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• W2047029365 abstract "6-O-Sulfated galactose residues have been demonstrated in the glycosaminoglycan-protein linkage region GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser isolated from shark cartilage chondroitin 6-sulfate (Sugahara, K., Ohi, Y., Harada, T., de Waard, P., and Vliegenthart, J. F. G. (1992) J. Biol. Chem. 267, 6027–6035). In this study, we investigated whether a recombinant human chondroitin 6-sulfotransferase-1 (C6ST-1) catalyzes the sulfation of C6 on both galactose residues in the linkage region using structurally defined acceptor substrates. The C6ST-1 was expressed as a soluble protein A chimeric form in COS-1 cells and purified using IgG-Sepharose. The purified C6ST-1 utilized the linkage tri-, tetra-, penta-, and hexasaccharide-serines and hexasaccharide alditols, including GlcUAβ1–3GalNAc(4-O-sulfate)β1–4GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xylβ1-O-Ser and ΔGlcUAβ1–3GalNAc(6-O-sulfate)β1–4GlcUAβ1–3Galβ1–3Gal(6-O-sulfate)β1–4Xyl-ol. Identification of the reaction products obtained with the linkage tetra-, penta-, and hexasaccharide-serines revealed that the C6ST-1 catalyzed the sulfation of C6 on both galactose residues in the linkage region. Notably, the linkage tetrasaccharide-peptide GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-(Gly)Ser-(Gly-Glu) was a good acceptor substrate for the C6ST-1, suggesting that the sulfation of the galactose residues can occur before the transfer of the first N-acetylhexosamine residue to the linkage tetrasaccharide. In contrast, no incorporation was observed into ΔGlcUAβ1–3GalNAc(4-O-sulfate)β1–4GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xyl-ol, indicating that an intact xylose is necessary for the transfer of a sulfate to the second sugar residue Gal from the reducing end. These findings clearly demonstrated that the recombinant C6ST-1 catalyzes the sulfation of C6 on both galactose residues in the linkage region in vitro. This is the first identification of the sulfotransferase responsible for the sulfation of galactose residues in the glycosaminoglycan-protein linkage region. 6-O-Sulfated galactose residues have been demonstrated in the glycosaminoglycan-protein linkage region GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser isolated from shark cartilage chondroitin 6-sulfate (Sugahara, K., Ohi, Y., Harada, T., de Waard, P., and Vliegenthart, J. F. G. (1992) J. Biol. Chem. 267, 6027–6035). In this study, we investigated whether a recombinant human chondroitin 6-sulfotransferase-1 (C6ST-1) catalyzes the sulfation of C6 on both galactose residues in the linkage region using structurally defined acceptor substrates. The C6ST-1 was expressed as a soluble protein A chimeric form in COS-1 cells and purified using IgG-Sepharose. The purified C6ST-1 utilized the linkage tri-, tetra-, penta-, and hexasaccharide-serines and hexasaccharide alditols, including GlcUAβ1–3GalNAc(4-O-sulfate)β1–4GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xylβ1-O-Ser and ΔGlcUAβ1–3GalNAc(6-O-sulfate)β1–4GlcUAβ1–3Galβ1–3Gal(6-O-sulfate)β1–4Xyl-ol. Identification of the reaction products obtained with the linkage tetra-, penta-, and hexasaccharide-serines revealed that the C6ST-1 catalyzed the sulfation of C6 on both galactose residues in the linkage region. Notably, the linkage tetrasaccharide-peptide GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-(Gly)Ser-(Gly-Glu) was a good acceptor substrate for the C6ST-1, suggesting that the sulfation of the galactose residues can occur before the transfer of the first N-acetylhexosamine residue to the linkage tetrasaccharide. In contrast, no incorporation was observed into ΔGlcUAβ1–3GalNAc(4-O-sulfate)β1–4GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xyl-ol, indicating that an intact xylose is necessary for the transfer of a sulfate to the second sugar residue Gal from the reducing end. These findings clearly demonstrated that the recombinant C6ST-1 catalyzes the sulfation of C6 on both galactose residues in the linkage region in vitro. This is the first identification of the sulfotransferase responsible for the sulfation of galactose residues in the glycosaminoglycan-protein linkage region. Sulfated glycosaminoglycans (GAGs), 3The abbreviations used are: GAG, glycosaminoglycan; C6ST, chondroitin 6-sulfotransferase; GlcUA, d-glucuronic acid; ΔHexUA, 4,5-unsaturated hexuronic acid or 4-deoxy-α-l-threo-hex-4-ene-pyranosyluronic acid; HPLC, high performance liquid chromatography; ΔDi-6S, Δ4,5HexUAα1–3GalNAc(6-O-sulfate); PAPS, 3′-phosphoadenosine 5′-phosphosulfate; Xyl-ol, xylitol. 