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 GlcUAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-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 GlcUAbeta1-3GalNAc(4-O-sulfate)beta1-4GlcUAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xylbeta1-O-Ser and DeltaGlcUAbeta1-3GalNAc(6-O-sulfate)beta1-4GlcUAbeta1-3Galbeta1-3Gal(6-O-sulfate)beta1-4Xyl-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 GlcUAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-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 DeltaGlcUAbeta1-3GalNAc(4-O-sulfate)beta1-4GlcUAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xyl-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.
Kitagawa
Hiroshi
H
Department of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan. Electronic address: kitagawa@kobepharma-u.ac.jp.
Tsutsumi
Kae
K
Department of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan.
Ikegami-Kuzuhara
Akemi
A
Department of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan.
Nadanaka
Satomi
S
Department of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan.
Goto
Fumitaka
F
RIKEN (Institute of Physical and Chemical Research), Wako-shi, Saitama, 351-01, Japan.
Ogawa
Tomoya
T
RIKEN (Institute of Physical and Chemical Research), Wako-shi, Saitama, 351-01, Japan.
Sugahara
Kazuyuki
K
Department of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan; Laboratory of Proteoglycan Signaling and Therapeutics, Graduate School of Life Science, Hokkaido University, Frontier Research Center for Post-Genomic Science and Technology, Sapporo, Hokkaido 001-0021, Japan.
D000818:Animals; D019556:COS Cells; D002236:Carbohydrate Conformation; D002384:Catalysis; D002522:Chlorocebus aethiops; D005690:Galactose / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009844:Oligosaccharides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000097811:Carbohydrate Sulfotransferases
D005690:Galactose; D006025:Glycosaminoglycans; D009844:Oligosaccharides; D011994:Recombinant Proteins; D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases