2-O-Phosphorylation of Xylose and 6-O-Sulfation of Galactose in the Protein Linkage Region of Glycosaminoglycans Influence the Glucuronyltransferase-I Activity Involved in the Linkage Region Synthesis

Abstract

Sulfated glycosaminoglycans (GAGs), including heparan sulfate and chondroitin sulfate, are synthesized on the so-called common GAG-protein linkage region (GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser) of core proteins, which is formed by the stepwise addition of monosaccharide residues by the respective specific glycosyltransferases. Glucuronyltransferase-I (GlcAT-I) is the key enzyme that completes the synthesis of this linkage region, which is a prerequisite for the conversion of core proteins to functional proteoglycans bearing GAGs. The Xyl and Gal residues in the linkage region can be modified by phosphorylation and sulfation, respectively, although the biological significance of these modifications remains to be clarified. Here we present evidence that these modifications can significantly influence the catalytic activity of GlcAT-I. Enzyme assays showed that the synthetic substrates, Gal-Gal-Xyl

Identification of Phosphatase That Dephosphorylates Xylose in the Glycosaminoglycan-Protein Linkage Region of Proteoglycans

“…Findings from in vitro experiments with authentic substrates indicate that phosphorylation of the Xyl residue prevents the transfer of a Gal residue by GalT-I (9). In contrast, GlcAT-I efficiently transfers a GlcUA residue to the phosphorylated trisaccharide in vitro (10). These results sug-gest that phosphorylation of the Xyl residue takes place after transfer of the first Gal residue by GalT-I and before transfer of the GlcUA residue by GlcAT-I.…”

“…One such modification is the phosphorylation of the Xyl residue at position 2 (1,6). This modification occurs in both HS and CS derived from cell-rich tissues (7,8) and appears to affect the transfer of Gal and GlcUA residues by GalT-I and GlcAT-I, respectively (9,10). Findings from in vitro experiments with authentic substrates indicate that phosphorylation of the Xyl residue prevents the transfer of a Gal residue by GalT-I (9).…”

“…Site-directed Mutagenesis-A soluble form of GlcAT-I was produced as described previously (10). A two-stage PCR mutagenesis method was used to construct an expression plasmid that encoded a soluble form of the GlcAT-I mutant.…”

O-Glycosylation of glycine-serine linkers in recombinant Fc-fusion proteins

“…[21][22][23][24] For example, sulfates have been found to be attached at two of the galactose sugar units via different linkages. 25 Sulfated galactose was only found in CS, but not in HS, and may play a regulatory role in CS biosynthesis. 25 Phosphate has also been identified to be attached at xylose by a specific xylose kinase, FAB20B.…”

“…25 Sulfated galactose was only found in CS, but not in HS, and may play a regulatory role in CS biosynthesis. 25 Phosphate has also been identified to be attached at xylose by a specific xylose kinase, FAB20B. 26 In a recent report, Nadanaka et al hypothesized that the phosphorylation of xylose by FAM20B facilitates the formation of the linkage region and thereby leads to enhanced GAG biosynthesis.…”