Sulfation of the Galactose Residues in the Glycosaminoglycan-Protein Linkage Region by Recombinant Human Chondroitin 6-O-Sulfotransferase-1

Abstract

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...

Human Genetic Disorders and Knockout Mice Deficient in Glycosaminoglycan

“…GAG-Xyl kinase, encoded by FAM20B , Xyl phosphatase, encoded by ACPL2 , and Gal-6- O -sulfotransferase, encoded by CHST3 (C6ST1), have so far been identified (Table 2) [33–35]. These modifications affect the glycosyltransferase reactions of GalT-I and GlcAT-I in vitro and may regulate the formation of GAG chains [36, 37].…”

Incomplete Elongation of the Chondroitin Sulfate Linkage Region on Aggrecan and Response to Interleukin-1β

“…Many enzymes involved in linkage structure formation, sulfation and/or phosphorylation, and chain polymerization of these structures have been carefully characterized, but we have yet to understand even the most rudimentary regulatory mechanism involved in this process [11,23,48,49]. Empirical evidence has shown that CS chain length, as well as the location and degree of modifications, can vary along the core protein of aggrecan [50,51].…”

“…The CS linkage region has been shown to have post-ribosomal modifications that include sulfation on the four and/or six carbons of each of the two galactosyl residues and phosphorylation on the 2-O position of the xylosyl residue [21][22][23][24][25]. On decorin, the xylose-2-phosphate has been shown to be a transient modification that may help provide a high degree of substrate specificity for the GlcAT I, which appears to engage the nascent trisaccharide linkage region and not the final sugar moiety (e.g., galactose 2) alone [26].…”

The Enzymatic Degradation of Heparan Sulfate

“…The conserved tetrasaccharide in the GAG-protein linkage can be modified by phosphorylation of the Xyl (by FAM20B) [11] and dephosphorylation of the Xyl (by 2-phosphoxylose phosphatase (XYLP)) [12]. The tetrasaccharide of CS or DS can also be modified by 6-O sulfation of Gal (by chondroitin 6-sulfotransferase I (C6ST-I)) [13] and 4-O sulfation of Gal [14]. These modifications to the tetrasaccharide in the GAGprotein linkage are often important for the regulation of the type and length of GAG chain synthesised.…”