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• W2106796522 abstract "Enzymatic elimination of heparan sulfate (HS) causes abnormal mesodermal and neural formation in Xenopusembryos, and HS plays an indispensable role in establishing the embryogenesis and tissue morphogenesis during early Xenopusdevelopment (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 fromXenopus 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. Enzymatic elimination of heparan sulfate (HS) causes abnormal mesodermal and neural formation in Xenopusembryos, and HS plays an indispensable role in establishing the embryogenesis and tissue morphogenesis during early Xenopusdevelopment (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 fromXenopus 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. Heparan sulfate proteoglycans (HS-PG) 1The 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; ΔHexA, 4-deoxy-α-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. 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. 1Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1678) Google Scholar). 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 (2Burgess W.H. Maciag T. Annu. Rev. Biochem. 1991; 58: 575-606Crossref Google Scholar). 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 (3Yayon A. Klagsbrum M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2086) Google Scholar). HS-PG has also been postulated to participate in mesoderm formation in earlyXenopus embryos (4Brickman M.C. Gerhart J.C. Dev. Biol. 1994; 164: 484-501Crossref PubMed Scopus (32) Google Scholar, 5Itoh K. Sokol S.Y. Development. 1994; 120: 2703-2711PubMed Google Scholar, 6Furuya S. Sera M. Tohno-oka R. Sugahara K. Shiokawa K. Hirabayashi Y. Dev. Growth Differ. 1995; 37: 337-346Crossref Scopus (10) Google Scholar). 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 (6Furuya S. Sera M. Tohno-oka R. Sugahara K. Shiokawa K. Hirabayashi Y. Dev. Growth Differ. 1995; 37: 337-346Crossref Scopus (10) Google Scholar). Furthermore, elimination of HS-PG by heparitinases induced abnormal mesodermal differentiation. Embryogenesis is thought to be regulated by signaling factors, such as FGFs (7Song J. Slack J.M.W. Mech. Dev. 1994; 48: 141-151Crossref PubMed Scopus (51) Google Scholar), bone morphogenetic proteins (8Wilson P.A. Hemmati-Brivanlou A. Nature. 1995; 376: 331-333Crossref PubMed Scopus (649) Google Scholar, 9Sasai Y. Lu B. Steinbeisser H. De Robertis E.M. Nature. 1995; 376: 333-336Crossref PubMed Scopus (540) Google Scholar), activin (10Smith J.C. Price B.M.J. Van Nimmen K. Huylebroeck D. Nature. 1990; 345: 729-731Crossref PubMed Scopus (565) Google Scholar, 11van den Eijnden-Van Raaij A.J.M. van Zoelent E.J.J. van Nimmen K. Koster C.H. Snoek G.T. Durston A.J. Huylebroeck D. Nature. 1990; 345: 732-734Crossref PubMed Scopus (188) Google Scholar), midkine (12Sekiguchi K. Yokota C. Asashima M. Kaname T. Fan Q.-W. Muramatsu T. Kadomatsu K. J. Biochem. 1995; 118: 94-100Crossref PubMed Scopus (33) Google Scholar), hepatocyte growth factor (13Nakamura H. Tashiro K. Nakamura T. Shiokawa K. Mech. Dev. 1995; 49: 123-131Crossref PubMed Scopus (26) Google Scholar), Vg1 (14Kessler D.S. Melton D.A. Development. 1995; 121: 2155-2164Crossref PubMed Google Scholar), Wnt (15McGrew L.L. Otte A.P. Moon R.T. Development. 1992; 115: 463-473Crossref PubMed Google Scholar, 16Christian J.L. McMahon J.A. McMahon A.P. Moon R.T. Development. 1991; 111: 1045-1055PubMed Google Scholar) and Sonic Hedgehog (17Ekker S.C. McGrew L.L. Lai C.J. Lee J.J. von Kessler D.P. Moon R.T. Beachy P.A. Development. 1995; 121: 2337-2347PubMed Google Scholar), 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 (7Song J. Slack J.M.W. Mech. Dev. 1994; 48: 141-151Crossref PubMed Scopus (51) Google Scholar). Disruption of the bFGF signaling pathway resulted in severe inhibition of invagination and neural tube closure in the posterior region of embryos (18Amaya E. Musci T.J. Kirschner M.W. Cell. 1991; 66: 257-270Abstract Full Text PDF PubMed Scopus (927) Google Scholar), 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. 19Michel U. Farnworth P. Findlay J.K. Mol. Cell. Endocrinol. 1993; 91: 1-11Crossref PubMed Scopus (136) Google Scholar), 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 (20Sugino K. Kurosawa N. Nakamura T. Takio K. Shimasaki S. Ling N. Titani K. Sugino H. J. Biol. Chem. 1993; 268: 15579-15587Abstract Full Text PDF PubMed Google Scholar). Both FS-288 and FS-315 neutralize the diverse actions of activin by forming a complex with activin (21Kogawa K. Nakamura T. Sugino K. Takio K. Titani K. Sugino H. Endocrinology. 