characterize the binding specificity of the Xenopus embryo HS to bFGF, the effects of various
kinds of GAGs on the binding were examined. The 3H-labeled
Xenopus embryo HS was incubated with rh-bFGF in the presence
of various GAGs and dextran sulfate (Table
II). Dextran sulfate, HP, and HS
preparations except for the undersulfated HS produced by the
Engelbreth-Holm-Swarm mouse tumor inhibited the binding, whereas
various chondroitin sulfate isoforms exhibited no significant
inhibition, supporting the specific binding of the
Xenopus embryo HS to bFGF.

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Fig. 1.
Binding of Xenopus embryo
[3H]HS to bFGF. Xenopus embryo
[3H]HS (30 ng, 4.0 × 104 dpm) was
incubated with various amounts of rh-bFGF. The radioactivity bound to
bFGF was quantified by the filter binding assay as described under
"Experimental Procedures." Values were obtained from the average of
two separate experiments, and are expressed as percentages of the
radioactivity added to the incubation.
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Table II
Inhibition of the binding of Xenopus embryo HS to rh-bFGF by
various glycosaminoglycans
rh-bFGF (0.1 µg) was incubated with Xenopus embryo
[3H]HS (30 ng, 4.0 × 104 dpm) in the presence
of an unlabeled GAG or dextran sulfate (Mr = 5000)
at a concentration of 0.4 or 2.0 µg/ml. The radioactivity bound to
rh-bFGF was quantitated by liquid scintillation counting. Negative
controls without rh-bFGF showed a background level of approximately 300 dpm. The values were expressed as the percentages of the positive
control values.
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Binding of Xenopus Embryo HS to FS--
The binding ability of the
Xenopus embryo HS to the two FS isoforms originating from
alternatively spliced mRNA were compared using the nitrocellulose
filter binding assay (Fig. 2). The
Xenopus embryo HS showed a high affinity for rhFS-288, the
human recombinant FS short form, but no appreciable affinity for the
long form, rhFS-315. Binding of the Xenopus embryo HS to
rhFS-288 was concentration-dependent (Fig. 2),
demonstrating direct interaction between the two. Although saturation
of the binding was not shown due to a limited availability of rhFS-288,
the binding specificity was demonstrated by the inhibition study as
described below. The above results are in good agreement with the
previous observation that the COOH-terminal truncated isoform (FS-288)
bound to the HS on rat granulosa cell surfaces (20). The FS isoforms
containing one N-glycosidic oligosaccharide chain, which
were isolated from porcine ovaries and designated as FS-288-1CHO and
FS-303-1CHO (20), were also examined for their affinities to the
Xenopus embryo HS. FS-303-1CHO is thought to be
derived from FS-315 by post-translation proteolytic cleavage of the 12 COOH-terminal amino acids, and it still contains the carboxyl-terminal
glutamic acid cluster. The short form, FS-288-1CHO, bound to the HS
whereas the long form, FS-303-1CHO, did not (data not shown),
suggesting that HS regulates the action of FS-288 but not that of
FS-303, during the Xenopus embryo development and that the
carbohydrate chain does not appreciably affect its interaction with
HS.

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Fig. 2.
Binding of Xenopus embryo
[3H]HS to FS. Xenopus embryo
[3H]HS (90 ng, 2.5 × 104 dpm) was
incubated with various amounts of rhFS-288 or rhFS-315. The
radioactivity bound to rhFS-288 or rhFS-315 was quantified by filter
binding assay as described under "Experimental Procedures." Values
were obtained from the average of two separate experiments, and are
expressed as percentages of the radioactivity added to the incubation.
Closed circles, FS-288; open circles,
FS-315.
