Involvement of the core protein in the first beta-N-acetylgalactosamine transfer to the glycosaminoglycan-protein linkage-region tetrasaccharide and in the subsequent polymerization: the critical determining step for chondroitin sulphate biosynthesis

• Published in: Biochemical journal 1999-06, Vol.340 ( Pt 2) (2), p.353-357
• Main Authors: Nadanaka, S, Kitagawa, H, Goto, F, Tamura, J, Neumann, K W, Ogawa, T, Sugahara, K
• Format: Article
• Language: English
• Subjects: Acetylgalactosamine - metabolism; Biopolymers; Carbohydrate Sequence; Cell-Free System; Chondroitin Sulfates - biosynthesis; Glycosaminoglycans - chemistry; Glycosaminoglycans - metabolism; Humans; Molecular Sequence Data; N-Acetylgalactosaminyltransferases - metabolism; Substrate Specificity; Thrombomodulin - chemistry; Thrombomodulin - metabolism; Tumor Cells, Cultured
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/0264-6021:3400353

alpha-Thrombomodulin (alpha-TM) with a truncated glycosaminoglycan-protein linkage tetrasaccharide, GlcAbeta1-3Galbeta1-3Galbeta1-4Xyl, 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 alpha-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 alpha-TM through a beta1,4-linkage. The substrate competition experiments with the chondro-hexasaccharide and alpha-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 alpha-TM using both UDP-glucuronic acid and UDP-N-acetylgalactosamine as sugar donors. Much longer chains were synthesized on alpha-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 beta1,4-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.