3The abbreviations used are: GAG, glycosaminoglycan; C6ST, chondroitin 6-sulfotransferase; GlcUA, d-glucuronic acid; ΔHexUA, 4,5-unsaturated hexuronic acid or 4-deoxy-α-l-threo-hex-4-ene-pyranosyluronic acid; HPLC, high performance liquid chromatography; ΔDi-6S, Δ4,5HexUAα1–3GalNAc(6-O-sulfate); PAPS, 3′-phosphoadenosine 5′-phosphosulfate; Xyl-ol, xylitol. including heparin/heparan sulfate, chondroitin sulfate, and dermatan sulfate, are covalently bound to Ser residues in the core proteins through the common carbohydrate-protein linkage structure, GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser. Heparin/heparan sulfate is synthesized once GlcNAc is transferred to the common linkage region, whereas chondroitin sulfate is formed if GalNAc is first added. Therefore, the first hexosamine transfer is critical in determining whether heparin/heparan sulfate or chondroitin/dermatan sulfate chains are selectively assembled on the common linkage region (for reviews, see Refs. 1Sugahara K. Kitagawa H. Curr. Opin. Struct. Biol. 2000; 10: 518-527Crossref PubMed Scopus (339) Google Scholar and 2Uyama T. Kitagawa H. Sugahara K. Kamerling J.P. Comprehensive Glycoscience. Elsevier, London2007: 79-104Crossref Scopus (37) Google Scholar). Although such mechanisms have long been proposed based on data from conventional structural and enzymological studies (2Uyama T. Kitagawa H. Sugahara K. Kamerling J.P. Comprehensive Glycoscience. Elsevier, London2007: 79-104Crossref Scopus (37) Google Scholar, 3Sugahara K. Yamashina I. de Waard P. van Halbeek H. Vliegenthart J.F.G. J. Biol. Chem. 1988; 263: 10168-10174Abstract Full Text PDF PubMed Google Scholar), the molecular mechanisms underlying the selective chain assembly of different GAG chains have not yet been clarified. We have been investigating the structure of the linkage region of various GAGs to search for possible structural differences, which may determine the characteristics of the GAG species to be synthesized. These structural studies revealed that sulfation of C6 on both Gal residues and C4 of Gal adjacent to GlcUA was characteristic of chondroitin sulfate (3Sugahara K. Yamashina I. de Waard P. van Halbeek H. Vliegenthart J.F.G. J. Biol. Chem. 1988; 263: 10168-10174Abstract Full Text PDF PubMed Google Scholar, 7de Waard P. Vliegenthart J.F.G. Harada T. Sugahara K. J. Biol. Chem. 1992; 267: 6036-6043Abstract Full Text PDF PubMed Google Scholar). Sulfation of C4 of the Gal residue was also demonstrated in the linkage region of bovine aorta dermatan sulfate (8Sugahara K. Ohkita Y. Shibata Y. Yoshida K. Ikegami A. J. Biol. Chem. 1995; 270: 7204-7212Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Sulfated Gal residues have not been found to date in the linkage region of heparin or heparan sulfate (9Sugahara 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, 10Sugahara K. Tohno-oka R. Yamada S. Khoo K.-H. Morris H.R. Dell A. Glycobiology. 1994; 4: 535-544Crossref PubMed Scopus (59) Google Scholar). Thus, the possibility exists that the sulfated Gal structures contribute to the segregation of chondroitin/dermatan sulfate and heparin/heparan sulfate in relation to the sorting mechanisms in the biosynthesis of different GAGs. Although a considerable amount of information is available concerning the sulfation profile of the Gal residues in the linkage region, no sulfotransferase responsible for the sulfation of the Gal residues has yet been identified. cDNA encoding chondroitin 6-sulfotransferase-1 (C6ST-1), one of the sulfotransferases involved in the biosynthesis of GAGs, which catalyzes the transfer of sulfate from PAPS to position 6 of the GalNAc residue, has been cloned from chicken, mouse, and human (11Fukuta M. Uchimura K. Nakashima K. Kato M. Kimata K. Shinomura T. Habuchi O. J. Biol. Chem. 1995; 270: 18575-18580Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 14Tsutsumi K. Shimakawa H. Kitagawa H. Sugahara K. FEBS Lett. 1998; 441: 235-241Crossref PubMed Scopus (49) Google Scholar). It was reported that C6ST catalyzes the sulfation of position 6 of a Gal residue in keratan sulfate as well as of a GalNAc residue in chondroitin (11Fukuta M. Uchimura K. Nakashima K. Kato M. Kimata K. Shinomura T. Habuchi O. J. Biol. Chem. 1995; 270: 18575-18580Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 13Fukuta M. Kobayashi Y. Uchimura K. Kimata K. Habuchi O. Biochim. Biophys. Acta. 1998; 1399: 57-61Crossref PubMed Scopus (79) Google Scholar). These findings prompted us to investigate whether C6ST-1 catalyzes the sulfation of C6 on both Gal residues in the linkage region using structurally defined acceptor