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Inhibition of the Binding of Xenopus Embryo HS to FS by Various
GAGs--
To characterize the binding specificity of the
Xenopus embryo HS to FS, the effects of various kinds of
GAGs on the binding were examined. The 3H-labeled
Xenopus embryo HS (1.8 µg/ml) was incubated with rhFS-288 in the presence of increasing amounts of various GAGs (0.5-4.0 µg/ml). Unlabeled Xenopus embryo HS precluded the binding
of the 3H-labeled Xenopus embryo HS to rhFS-288,
and 50% inhibition was observed at a concentration of 1.0 µg/ml
(Fig. 3). Bovine kidney HS, bovine
intestinal HS, and porcine intestinal HP inhibited the binding also,
whereas chondroitin sulfate A exhibited no significant inhibition even
at 4.0 µg/ml. Undersulfated HS produced by the Engelbreth-Holm-Swarm
mouse tumor exhibited weak inhibition (data not shown). Porcine
intestinal HP was more potent than the HS preparations in these
inhibition assays. The results indicate that HP and the HS except for
the undersulfated HS preparation bear specific binding domains for
rhFS-288, and that sulfate groups are essential for FS binding.

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Fig. 3.
Inhibition of the binding of Xenopus
embryo HS to FS-288 by various GAGs. rhFS-288 (0.6 µg) was
incubated with Xenopus embryo [3H]HS (90 ng,
2.5 × 104 dpm) along with various amounts of
unlabeled Xenopus embryo HS (closed circles),
bovine kidney HS (open circles), bovine intestinal HS
(open triangles), porcine intestinal HP (open
squares), or chondroitin sulfate A (closed squares).
The radioactivity bound to rhFS-288 was quantified by filter binding
assay as described under "Experimental Procedures." The data are
expressed as percentages of the control value obtained in the absence
of added GAGs.
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Inhibition of the Binding of Bovine Intestinal HS to FS-288 by
Various GAGs--
To further investigate the structural features of HS
responsible for its binding to FS-288 and to clarify whether bFGF and FS-288 recognize the same or distinct sequences within the HS chain(s),
we examined the effects of various kinds of oligo- and polysaccharides
including size-defined sulfated oligosaccharides and chemically
modified GAGs on the binding of HS to FS-288. Since, however, the
specific radioactivity of the 3H-labeled Xenopus
embryo HS was considerably low (2.3 × 105 dpm/µg)
and the available amount was limited, bovine intestinal HS was used for
further studies. FS is expressed in intestine (19), supporting the
possibility that intestinal HS interacts with FS. Commercial bovine
intestinal HS was radiolabeled with [3H](CH3CO)2O after
N-deacetylation with hydrazine. The resultant 3H-labeled HS with a specific radioactivity (6.2 × 105 dpm/µg) was incubated with rhFS-288 and the binding
ability was evaluated. Direct binding was observed in a
concentration-dependent manner as shown in Fig.
4. The amount of bovine intestinal HS (approx. 7 ng) bound to 0.5 µg of rhFS-288 was comparable to that of
the Xenopus embryo HS bound to the same ligand. Although
saturation of the binding was not shown due to the limited availability
of rhFS-288, the binding specificity was demonstrated by the inhibition studies described below. The effects of various kinds of sulfated oligo- and polysaccharides were next examined on the binding of the
3H-labeled bovine intestinal HS to rhFS-288. The bovine
intestinal [3H]HS was incubated with rhFS-288 in the
presence of increasing amounts of various GAGs as shown in Fig.
5. Bovine intestinal HS and porcine
intestinal HP inhibited clearly the 3H-labeled HS binding,
with 50% inhibition at 6.0 and 0.6 µg/ml, respectively, whereas
bovine kidney HS showed only weak inhibition. Most of the chondroitin
sulfate isoforms exhibited weak or no inhibition, whereas chondroitin
sulfate E unexpectedly had a strong effect with 50% inhibition at 1.2 µg/ml. The IC50 value was approximately equivalent to
that of porcine intestinal HP. The inhibition was in strong contrast
with the lack of inhibition by chondroitin sulfate E in the bFGF
system, supporting the specificity of the observed inhibition and
different structural requirements of HS for the binding to bFGF and
FS.

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Fig. 4.
Binding of bovine intestinal
[3H]HS to FS-288. Bovine intestinal
[3H]HS (50 ng, 3.5 × 104 dpm) was
incubated with various amounts of rhFS-288. The radioactivity bound to
rhFS-288 was quantified by filter binding assay as described under
"Experimental Procedures." Values were obtained from the average of
two separate experiments and are expressed as percentages of the
radioactivity added to the incubation.
