Molecular Characterization of Xenopus Embryo Heparan Sulfate
DIFFERENTIAL STRUCTURAL REQUIREMENTS FOR THE SPECIFIC BINDING TO BASIC FIBROBLAST GROWTH FACTOR AND FOLLISTATIN*

Yukari YamaneDagger , Rie Tohno-okaDagger , Shuhei YamadaDagger , Shigeki Furuya§, Koichiro Shiokawa, Yoshio Hirabayashi§, Hiromu Suginopar , and Kazuyuki SugaharaDagger **

From the Dagger  Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658, the § Laboratory for Cellular Glycobiology, Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, the  Laboratory for Molecular Embryology, Zoological Institute, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, and the par  Institute for Enzyme Research, University of Tokushima, Kuramoto, Tokushima 770, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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-beta 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

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.

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.

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.

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.

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.

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.

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 (beta A and beta B) and therefore exists in three different isoforms; activin A (beta A/beta A), activin B (beta B/beta B), and activin AB (beta A/beta 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.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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-gamma (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)beta 1-3GalNAc(4-O-,6-O-disulfate) which is equivalent in charge density to the typical trisulfated disaccharide unit IdceA(2-O-sulfate)alpha 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.

    ACKNOWLEDGEMENTS

We thank Chie Tohriyama, Astuko Nagata, and Hiromi Hashiguchi (Kobe Pharmaceutical University) for the preparation of HS from bovine liver.

    FOOTNOTES

* This work was supported in part (at Kobe Pharmaceutical University) by the Science Research Promotion Fund from Japan Private School Promotion Foundation, a grant from the Hyogo Science and Technology Association, a grant from the Japan Health Sciences Foundation, and Grants-in-aid for Exploratory Research 08877338, Scientific Research (B) 09470509, and Scientific Research on Priority Areas 05274107 from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed. Tel.: 81-78-441-7570; Fax: 81-78-441-7569; E-mail: k-sugar{at}kobepharma-u.ac.jp.

1 The abbreviations used are: HS-PG, heparan sulfate proteoglycan; HS, heparan sulfate; FGF, fibroblast growth factor; bFGF, basic fibroblast growth factor; FS, follistatin; 2-AB, 2-aminobenzamide; GAG, glycosaminoglycan; HP, heparin; Delta HexA, 4-deoxy-alpha -L-threo-hex-4-enepyranosyluronic acid; HexA, hexuronic acid; IdceA, L-iduronic acid; 2S, 2-O-sulfate; 6S, 6-O-sulfate; NS, 2-N-sulfate; rh-bFGF, recombinant human basic fibroblast growth factor; rhFS, recombinant human follistatin; HPLC, high performance liquid chromatography.

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Abstract
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Discussion
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