substrates. To carry out this analysis, we took advantage of the fact that the soluble form of the human C6ST-1 cDNA had successfully been expressed, and the purified recombinant enzyme was well characterized (14Tsutsumi K. Shimakawa H. Kitagawa H. Sugahara K. FEBS Lett. 1998; 441: 235-241Crossref PubMed Scopus (49) Google Scholar). In this study, we present evidence that the recombinant human C6ST-1 catalyzes the sulfation of C6 on both Gal residues in the linkage region in vitro. Materials—[35S]PAPS and unlabeled PAPS were purchased from PerkinElmer Life Sciences and Sigma, respectively. Five unsaturated standard disaccharides derived from chondroitin sulfate, Δ4,5HexUAα1–3GalNAc (ΔDi-0S), Δ4,5HexUAα1–3GalNAc(4-O-sulfate) (ΔDi-4S), Δ4,5HexUAα1–3GalNAc(6-O-sulfate) (ΔDi-6S), Δ4,5HexUAα1–3GalNAc(4,6-O-disulfate) (ΔDi-diSE), and Δ4,5HexUA (2-O-sulfate)α1–3GalNAc(6-O-sulfate) (ΔDi-diSD), and chondroitinase AC-II (EC 4.2.2.5), were purchased from Seikagaku Corp. (Tokyo, Japan). The following linkage tri-, tetra-, penta-, and hexasaccharide-serines and a tetrasaccharide-peptide were chemically synthesized (15Goto F. Ogawa T. Tetrahedron Lett. 1992; 33: 5099-5102Crossref Scopus (28) Google Scholar, 16Goto F. Ogawa T. Tetrahedron Lett. 1992; 33: 6841-6844Crossref Scopus (12) Google Scholar, 17Goto F. Ogawa T. Pure Appl. Chem. 1993; 65: 793-801Crossref Scopus (33) Google Scholar): Galβ1–3Galβ1–4Xylβ1-O-Ser, GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser, GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xylβ1-O-Ser, GalNAcβ1–4GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser, GlcNAcα1–4GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser, GlcUAβ1–3GalNAcβ1–4GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser, GlcUAβ1–3GalNAc(4-O-sulfate)β1–4GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xylβ1-O-Ser, and GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-(Gly)Ser-(Gly-Glu). The purity and concentration of these chemically synthesized substrates were checked by 1H NMR spectroscopy (15Goto F. Ogawa T. Tetrahedron Lett. 1992; 33: 5099-5102Crossref Scopus (28) Google Scholar, 17Goto F. Ogawa T. Pure Appl. Chem. 1993; 65: 793-801Crossref Scopus (33) Google Scholar) and high performance liquid chromatography (HPLC) on an amine-bound silica column as described (18Sugahara K. Okumura Y. Yamashina I. Biochem. Biophys. Res. Commun. 1989; 162: 189-197Crossref PubMed Scopus (77) Google Scholar), respectively. The following unsaturated linkage tetrasaccharide- and hexasaccharide-serines, and hexasaccharide alditols were isolated from porcine intestinal heparin (9Sugahara 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), rat chondrosarcoma chondroitin 4-sulfate (3Sugahara K. Yamashina I. de Waard P. van Halbeek H. Vliegenthart J.F.G. J. Biol. Chem. 1988; 263: 10168-10174Abstract Full Text PDF PubMed Google Scholar), whale cartilage chondroitin 4-sulfate (4Sugahara K. Masuda M. Harada T. Yamashina I. de Waard P. Vliegenthart J.F.G. Eur. J. Biochem. 1991; 202: 805-811Crossref PubMed Scopus (59) Google Scholar), and shark cartilage chondroitin 6-sulfate (6Sugahara K. Ohi Y. Harada T. de Waard P. Vliegenthart J.F.G. J. Biol. Chem. 1992; 267: 6027-6035Abstract Full Text PDF PubMed Google Scholar, 7de Waard P. Vliegenthart J.F.G. Harada T. Sugahara K. J. Biol. Chem. 1992; 267: 6036-6043Abstract Full Text PDF PubMed Google Scholar) as described: Δ4,5HexUAα1–3Galβ1–3Galβ1–4Xylβ1-O-Ser, Δ4,5HexUAα1–4GlcNAc(6-O-sulfate)α1–4GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser, Δ4,5HexUAα1–3GalNAc(4-O-sulfate)β1–4GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xylβ1-O-Ser, Δ4,5HexUAα1–3GalNAc(4-O-sulfate)β1–4GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser, Δ4,5HexUAα1–3GalNAc(4-O-sulfate)β1–4GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xyl-ol, Δ4,5HexUAα1–3GalNAc(6-O-sulfate)β1–4GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate)-ol, Δ4,5HexUAα1–3GalNAc(6-O-sulfate)β1–4GlcUAβ1–3Galβ1–3Gal(6-O-sulfate)β1–4Xyl-ol, and Δ4,5HexUAα1–3GalNAc(6-O-sulfate)β1–4GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser. They were structurally characterized enzymatically and also by 500-MHz 1H NMR spectroscopy (3Sugahara K. Yamashina I. de Waard P. van Halbeek H. Vliegenthart J.F.G. J. Biol. Chem. 1988; 263: 10168-10174Abstract Full Text PDF PubMed Google Scholar, 4Sugahara K. Masuda M. Harada T. Yamashina I. de Waard P. Vliegenthart J.F.G. Eur. J. Biochem. 