α-Thrombomodulin (α-TM) with a truncated glycosaminoglycan-protein linkage tetrasaccharide, GlcAβ1-3Galβ1-3Galβ1-4Xyl, was tested as an acceptor together with a sugar donor, UDP-N-[3H]acetylgalactosamine, using a cell-free enzyme system prepared from the serum-free culture medium of a human melanoma cell line. The truncated tetrasaccharide on α-TM served as an acceptor, whereas the linkage tetrasaccharide-serine did not. Our characterization of the radioactively labelled product by enzymic digestion revealed that the N-[3H]acetylgalactosamine residue was transferred to α-TM through a β1,4-linkage. The substrate competition experiments with the chondro-hexasaccharide and α-TM reinforced our speculation that a common N-acetylgalactosaminyltransferase catalysed the transfer of N-acetylgalactosamine to both the linkage tetrasaccharide and the longer chondroitin oligosaccharides. Moreover, chondroitin polymerization was demonstrated on the tetrasaccharide of α-TM using both UDP-glucuronic acid and UDP-N-acetylgalactosamine as sugar donors. Much longer chains were synthesized on α-TM than on the linkage penta- and hexa-saccharide-serines. Together, these results indicated that the core protein is required for the transfer of the first N-acetylgalactosamine residue through a β1,4-linkage and also for subsequent efficient chain polymerization reactions, and that the critical determining step for chondroitin sulphate biosynthesis is the transfer of the first N-acetylgalactosamine residue.

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