Sulfation of the galactose residues in the glycosaminoglycan-protein linkage region by recombinant human chondroitin 6-O-sulfotransferase-1

6-O-Sulfated galactose residues have been demonstrated in the glycosaminoglycan-protein linkage region GlcUA beta 1-3Gal beta 1-3Gal-4Xyl beta 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 beta 1-3GalNAc(4-O-sulfate)beta 1-4GlcUA beta 1-3Gal(4-O-sulfate) beta 1-3Gal beta 1-4Xy1 beta 1-O-Ser and Delta GlcUA beta 1-3Gal-NAc(6-O-sulfate)beta 1-4GlcUA beta 1-3Gal beta 1-3Gal(6-O-sulfate)beta 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 beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 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 Delta GlcUA beta 1 3GalNAc(4-O-sulfate)beta 1-4GlcUA beta 1-3Gal(4-O-sulfate)beta 1-3Gal beta 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 glycosaminoglycan-protein linkage region.