1991; 202: 805-811Crossref PubMed Scopus (59) Google Scholar, 6Sugahara K. Ohi Y. Harada T. de Waard P. Vliegenthart J.F.G. J. Biol. Chem. 1992; 267: 6027-6035Abstract Full Text PDF PubMed Google Scholar, 7de Waard P. Vliegenthart J.F.G. Harada T. Sugahara K. J. Biol. Chem. 1992; 267: 6036-6043Abstract Full Text PDF PubMed Google Scholar, 9Sugahara 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). HiLoad™16/60 Superdex 30 (preparation grade) was obtained from Amersham Biosciences (Uppsala, Sweden). All other reagents and chemicals were of the highest quality. Expression of the Soluble Form of the Human C6ST and Enzyme Assay—Construction of the soluble form of the human C6ST-1 fused with the cleavable insulin signal sequence and the protein A IgG-binding domain was carried out as described (14Tsutsumi K. Shimakawa H. Kitagawa H. Sugahara K. FEBS Lett. 1998; 441: 235-241Crossref PubMed Scopus (49) Google Scholar). The expression plasmid (11 μg) was transfected into COS-1 cells on 100-mm plates using Lipofectamine (Invitrogen) according to the instructions provided by the manufacturer. Two days after transfection, 1 ml of the culture medium (∼0.5 μg of protein) was collected and incubated with 10 μl of IgG-Sepharose (Amersham Biosciences) for 1 h at 4 °C. The beads recovered by centrifugation were washed with and then resuspended in the assay buffer and tested for sulfotransferase activity using various linkage oligosaccharides (1 nmol each) as sulfate acceptor substrates and [35S]PAPS as a sulfate donor as described (14Tsutsumi K. Shimakawa H. Kitagawa H. Sugahara K. FEBS Lett. 1998; 441: 235-241Crossref PubMed Scopus (49) Google Scholar). Characterization of the Reaction Products—The reaction products were isolated by gel filtration on a column of Superdex 30 (1.6 × 60 cm) equilibrated with 0.25 m NH4HCO3, 7% 1-propanol. The radioactive peak containing the product was pooled and evaporated dry. The isolated products were digested for 1 h at 37 °C with 20 mIU of chondroitinase AC-II in a total volume of 30 μl of 50 mm sodium acetate buffer, pH 6.0. The digest was analyzed by anion exchange HPLC on an amine-bound silica PA03 column (YMC Co., Kyoto, Japan) as described (18Sugahara K. Okumura Y. Yamashina I. Biochem. Biophys. Res. Commun. 1989; 162: 189-197Crossref PubMed Scopus (77) Google Scholar). To identify the disaccharide and the linkage tetrasaccharide-serine or alditol, the chondroitinase AC-II digest was co-chromatographed with five unsaturated standard disaccharides and two structurally defined tetrasaccharide-serines isolated from shark and whale cartilage (4Sugahara K. Masuda M. Harada T. Yamashina I. de Waard P. Vliegenthart J.F.G. Eur. J. Biochem. 1991; 202: 805-811Crossref PubMed Scopus (59) Google Scholar, 5Sugahara K. Mizuno N. Okumura Y. Kawasaki T. Eur. J. Biochem. 1992; 204: 401-406Crossref PubMed Scopus (34) Google Scholar, 6Sugahara K. Ohi Y. Harada T. de Waard P. Vliegenthart J.F.G. J. Biol. Chem. 1992; 267: 6027-6035Abstract Full Text PDF PubMed Google Scholar, 7de Waard P. Vliegenthart J.F.G. Harada T. Sugahara K. J. Biol. Chem. 1992; 267: 6036-6043Abstract Full Text PDF PubMed Google Scholar): ΔHexUAα1–3Galβ1–3Gal(6-O-sulfate)β1–4Xylβ1-O-Ser and ΔHexUAα1–3Gal(6-O-sulfate)β1–3Gal(6-O-sulfate)β1–4Xylβ1-O-Ser. Sulfation of C6 on Both Gal Residues in the GAG-Protein Linkage Region—To facilitate the functional analysis of the human C6ST-1, a soluble form of the protein was generated by replacing the first 48 amino acids of the C6ST-1 with a cleavable insulin signal sequence and a protein A IgG-binding domain as described (14Tsutsumi K. Shimakawa H. Kitagawa H. Sugahara K. FEBS Lett. 1998; 441: 235-241Crossref PubMed Scopus (49) Google Scholar), and then the soluble C6ST-1 was expressed in COS-1 cells as a recombinant enzyme fused with the protein A IgG-binding domain. The fused enzyme expressed in the medium was absorbed on IgG-Sepharose beads to eliminate endogenous C6ST-1, and then the enzyme-bound beads corresponding to ∼0.5 μg of protein were used as an enzyme source. The bound fusion protein was assayed for sulfotransferase activity using the nonsulfated linkage penta- and hexasaccharide-serines as sulfate acceptors and [35S]PAPS as a sulfate donor. The reaction products were isolated by gel filtration on a column of Superdex 30, digested by chondroitinase AC-II, and then analyzed by anion exchange HPLC as shown in Fig. 1, A and B. The digests of the products obtained with the pentasaccharide-serine (GalNAc-GlcUA-Gal-Gal-Xyl-Ser) as an acceptor substrate showed three radioactive peaks at the elution positions of free GalNAc(6-O-sulfate), ΔHexUA-Gal-Gal(6-O-sulfate)-Xyl-Ser, and ΔHexUA-Gal(6-O-sulfate)-Gal(6-O-sulfate)-Xyl-Ser in a ratio of 10:4:1, respectively (Fig. 1A). In addition, the digests of the