1991; 128: 1434-1440Crossref PubMed Scopus (210) Google Scholar,22de Winter J.P. ten Dijke P. de Vries C.J.M. van Achterberg T.A.E. Sugino H. de Waele P. Huylebroeck D. Vershueren K. van den Eijnden-van Raaij A.J.M. Mol. Cell. Endocrinol. 1996; 116: 105-114Crossref PubMed Scopus (173) Google Scholar), a member of the transforming growth factor-β 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 (20Sugino K. Kurosawa N. Nakamura T. Takio K. Shimasaki S. Ling N. Titani K. Sugino H. J. Biol. Chem. 1993; 268: 15579-15587Abstract Full Text PDF PubMed Google Scholar). 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 (23Hemmati-Brivanlou A. Kelly O.G. Melton D.A. Cell. 1994; 77: 283-295Abstract Full Text PDF PubMed Scopus (653) Google Scholar, 24Koga C. Tashiro K. Shiokawa K. Roux's Arch. Dev. Biol. 1995; 204: 172-179Crossref Scopus (7) Google Scholar). Since HS-PG is also expressed in the notochord (6Furuya S. Sera M. Tohno-oka R. Sugahara K. Shiokawa K. Hirabayashi Y. Dev. Growth Differ. 1995; 37: 337-346Crossref Scopus (10) Google Scholar), 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 (25Nakamura T. Sugino K. Titani K. Sugino H. J. Biol. Chem. 1991; 266: 19432-19437Abstract Full Text PDF PubMed Google Scholar), suggesting that FS has a high affinity for the cell surface HS-PG. In this study, the binding specificities of bFGF and FS toXenopus 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 (26Habuchi H. Suzuki S. Saito T. Tamura T. Harada T. Yoshida K. Kimata K. Biochem. J. 1992; 285: 805-813Crossref PubMed Scopus (163) Google Scholar, 27Turnbull J.E. Fernig D.G. Ke Y. Wilkinson M.C. Gallagher J.T. J. Biol. Chem. 1992; 267: 10337-10341Abstract Full Text PDF PubMed Google Scholar, 28Maccarana M. Casu B. Lindahl U. J. Biol. Chem. 1993; 268: 23898-23905Abstract Full Text PDF PubMed Google Scholar, 29Ishihara M. Shaklee P.N. Yang Z. Liang W. Wei Z. Stack R.J. Holme K. Glycobiology. 1994; 4: 451-458Crossref PubMed Scopus (91) Google Scholar), and the minimum pentasaccharide sequence required for bFGF binding (28Maccarana M. Casu B. Lindahl U. J. Biol. Chem. 1993; 268: 23898-23905Abstract Full Text PDF PubMed Google Scholar) has been elucidated, although the biologically functional domain is larger than the binding domain (30Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar). 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. Heparan sulfate (HS) from Xenopusembryos (stage 30) was prepared by exhaustive chondroitinase ABC digestion of the sulfated GAG fraction that was purified from the early tailbud embryos as reported (6Furuya S. Sera M. Tohno-oka R. Sugahara K. Shiokawa K. Hirabayashi Y. Dev. Growth Differ. 1995; 37: 337-346Crossref Scopus (10) Google Scholar). 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 (31Sugahara K. Okumura Y. Yamashina I. Biochem. Biophys. Res. Commun. 1989; 162: 189-197Crossref PubMed Scopus (77) Google Scholar), and further purified by chromatography on a DEAE-cellulose column through stepwise elution with 1.0 m LiCl (fraction 1) and 2.0m 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 (31Sugahara K. Okumura Y. Yamashina I. Biochem. Biophys. Res. Commun. 1989; 162: 189-197Crossref PubMed Scopus (77) Google Scholar). Human recombinant bFGF (rh-bFGF) was a gift from Dr. Koichi Igarashi (Discovery Research Loboratories II, Takeda Chemical Ind. Ltd., Tsukuba, Japan) (32Iwane M. Kurokawa T. Sasada R. Seno M. Nakagawa S. Igarashi K. Biochem. Biophys. Res. Commun. 1987; 146: 470-477Crossref PubMed Scopus (83) Google Scholar). 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). 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 (33Kinoshita, A., Sugahara, K., Abstracts of XIXth Japanese Carbohydrate Symposium, August 5–7, 1997, Nishinomiya, Japan, 1997, 106.Google Scholar), 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 0.25m 2-AB solution in glacial acetic acid/dimethyl sulfoxide (7/3, v/v) and 50 μl of a 1.0 m NaCNBH3solution 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 (34Sugahara K. Yamada S. Yoshida K. de Waard P. Vliegenthart J.F.G. J. Biol. Chem. 1992; 267: 1528-1533Abstract Full Text PDF PubMed Google Scholar), 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. N-[3H]Acetyl labeling of HS was carried out basically according to the procedure of Shaklee and Conrad (35Shaklee P.N. Conrad H.E. Biochem. J. 1986; 235: 225-236Crossref PubMed Scopus (32) Google Scholar). 