6-O-Sulfated galactose residues have been demonstrated in the glycosaminoglycan-protein linkage region GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-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 GlcUA␤1-3GalNAc(4-O-sulfate)␤1-4GlcUA␤1-3Gal(4-Osulfate)␤1-3Gal␤1-4Xyl␤1-O-Ser and ⌬GlcUA␤1-3Gal-NAc(6-O-sulfate)␤1-4GlcUA␤1-3Gal␤1-3Gal(6-O-sulfate)␤1-4Xyl-ol. Identification of the reaction products obtained with the linkage tetra-, penta-, and hexasaccharideserines revealed that the C6ST-1 catalyzed the sulfation of C6 on both galactose residues in the linkage region. Notably, the linkage tetrasaccharide-peptide GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-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 ⌬GlcUA␤1-3GalNAc(4-O-sulfate)␤1-4GlcUA␤1-3Gal(4-O-sulfate)␤1-3Gal␤1-4Xyl-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 glycosaminoglycanprotein linkage region. Sulfated glycosaminoglycans (GAGs),3 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, GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-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. 1 and 2). Although such mechanisms have long been proposed based on data from conventional structural and enzymological studies (2, 3), 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 ch...

1 We investigated the ability of a newly synthesized sugar derivative, OJ-R9188, {N-(2-tetradecylhexadecanoyl)-O-(L-alpha-fucofuranosyl)-D-seryl}-L-glutamic acid 1-methylamide 5-L-arginine salt, to block binding of selectins to their ligands in vitro and inhibit the in®ltration of leukocytes in vivo. 2 OJ-R9188 prevented the binding of human E-, P-and L-selectin-IgG fusion proteins to immobilized sialyl Lewis x (sLe x )-pentasaccharide glycolipid, with IC 50 values of 4.3, 1.3, and 1.2 mM, respectively.3 In a mouse model of thioglycollate-induced peritonitis, OJ-R9188 at 10 mg kg 71 , i.v. inhibited neutrophil accumulation in the peritoneal cavity. In the IgE-mediated skin reaction, OJ-R9188 at 3 and 10 mg kg 71 , i.v. signi®cantly inhibited extravasation of neutrophils and eosinophils into the in¯ammatory sites and at 10 mg kg 71 , i.v. also inhibited in®ltration caused by picryl chlorideinduced delayed-type hypersensitivity in mice. These results suggest that OJ-R9188 may be a useful selectin blocker, with activity against human and mouse E-, P-and L-selectins in vitro and in vivo, and that blocking selectin-sLe x binding is a promising strategy for the treatment of allergic skin diseases.