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Fig. 5.
Inhibition of the binding of bovine
intestinal HS to FS-288 by various GAGs. rhFS-288 (0.3 µg) was
incubated with bovine intestinal [3H]HS (50 ng, 3.5 × 104 dpm) along with various amounts of unlabeled bovine
intestinal HS (closed circles), bovine kidney HS (open
circles), porcine intestinal HP (closed squares),
chondroitin sulfate A, C, and D (open squares), chondroitin
sulfate B (closed triangles), and chondroitin sulfate E
(open triangles). The radioactivity bound to rhFS-288 was
quantified by filter binding assay as described under "Experimental
Procedures." Data are expressed as percentages of the control value
obtained in the absence of the added GAG.
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The effects of chemically modified HP preparations on the binding
of the bovine intestinal [3H]HS with rhFS-288 were also
investigated (Fig. 6). Three HP
derivatives including completely desulfated and N-acetylated
(CDSNAc), completely desulfated and N-sulfated
(CNDNS), and N-desulfated and
N-acetylated (NDSNAc) HPs were used. None of the
HP derivatives showed competition with the binding of the
[3H]HS with rhFS-288, indicating the importance of both
O- and N-sulfate groups.

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Fig. 6.
Inhibition of the binding of bovine
intestinal HS to FS-288 by chemically modified HP derivatives.
rhFS-288 (0.3 µg) was incubated with bovine intestinal
[3H]HS (50 ng, 3.5 × 104 dpm) in the
presence of completely desulfated and N-acetylated (CDSNAc-) HP, completely desulfated and
N-sulfated (CNDNS-) HP, N-desulfated
and N-acetylated (NDSNAc-) HP, or unmodified HP
(50 ng). The radioactivity bound to rhFS-288 was quantified by filter binding assay as described under "Experimental Procedures." Values were obtained from the average of two separate experiments and are
expressed as percentages of the control value obtained in the absence
of added HP or chemically modified HP derivatives.
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Determination of the Minimum Size Requirement of HP for Interaction
with FS-288--
HP showed the highest inhibition activity among the
polysaccharides tested against the binding of the bovine intestinal
[3H]HS to rhFS-288. To investigate the minimum size
requirement of the HP saccharide sequence for the interaction,
competition experiments were carried out, in which even-numbered HP
oligosaccharide fractions were allowed to compete with the bovine
intestinal [3H]HS for binding to rhFS-288. As shown in
Fig. 7B, oligosaccharide fractions longer than dodecasaccharides were able to compete with the
[3H]HS, whereas shorter oligosaccharide (di- to
decasaccharide) fractions were not. The tetradecasaccharide fraction
showed stronger inhibition than the dodecasaccharide fraction, and the
hexadecasaccharide fraction showed a comparable inhibition to that of
the intact parent HP fraction. These results support that the
dodecasaccharide represents the minimum size required to interact with
FS-288. Fig. 7A shows the results of the competition
experiments where the minimum size requirement for the bFGF binding was
examined. The smallest size showing appreciable inhibition was a
hexasaccharide, being consistent with previous reports (28, 38). These
results suggest that the minimum size required for interaction with
FS-288 is larger than that for the interaction with bFGF and that the structural requirement of HS for the FS-288 binding is different from
that for the bFGF binding.

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Fig. 7.
Inhibition of the binding of bovine
intestinal HS to bFGF and FS-288 by HP oligosaccharides. A,
rh-bFGF (0.1 µg) was incubated with Xenopus embryo
[3H]HS (30 ng, 4.0 × 104 dpm) in the
presence of unlabeled HP or HP oligosaccharides of various sizes (0.1 µg). B, rhFS-288 (0.3 µg) was incubated with bovine
intestinal [3H]HS (50 ng, 3.5 × 104
dpm) in the presence of unlabeled HP or HP oligosaccharides of various
sizes (50 ng). The radioactivity bound to rh-bFGF and rhFS-288 was
quantified by filter binding assay as described under "Experimental
Procedures." Values were obtained from the average of two separate
experiments and are expressed as percentages of the control value
obtained in the absence of added HP or HP oligosaccharides.
