N-Acetylgalactosamine (GalNAc) Transfer to the Common Carbohydrate-Protein Linkage Region of Sulfated Glycosaminoglycans

During the course of a study of elucidate the role of modification of the common polysaccharide-protein linkage structure, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, in biosynthetic sorting mechanisms of the different sulfated glycosaminoglycan chains, a novel N-acetylgalactosamine (GalNAc) transferase was discovered in fetal bovine serum. The enzyme catalyzed the transfer of [3H]GalNAc from UDP-[3H]GalNAc to linkage tetrasaccharide and hexasaccharide serines synthesized chemically and to various regular oligosaccharides containing terminal D-glucuronic acid (GlcA), which were prepared from chondroitin and chondroitin sulfate using testicular hyaluronidase digestion. The labeled products obtained with the linkage tetra- and hexasaccharide serines and with the tetrasaccharide (GlcA beta 1-3GalNAc)2 were resistant to digestion with chondroitinase AC-II and beta-N-acetylhexosaminidase but sensitive to alpha-N-acetylgalactosaminidase digestion, indicating that the enzyme is an alpha-N-acetylgalactosaminyltransferase. This finding is in contrast to that of Rohrmann et al. (Rohrmann, K., Niemann, R., and Buddecke, E. (1985) Eur. J. Biochem., 148, 463-469), who reported that a corresponding product was susceptible to digestion with beta-N-acetylhexosaminidase. The presence of a sulfate group at C4 of the penultimate GalNAc or Gal units markedly inhibited the transfer of GalNAc to the terminal GlcA, while a sulfate group at C6 of the GalNAc had little effect on the transfer. Moreover, a slight but significant transfer of [3H]GalNAc was observed to an oligosaccharide containing terminal 2-O-sulfated GlcA as acceptor, whereas no incorporation was detected into oligosaccharides containing terminal unsaturated or 3-O-sulfated GlcA units. These results suggest that this novel serum enzyme is a UDP-GalNAc:chondro-oligosaccharide alpha 1-3- or 1-4-N-acetylgalactosaminyltransferase. The possibility of involvement of this enzyme in glycosaminoglycan biosynthesis is discussed.