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 0.25m NH4HCO3, 7% propanol. The polysaccharide fraction was desalted by repeated evaporation with water and dissolved in 200 μl of 10% methanol containing 0.05m 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 mNa2CO3. 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 mNa2CO3. The reaction mixture was applied to a column (1 × 46 cm) of Sephadex G-50, which was eluted with 0.25m NH4HCO3, 7% propanol. Labeled materials excluded from the gel were pooled, concentrated to dryness, and desalted by repeated evaporation with water. 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. 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 mmCa(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. Uronic acid was determined by the carbazole method (36Bitter M. Muir H. Anal. Biochem. 1962; 4: 330-334Crossref PubMed Scopus (5205) Google Scholar). Our previous results suggested that HS played an indispensable role in establishing the fundamental body plan during the earlyXenopus development (6Furuya S. Sera M. Tohno-oka R. Sugahara K. Shiokawa K. Hirabayashi Y. Dev. Growth Differ. 1995; 37: 337-346Crossref Scopus (10) Google Scholar). 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. 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 (33Kinoshita, A., Sugahara, K., Abstracts of XIXth Japanese Carbohydrate Symposium, August 5–7, 1997, Nishinomiya, Japan, 1997, 106.Google Scholar). 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 (37Lyon M. Deakin J.A. Gallagher J.T. J. Biol. Chem. 1994; 269: 11208-11215Abstract Full Text PDF PubMed Google Scholar). 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.Table IDisaccharide composition analysis of Xenopus embryo HSDisaccharideProportion%ΔHexAα1–4GlcNAc38.7ΔHexAα1–4GlcNAc(6S)6.0ΔHexAα1–4GlcN(NS)26.1ΔHexAα1–4GlcN(NS,6S)4.8ΔHexA(2S)α1–4GlcN(NS)17.9ΔHexA(2S)α1–4GlcN(NS,6S)6.5 Open table in a new tab 3H labeling of the HS preparation was conducted byN-deacetylation with hydrazine followed byN-reacetylation with [3H](CH3CO)2O. The3H-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 Xenopusembryo 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 (28Maccarana M. Casu B. Lindahl U. J. Biol. Chem. 1993; 268: 23898-23905Abstract Full Text PDF PubMed Google Scholar). 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-labeledXenopus 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.Table IIInhibition of the binding of Xenopus embryo HS to rh-bFGF by various glycosaminoglycansPolysaccharides[3H]HS binding at concentrations:0.4 μg/ml2.0 μg/ml%None100Xenopus embryo HS6117Bovine liver GAG fraction 12-2001Fractions 1 and 2 contained HS and chondroitin sulfate in a ratio of 1:1 and 7:3, respectively.177Bovine liver GAG fraction 22-2001Fractions 1 and 2 contained HS and chondroitin sulfate in a ratio of 1:1 and 7:3, respectively.03Bovine kidney HS5320EHS tumor mouse GAG fraction2-bThis GAG preparation was isolated from the Engelbreth-Holm-Swarm mouse tumor and was a mixture of HS and chondroitin sulfate in a ratio of 22:3.9274Bovine lung HS4816Bovine intestinal HS6820Porcine intestinal HP122Chondroitin sulfate A—2-2003—, not determined.94Chondroitin sulfate B—77Chondroitin sulfate C—100Chondroitin sulfate D—100Chondroitin sulfate E—82Chondroitin sulfate K—91Dextran sulfate (Mr = 5000)—02-2001 Fractions 1 and 2 contained HS and chondroitin sulfate in a ratio of 1:1 and 7:3, respectively.2-b This GAG preparation was isolated from the Engelbreth-Holm-Swarm mouse tumor and was a mixture of HS and chondroitin sulfate in a ratio of 22:3.2-2003 —, not determined. Open table in a new tab 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 (20Sugino K. Kurosawa N. Nakamura T. Takio K. Shimasaki S. Ling N. Titani K. Sugino H. J. Biol. Chem. 1993; 268: 15579-15587Abstract Full Text PDF PubMed Google Scholar). 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 (20Sugino K. Kurosawa N. Nakamura T. Takio K. Shimasaki S. Ling N. Titani K. Sugino H. J. Biol. Chem. 1993; 268: 15579-15587Abstract Full Text PDF PubMed Google Scholar), 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. 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-labeledXenopus 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" @default.
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• W2106796522 date "1998-03-01" @default.
• W2106796522 modified "2023-09-30" @default.
• W2106796522 title "Molecular Characterization of Xenopus Embryo Heparan Sulfate" @default.
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• W2106796522 doi "https://doi.org/10.1074/jbc.273.13.7375" @default.
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