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.
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.
Nadanaka
S
S
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan.
Kitagawa
H
H
Goto
F
F
Tamura
J
J
Neumann
K W
KW
Ogawa
T
T
Sugahara
K
K
D000116:Acetylgalactosamine / Q000378:metabolism*; D001704:Biopolymers; D002240:Carbohydrate Sequence; D002474:Cell-Free System; D002809:Chondroitin Sulfates / Q000096:biosynthesis*; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism; D013379:Substrate Specificity; D018180:Thrombomodulin / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D001704:Biopolymers; D006025:Glycosaminoglycans; D018180:Thrombomodulin; D002809:Chondroitin Sulfates; D017350:N-Acetylgalactosaminyltransferases; D000116:Acetylgalactosamine