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Effect of Activin on the Interaction between FS and
HS--
Activin was preincubated with FS, then incubated with
[3H]HS, and the reaction mixture was subjected to a
filter binding assay using a nitrocellulose membrane. Activin is a
covalently linked dimer of at least two different subunits
(
A and
B) and therefore exists in three
different isoforms; activin A (
A/
A),
activin B (
B/
B), and activin AB
(
A/
B) (for a review, see Ref. 39). Three
activin isoforms, A, B, and AB, were tested for the effects on the
interaction between rhFS-288 or -315 and the bovine intestinal [3H]HS. Although activin by itself had no affinity for
HS, it increased the amount of [3H]HS bound to FS (Fig.
8). These results suggest that the
formation of the activin-FS complex increased the binding affinity of
FS to HS, and that the conformation of the HS binding site of FS may
have been changed by the formation of the complex. We next performed
competition experiments similar to those performed in Fig. 7 and
investigated the minimum size requirement of HP saccharide sequence for
the interaction with the activin-FS complex. The minimum size was a
dodecasaccharide (results not shown) as for the interaction with FS
alone.

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Fig. 8.
Effect of activin on the binding of bovine
intestinal HS to FS. rhFS-288 or -315 (0.3 µg) was preincubated
with purified porcine activin A, B, or AB (0.6 µg), and the reaction
mixture was then incubated with bovine intestinal [3H]HS
(50 ng, 3.5 × 104 dpm). The radioactivity bound to
the activin-FS complex was quantified by filter binding assay as
described under "Experimental Procedures." Values were obtained
from the average of two separate experiments.
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DISCUSSION |
A number of signaling factors, such as FGFs, activin, and FS, are
thought to be involved in the regulation of embryogenesis. In the
present study, we isolated Xenopus embryo HS, which has been
proposed to play an indispensable role in establishing the fundamental
body plan during the early development (4-6), and investigated its
interactions with bFGF, FS, and activin. Significant binding of the
isolated HS to bFGF and FS but not to activin was demonstrated. The
present results support the concept that HS-PG is involved in the
regulation of the Xenopus embryogenesis through interacting
with signaling factors, including bFGF and FS.
The specific interactions between HS and various growth
factors/cytokines have attracted much attention, with bFGF having been
investigated the most. bFGF-binding structures within HS/HP molecules
have been suggested to contain consecutive -IdceA(2S)-GlcN(NS)- disaccharide units (26, 27). The shortest possible, least sulfated HS
oligosaccharide capable of binding bFGF was defined by Maccarana
et al. (28) as the pentasaccharide sequence,
-GlcA-GlcN(NS)-HexA-GlcN(NS)-IdceA(2S)-, although additional adjacent
saccharide sequence(s) are required for the expression of the
biological functions of the growth factor (30).
Among interactions between numerous other growth factors/cytokines and
HS/HP, only those for a few factors including hepatocyte growth factor
(40, 41), interferon-
(42), acidic FGF (43, 44), FGF-4 (44),
platelet factor 4 (45, 46), and platelet-derived growth factor (47)
have been partially characterized. In the present study, detailed
structural requirements of the HS-FS binding were investigated for the
first time. The results demonstrated that sulfate groups are essential
for the interaction with FS and that the O-sulfate and
N-sulfate groups are of equal importance. Previously,
Nakamura et al. (25) demonstrated that NDSNAc HP prevented
binding in a concentration-dependent manner, suggesting the
importance of O-sulfate but not N-sulfate groups.
This discrepancy may have resulted from differences in the experimental
conditions. They used a cell culture system and analyzed the
interaction between the HS-PG expressed on the cell surface and the
complex of 125I-labeled activin-FS, whereas we used
purified [3H]HS and FS in an in vitro system.