products obtained with the hexasaccharide-serine (GlcUA-GalNAc-GlcUA-Gal-Gal-Xyl-Ser) as an acceptor substrate showed three radioactive peaks at the elution positions of ΔHexUA-Gal-Gal(6-O-sulfate)-Xyl-Ser, GlcUA-GalNAc(6-O-sulfate), and ΔHexUA-Gal(6-O-sulfate)-Gal(6-O-sulfate)-Xyl-Ser in a ratio of 5:2:1, respectively (Fig. 1B). These results indicate that the recombinant C6ST-1 can catalyze the transfer of a sulfate group from PAPS to both Gal residues in the GAG-protein linkage region of chondroitin sulfate proteoglycans in vitro. It should be noted that no authentic ΔHexUA-Gal(6-O-sulfate)-Gal-Xyl-Ser is available, and therefore it could not be discriminated from ΔHexUA-Gal-Gal(6-O-sulfate)-Xyl-Ser by the present method. Substrate Specificity of the Recombinant C6ST-1 toward a Variety of Structurally Defined Linkage Oligosaccharides—To further characterize the substrate specificity of the recombinant C6ST-1, a variety of structurally defined linkage oligosaccharides (Table 1) were tested as acceptor substrates. They consisted of tri-, tetra-, penta-, and hexasaccharide-serines and a tetrasaccharide-peptide, all of which were chemically synthesized, as well as various tetra- and hexasaccharides in the forms of alditols and glycoserines, which were isolated from chondroitin sulfate and heparin. These results obtained at a fixed substrate concentration of 20 μm are summarized in Table 1.TABLE 1Sulfotransferase activities of the recombinant C6ST-1 toward oligosaccharides isolated from the GAG-protein linkage region of chondroitin sulfate and heparin and the putative reaction productsAcceptor (putative reaction product)Relative rate (%)Gal-Ga-Xyl-Ser4(Gal(6S)-Gal(6S)Xyl-Ser)GlcUA-Gal-Ga-Xyl-Ser8(GlcUA-Gal(6S-Gal(6S)Xyl-Ser)GlcUA-Gal(4S)-Ga-Xyl-Ser20(GlcUA-Gal(4S-Gal(6S)Xyl-Ser)GlcUA-Gal-GalXyl-(Gly)Ser-Gly-Glu)100(GlcUA-Gal(6S)Gal(6S)-Xyl-(Gly)Ser-(ly-Glu))ΔHexUA-Gal-Ga-Xyl-Ser24(ΔHexUA-Ga(6S)-Gal(6S)Xyl-Ser)GalNAc-GlUA-Gal-Ga-Xyl-Ser84(GalNAc(6S-GlcUA-Gal6S)-Gal(6S)Xyl-Ser)GlcNAc-GlUA-Gal-Ga-Xyl-Ser2(GlcNAcGlcUA-Gal6S)-Gal(6S)Xyl-Ser)GlcUA-GalNAc-GlUA-Gal-Ga-Xyl-Ser130(GlcUA-GalNA(6S)-GlcUA-Gal6S)-Gal(6S)Xyl-Ser)GlcUA-GaNAc(4S)-GlcU-Gal(4S)-Ga-Xyl-Ser250(GlcUA-GalNA(4S)-GlcUA-Gal4S)-Gal(6S)Xyl-Ser)ΔHexUA-GaNAc(4S)-GlcUAGal(4S)-Ga-Xyl-Ser270(ΔHexUA-GalNAc4S)-GlcUA-Gal4S)-Gal(6S)Xyl-Ser)ΔHexUA-GlNAc(4S)-GlcU-Gal(4S)-Gl-Xyl-ol0ΔHexUAGalNAc(4S)-GlUA-Gal-Ga-Xyl-Ser260(ΔHexUA-GalNAc4S)-GlcUA-Gal6S)-Gal(6S)Xyl-Ser)ΔHexUA-GlNAc(6S)-GlcU-Gal-Gal-Xl(2P)-ol290(ΔHexUA-GalNA(6S)-GlcUA-Ga(6S)-Gal-Xy(2P)-ol)ΔHexUA-GlNAc(6S)-GlcU-Gal-Gal(6)-Xyl-ol360(ΔHexUA-GalNc(6S)-GlcUA-Ga(6S)-Gal(6S-Xyl-ol)ΔHexUAGlcNAc(6S)-GlUA-Gal-GalXyl-Ser17(ΔHexUA-GalNAc6S)-GlcUA-Gal(S)-Gal(6S)Xyl-Ser) Open table in a new tab The purified human C6ST-1 utilized the linkage tri- and tetrasaccharide-serines (Gal-Gal-Xyl-Ser and GlcUA-Gal-Gal-Xyl-Ser) as well as penta- and hexasaccharide-serines (Gal-NAc-GlcUA-Gal-Gal-Xyl-Ser and GlcUA-GalNAc-GlcUA-Gal-Gal-Xyl-Ser). To confirm that the C6ST-1 catalyzed the sulfation of C6 on both Gal residues in the tetrasaccharide-serine, the reaction products obtained with ΔHexUA-Gal-Gal-Xyl-Ser as an acceptor were analyzed by HPLC, as shown in Fig. 1C. The products showed two radioactive peaks at the elution positions of ΔHexUA-Gal-Gal(6-O-sulfate)-Xyl-Ser and ΔHexUA-Gal(6-O-sulfate)-Gal(6-O-sulfate)-Xyl-Ser in a ratio of 4:1, respectively. These results suggest that the sulfation of the Gal residues can occur before the transfer of the first N-acetylhexosamine residue to the linkage tetrasaccharide. In addition, the tetrasaccharide-peptide showed stronger acceptor activity than the corresponding tetrasaccharide-serine, indicating that the acidic peptide had a positive influence. Notably, GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xylβ1-O-Ser and GlcUAβ1–3GalNAc(4-O-sulfate)β1–4GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xylβ1-O-Ser were also utilized as acceptor substrates, implying that the C6ST-1 can form the disulfated linkage structure, GlcUAβ1–3Gal(4-O-sulfate)β1–3Gal(6-O-sulfate)β1–4Xyl, previously found in shark cartilage chondroitin sulfate proteoglycans (7de Waard P. Vliegenthart J.F.G. Harada T. Sugahara K. J. Biol. Chem. 1992; 267: 6036-6043Abstract Full Text PDF PubMed Google Scholar). Besides, ΔHexUAα1–3GalNAc(6-O-sulfate)β1–4GlcUAβ1–3Galβ1-3Gal(6-O-sulfate)β1–4Xyl-ol was also utilized as an acceptor substrate, whereas no incorporation of sulfate was observed into ΔHexUAα1–3GalNAc(4-O-sulfate)β1–4GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xyl-ol. These results suggest that the intact xylose residue is necessary for the transfer of a