We determined whether the two major structural modifications, i.e. phosphorylation and sulfation of the glycosaminoglycan-protein linkage region (GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1), govern the specificity of the glycosyltransferases responsible for the biosynthesis of the tetrasaccharide primer. We analyzed the influence of C-2 phosphorylation of Xyl residue on human ␤1,4-galactosyltransferase 7 (GalT-I), which catalyzes the transfer of Gal onto Xyl, and we evaluated the consequences of C-4/C-6 sulfation of Gal␤1-3Gal (Gal2-Gal1) on the activity and specificity of ␤1,3-glucuronosyltransferase I (GlcAT-I) responsible for the completion of the glycosaminoglycan primer sequence. For this purpose, a series of phosphorylated xylosides and sulfated C-4 and C-6 analogs of Gal␤1-3Gal was synthesized and tested as potential substrates for the recombinant enzymes. Our results revealed that the phosphorylation of Xyl on the C-2 position prevents GalT-I activity, suggesting that this modification may occur once Gal is attached to the Xyl residue of the nascent oligosaccharide linkage. On the other hand, we showed that sulfation on C-6 position of Gal1 of the Gal␤1-3Gal analog markedly enhanced GlcAT-I catalytic efficiency and we demonstrated the importance of Trp 243 and Lys 317 residues of Gal1 binding site for enzyme activity. In contrast, we found that GlcAT-I was unable to use digalactosides as acceptor substrates when Gal1 was sulfated on C-4 position or when Gal2 was sulfated on both C-4 and C-6 positions. Altogether, we demonstrated that oligosaccharide modifications of the linkage region control the specificity of the glycosyltransferases, a process that may regulate maturation and processing of glycosaminoglycan chains.

Proteoglycans (PGs) are macromolecules composed of glycosaminoglycan (GAG) side chains covalently bound to a core protein. There is currently great interest in the elucidation of the biosynthetic pathways of PGs as they are increasingly implicated as important regulators of many fundamental biological processes[1]. These include regulation of cell proliferation and recognition, extracellular matrix deposition, and morphogenesis, which are the consequences of the characteristic GAG moieties interacting with a variety of protein ligands such as growth and/or differentiation factors, cytokines, and morphogens[2]. GAGs include chondroitin/dermatan sulfate (CS/DS) and heparan sulfate/heparin (HS/Hep), which are classified as galactosaminoglycans and glucosaminoglycans, respectively[3]. Major components of these linear GAGs consist of hexosamine (GalNAc, GlcNAc) and hexuronic acid (GlcA or IdoA), which are arranged in alternating sequences to form the so-called repeating disaccharide region. These repeating units contain a number of sulfate substitutents that contribute to the structural and functional diversity of GAGs[4]. Both types of GAGs are covalently bound to serine residues in the core proteins through the common GAG-protein linkage structure GlcAß1,3Galß1,3Galß1,4Xylß1-O-Ser (GlcA-Gal2-Gal1-Xyl-O-Ser)[5,6]. The linkage region tetrasaccharide is formed by the sequential stepwise addition of each sugar residue by respective O-xylosyltransferase I[7], ß1,4-galactosyltransferase 7 (GalT-I)[8], ß1,3galactosyltransferase 6 (GalT-II)[9], and ß1,3-glucuronosyltransferase I (GlcAT-I)[10]. The transfer of