Inhibition experiments with HP-derived oligosaccharides indicated that
the minimal saccharide size required for the FS binding was a
dodecasaccharide. The pentasaccharide has been demonstrated to be the
minimum size required for interaction with bFGF (28), and a
hexasaccharide fraction was demonstrated to be in fact a minimum size
even-numbered oligosaccharide fraction in this study, indicating that
the structural requirement of HS for the FS-288 binding may vary from
that for bFGF. Furthermore, squid cartilage chondroitin sulfate E
inhibited the binding of HS to FS but not to bFGF, suggesting the
specificity of the binding to the former, also supporting the
difference in the recognition of the binding sequence between bFGF and
FS. Although it is unknown whether chondroitin sulfate E is a
physiological ligand of FS, it is expressed in cell-type and
tissue-type specific manners and in a developmentally regulated fashion
(48-51). Recent structural studies of squid cartilage chondroitin
sulfate E revealed the highly sulfated novel disaccharide unit
GlcA(3-O-sulfate)
1-3GalNAc(4-O-,6-O-disulfate) which is equivalent in charge density to the typical trisulfated disaccharide unit
IdceA(2-O-sulfate)
1-4GlcN(2-N-,6-O-disulfate) of HP (52). Furthermore, chondroitin sulfate E has been
demonstrated to bind to a HP-binding growth/differentiation factor
midkine (53, 54), suggesting biological activities of chondroitin sulfate E. It remains to be determined whether chondroitin sulfate E is
expressed at any developmental stage of Xenopus
embryogenesis and indeed plays an important role during differentiation
by interacting with FS.
The ability of FS to bind to the pleiotropic growth/differentiation
factor, activin, and thereby to neutralize activin actions, makes FS a
potentially important regulatory factor that modulates differentiation and developmental processes (19). Although FS by itself has recently been shown to exhibit definable effects as a
growth and/or differentiation factor independent of activin (for a
review, see Ref. 55), its receptor has not been identified and most of
the physiological activities of FS are expressed in general through
neutralization of activin actions. The physiological significance of a
formation of the FS-HS complex on the cell surface is not well
understood. Since the activin binding to the cell surface has been
demonstrated to be promoted much more markedly by FS-288 than by FS-315
and abolished by exogenous HS (20, 25), it is possible that HS plays a
regulatory role in the activin signaling through the interaction with
FS. Hashimoto et al. (56) recently showed that FS associated
with HS at the cell surface accelerated the internalization and
subsequent degradation of activin by lysosomal enzymes. Hence, HS may
play a role in the clearance of activin. The present results indicated
that the formation of an activin-FS complex increased the affinity of
FS to HS. Since the activin-FS complex contains one activin and two FS
molecules (19), the dimeric structure may have a higher affinity to HS than a FS monomer through conformational changes of the protein. The
effects of activin were observed not only for FS-288 but also for
FS-315, indicating that they are independent of the unique stretch of
acidic amino acids which is located at the carboxyl terminus of FS-315
and prevents HS binding (20). The minimal saccharide size of HS
required for binding to FS was a dodecasaccharide irrespective of the
formation of the FS-activin complex. The mechanism by which the binding
of FS to HS was accelerated by activin still remains unclear.
Although the interactions of various growth factors/cytokines with HS
have been investigated, most such studies have dealt with a single
protein. We demonstrated some differences in the binding specificities
of Xenopus embryo HS to two protein ligands bFGF and FS-288.
These differences in binding specificities may be associated with
functional differences. In the early Xenopus development,
the expression of bFGF and its mRNA is turned on both from anterior
and posterior regions in the mid-neurula stage and greatly increases
during the late neurula and tailbud stages (7), whereas FS is expressed
predominantly in the Spemann organizer and the notochord (23, 24). The
bFGF signaling pathway plays an important role in the formation of the
posterior and mesoderm, while FS plays a role in neural induction,
indicating that their roles in the early Xenopus development
are also different. Nurcombe et al. (57) demonstrated that a
single species of HS-PG undergoes a rapid, tightly controlled change in
growth factor-binding specificity concomitant with the temporal
expression of acidic FGF and bFGF during murine neural development. The
Xenopus HS chain may also undergo dynamic structural changes
and switch binding properties coordinated with the timing of the
expression of the various growth factors such as bFGF and FS to control
their activities during the early embryogenesis.
We thank Chie Tohriyama, Astuko Nagata, and
Hiromi Hashiguchi (Kobe Pharmaceutical University) for the preparation
of HS from bovine liver.