sulfate group to the second sugar residue Gal from the reducing end. Hence, when a phosphorylated hexasaccharide alditol ΔHexUAα1–3GalNAc(6-O-sulfate)β1–4GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate)-ol was used as an acceptor, it acted as an acceptor (Table 1), and the Gal residue substituted by a GlcUA residue was most likely sulfated. Although linkage region structures that contain both 6-O-sulfated Gal and 2-O-phosphorylated Xyl residues have never been demonstrated in natural chondroitin sulfate chains, the results indicate that the recombinant C6ST-1 can transfer a sulfate at least to the Gal residue substituted by a GlcUA residue in the linkage region with the 2-O-phosphorylated Xyl residue. In addition, as shown in Table 1, sulfated compounds, GlcUA-Gal(4S)-Gal-Xyl-Ser and GlcUA-GalNAc(4S)-GlcUA-Gal(4S)-Gal-Xyl-Ser, were better substrates than the corresponding nonsulfated compounds, GlcUA-Gal-Gal-Xyl-Ser and GlcUA-GalNAc-GlcUA-Gal-Gal-Xyl-Ser, respectively. Thus, the prior sulfation of the acceptor substrates enhances the acceptor activity. The GlcNAc-containing pentasaccharide-serine GlcNAcα1–4GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser showed a little activity, indicating that sulfation of the Gal residue(s) in the linkage region could take place at least in vitro even after the synthesis of the repeating disaccharide region was initiated toward heparan sulfate biosynthesis. Low yet significant 35S incorporation was also observed when ΔHexUAα1–4GlcNAc(6-O-sulfate)α1–4GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser derived from the linkage region of porcine intestinal heparin was used as an acceptor. Although sulfated Gal residues have not been demonstrated in the linkage region isolated from heparin or heparan sulfate, these results indicate that the recombinant C6ST-1 is capable of transferring sulfate at least in vitro to the linkage region of heparin or heparan sulfate that already contains the first GlcNAc residue despite the low efficiency. Effects of Acceptor Concentrations on the Recombinant C6ST-1 Activity—To investigate the effect of the concentration of an acceptor substrate, the C6ST-1 activity was determined using various concentrations of a synthetic disulfated hexasaccharide-serine, GlcUAβ1–3GalNAc(4-O-sulfate)β1–4GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xylβ1-O-Ser, which can accept only one sulfate group by the C6ST-1. As shown in Fig. 2, the apparent Km value for the hexasaccharide-serine was 50 μm. In this paper, we clearly demonstrated that the recombinant human C6ST-1 catalyzes the sulfation of C6 on both Gal residues in the linkage region in vitro. This is the first identification of the sulfotransferase responsible for the sulfation of Gal residues in the GAG-protein linkage region. To date, three distinct sulfation patterns of 6-O-sulfated Gal residue(s) in the linkage region, GlcUAβ1–3Galβ1–3Gal(6-O-sulfate)β1–4Xyl, GlcUAβ1–3Gal(6-O-sulfate)β1–3Gal(6-O-sulfate)β1–4Xyl, and GlcUAβ1–3Gal(4-O-sulfate)β1–3Gal(6-O-sulfate)β1–4Xyl, have been found in chondroitin sulfates from various sources, including whale cartilage (4Sugahara K. Masuda M. Harada T. Yamashina I. de Waard P. Vliegenthart J.F.G. Eur. J. Biochem. 1991; 202: 805-811Crossref PubMed Scopus (59) Google Scholar), shark cartilage (6Sugahara K. Ohi Y. Harada T. de Waard P. Vliegenthart J.F.G. J. Biol. Chem. 1992; 267: 6027-6035Abstract Full Text PDF PubMed Google Scholar, 7de Waard P. Vliegenthart J.F.G. Harada T. Sugahara K. J. Biol. Chem. 1992; 267: 6036-6043Abstract Full Text PDF PubMed Google Scholar), and rat chondrosarcoma (3Sugahara K. Yamashina I. de Waard P. van Halbeek H. Vliegenthart J.F.G. J. Biol. Chem. 1988; 263: 10168-10174Abstract Full Text PDF PubMed Google Scholar). The present findings indicate that the recombinant human C6ST-1 can synthesize all of these three structures. In addition, since the Km value of C6ST-1 for the disulfated hexasaccharide-serine, GlcUAβ1–3GalNAc(4-O-sulfate)β1–4GlcUAβ1–3Gal(4-O-sulfate)β1–3Galβ1–4Xylβ1-O-Ser, was low (50 μm), as revealed in this study, it is most likely that C6ST-1 catalyzes the sulfation of C6 on Gal residues in the linkage region in vivo as well. Moreover, ΔHexUA-GalNAc(4S)-GlcUA-Gal-Gal-Xyl-Ser, which is a good acceptor substrate for the C6ST-1, did not serve as acceptor at all for the other chondroitin 6-O-sulfotransferase, C6ST-2 (19Kitagawa H. Fujita M. Ito N. Sugahara K. J. Biol. Chem. 2000; 275: 21075-21080Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) (data not shown). Although we cannot rule out the existence of other sulfotransferase(s) responsible for the sulfation of Gal residues in the GAG-protein linkage region, C6ST-1 seems to be mainly responsible for the sulfation of Gal residues, since mouse and human C6ST-1 genes are widely expressed in many tissues (12Uchimura K. Kadomatsu K. Fan Q.