Identification and characterization of a novel UDP-GalNAc : GlcA beta-R alpha 1,4-N-acetylgalactosaminyltransferase from a human sarcoma cell line

We recently discovered a novel alpha-N-acetylgalactosaminyltransferase in f etal bovine serum (Kitagawa et al,, J, Biol, Chem., 270, 22190-22195, 1995) and also in mouse mastcytoma cells (Lidholt et al,, Glycoconjugate J., 14, 737-742, 1997), which catalyzed the transfer of an alpha-GalNAc residue to the linkage tetrasaccharide-serine, GlcA beta 1-3Gal beta 1-3Gal beta 1-4X yl beta 1-O-Ser, derived from proteoglycans, In this study, we characterize d this enzyme using a preparation obtained from the serum-free culture medi um of a human sarcoma (malignant fibrous histiocytoma) cell line by phenyl- Sepharose chromatography, Structural characterization by H-1 NMR spectrosco py of the reaction product using the linkage tetrasaccharide-serine, GlcA b eta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, as a substrate demonstrate d that the enzyme was a UDP-GalNAc:GlcA beta 1-R alpha 1,4-N-acetylgalactos aminyltransferase. This is the first identification of an alpha 1,4-N-acety lgalactosaminyltransferase, Using N-acetylchondrosine GlcA beta 1-3GalNAc a s an alternative substrate, the enzyme required divalent cations for the tr ansferase reaction, with maximal activity at 20 mM Mn2+ and exhibited a dua l optimum at pH 6.5 and pH 7.4 depending upon the buffers used, with the hi ghest activity in a 50 mM 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.5. The apparent Km values obtained for N-acetylchondrosine, the linkage t etrasaccharide-serine, and UDP-GalNAc were 1060 mu M, 188 mu M, and 27 mu M , respectively This suggested that the linkage tetrasaccharide-serine mas a good acceptor substrate for the enzyme, In addition, the enzyme utilized g lucuronylneolactotetraosylceramide GlcA beta 1-3Gal beta 1-4GlcNAc beta 1-3 Gal beta 1-4Glc beta 1-1Cer but not sulfoglucuronylneolactotetraosylceramid e GlcA(3-O-sulfate)beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer as acceptor substrates, The possibility of involvement of this enzym e in the biosynthesis of glycosaminoglycan as well as other GlcA-containing glycoconjugates is discussed.