-W. Muramatsu H. Kurosawa N. Kaname T. Yamamura K. Fukuta M. Habuchi O. Muramatsu T. Glycobiology. 1998; 8: 489-496Crossref PubMed Scopus (43) Google Scholar, 13Fukuta M. Kobayashi Y. Uchimura K. Kimata K. Habuchi O. Biochim. Biophys. Acta. 1998; 1399: 57-61Crossref PubMed Scopus (79) Google Scholar). Although the biological significance of the sulfation of the linkage region is as yet unknown, the sulfated residues may play important roles in the biosynthesis of GAGs (e.g. by contributing to the segregation of galactosaminoglycans (chondroitin sulfate and dermatan sulfate) and glucosaminoglycans (heparin and heparan sulfate) or as a marker for the intracellular transport of chondroitin sulfate chains to the Golgi compartment for biosynthetic processing or elongation and maturation of the repeating disaccharide units of chondroitin sulfate chains, since sulfated Gal residues have not been demonstrated in the linkage region isolated from heparin or heparan sulfate). On the basis of the present finding that the recombinant human C6ST-1 utilized the linkage tri- and tetrasaccharide-serines, it is possible that the sulfation of the Gal residues occurs before the transfer of the first N-acetylhexosamine residue to the linkage tetrasaccharide. In addition, the tetrasaccharide-peptide showed greater acceptor activity than the corresponding tetrasaccharide-serine, indicating that the peptide portion is recognized by C6ST-1 with a positive influence and that the Gal residues are sulfated much more efficiently when the linkage region is bound to the core protein as a naturally occurring nascent proteoglycan. Moreover, it should be noted that C6ST-1 activity was found in medial and trans-Golgi fractions and that glucuronyltransferase I involved in the biosynthesis of the GAG-protein linkage region had a dual Golgi distribution similar to that of chondroitin-polymerizing glucuronyltransferase II in both medial and trans-Golgi/trans-Golgi networks and distinct from the distribution of galactosyltransferases involved in the biosynthesis of the GAG-protein linkage region, which were found exclusively in cis-Golgi fractions (20Sugumaran G. Katsman M. Silbert J.E. Biochem. J. 1998; 329: 203-208Crossref PubMed Scopus (16) Google Scholar). It is possible, therefore, that the sulfation of the linkage region might occur before the transfer of the first N-acetylhexosamine residue to the linkage tetrasaccharide and could be a signal for differential assembly of chondroitin versus heparan chains on nascent proteoglycans as previously proposed (3Sugahara K. Yamashina I. de Waard P. van Halbeek H. Vliegenthart J.F.G. J. Biol. Chem. 1988; 263: 10168-10174Abstract Full Text PDF PubMed Google Scholar, 7de Waard P. Vliegenthart J.F.G. Harada T. Sugahara K. J. Biol. Chem. 1992; 267: 6036-6043Abstract Full Text PDF PubMed Google Scholar). In addition, we recently demonstrated that sulfation of the Gal residues can significantly influence the catalytic activity of glucuronyltransferase I (21Tone Y. Pedersen L.C. Yamamoto T. Izumikawa T. Kitagawa H. Nishihara J. Tamura J. Negishi M. Sugahara K. J. Biol. Chem. 2008; 283: 16801-16807Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Enzyme assays showed that the synthetic compounds, Galβ1–3Galβ1–4Xyl(2-O-phosphate)β1-O-Ser, Galβ1–3Gal(6-O-sulfate)β1–4Xylβ1-O-Ser, and Galβ1–3Gal(6-O-sulfate)β1–4Xyl(2-O-phosphate)β1-O-Ser, served as better substrates for the truncated form of the recombinant human glucuronyltransferase I than the unmodified trisaccharide serine, suggesting that both the 6-O-sulfate group on Gal(I) and the 2-O-phosphate group on Xyl enhance the acceptor activity. The results indicate the possible involvement of these modifications in the processing and maturation of the growing linkage region oligosaccharide required for the assembly of GAG chains. The 2-O-phosphorylation of the Xyl residue has been reported to influence the synthesis of the linkage region by promoting the transfer of GlcUA to the Gal residue (22Moses J. Oldberg A. Fransson L.A. Eur. J. Biochem. 1999; 260: 879-884Crossref PubMed Scopus (28) Google Scholar). Considering that various penta- and hexasaccharide-serines were efficiently utilized as acceptor substrates for C6ST-1, sulfation of the Gal residue(s) in the linkage region can occur even after the nascent proteoglycan has already been directed to the specific site for GAG polymerization and sulfation. In addition, the present study demonstrated that sulfated compounds, GlcUA-Gal(4S)-Gal-Xyl-Ser and GlcUA-GalNAc(4S)-GlcUA-Gal(4S)-Gal-Xyl-Ser, were better substrates than the corresponding nonsulfated compounds, GlcUA-Gal-Gal-Xyl-Ser and GlcUA-GalNAc-GlcUA-Gal-Gal-Xyl-Ser, respectively (Table 1). Thus, the prior sulfation of the acceptor substrates has significant influence upon further sulfation of the linkage region. Moreover, the sulfated linkage region may also contain signals that influence the subsequent polymerization and sulfation of the repeating disaccharide region. In fact, sulfation in the vicinity of the linkage region is reported to have a powerful influence upon chain elongation reactions (23Kitagawa H. Tsutsumi K. Ujikawa M. Goto F. Tamura J. Neumann K.W. Ogawa T. Sugahara K. Glycobiology. 1997; 7: 531-537Crossref PubMed Scopus (54) Google Scholar). Surprisingly, the GlcNAc-containing penta- and hexasaccharide-serines, GlcNAcα1–4GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser and ΔHexUAα1–4GlcNAc(6-O-sulfate)α1–4GlcUAβ1–3Galβ1–3Galβ1–4Xylβ1-O-Ser, served as acceptors for C6ST-1 despite the low efficiency, implying that sulfation of the Gal residue(s) in the linkage region could take place at least in vitro even after the synthesis of the repeating disaccharide region for initiation of heparan sulfate biosynthesis. In view of the fact that sulfated Gal residues have not been demonstrated for the linkage region isolated from heparin or heparan sulfate, it is possible that C6ST-1 interacts with the core protein and that the core protein portion of the nascent heparin/heparan sulfate proteoglycan has inhibitory effects on C6ST-1 activity. In fact, since the tetrasaccharide-peptide showed greater acceptor activity than the corresponding tetrasaccharide-serine, as described above, C6ST-1 obviously recognizes the peptide portion. In the future, if various tetrasaccharide-peptides with the specific amino acid sequence found only in heparin/heparan sulfate proteoglycans become available, it is of importance to determine how C6ST-1 interacts with the specific residues in the core protein. Chondroitin sulfate has recently been implicated in various intriguing biological phenomena, such as the regulation of growth factor functions (24Deepa S.S. Umehara Y. Higashiyama S. Itoh N. Sugahara K. J. Biol. Chem. 2002; 277: 43707-43716Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), cell division (25Sugahara K. Mikami T. Uyama U. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (572) Google Scholar), neuritogenesis (25Sugahara K. Mikami T. Uyama U. Mizuguchi S. Nomura K. Kitagawa H. Curr. Opin. Struct. Biol. 2003; 13: 612-620Crossref PubMed Scopus (572) Google Scholar, 26Li F. Shetty A.K. Sugahara K. J. Biol. Chem.,. 2007; 282: 2956-2966Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), central nervous system development (27Sugahara K. Mikami T. Curr. Opin. Struct. Biol. 2007; 17: 536-545Crossref PubMed Scopus (217) Google Scholar), and maintenance of neural stem cells (28Akita K. von Holst A. Furukawa Y. Mikami T. Sugahara K. Faissner A. Stem Cells. 2007; 26: 798-809Crossref PubMed Scopus (90) Google Scholar). In addition, a missense mutation (R304Q or L286P) of C6ST-1 has been demonstrated to abolish C6ST activity almost completely and cause severe human chondrodysplasia with major involvement of the spine (29Thiele H. Sakano M. Kitagawa H. Sugahara K. Rajab A. Höhne W. Leschik G. Nürnberg P. Mundlos S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10155-10160Crossref PubMed Scopus (143) Google Scholar, 30van Roij M.H.H. Mizumoto S. Yamada S. Morgan T. Tan-Sindhunata M.B. Meijers-Heijboer H. Verbeke J.I.L.M. Markie D. Sugahara K. Robertson S.P. Am. J. Med. Genet. 2008; 146: 2376-2384Crossref Scopus (44) Google Scholar). To further address the biological significance of the sulfation of the linkage region, it is necessary to examine in detail the structure of the GAGs in the affected tissues of the patients and how the type and/or the sulfation profiles of the GAGs to be synthesized are influenced by C6ST-1 expression using C6ST-1 cDNA-transfected cells, which originally synthesize chondroitin 4-sulfate as a major component, such as Chinese hamster ovary cells or rat chondrosarcoma cells. We thank Naomi Ikebe for technical assistance." @default.
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• W2047029365 title "Sulfation of the Galactose Residues in the Glycosaminoglycan-Protein Linkage Region by Recombinant Human Chondroitin 6-O-Sulfotransferase-1" @default.
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