Characterization and partial purification of β- N-acetylgalactosaminyltransferase from urine of Sd(a+) individuals

Abstract

Urine from Sd(a+) individuals was found to contain a β- N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine (GalNAc) from UDP-GalNAc to 3′-sialyllactose and glycoproteins carrying the terminal NeuAcα-3Galβ group. This enzyme has been purified 174-fold by affinity chromatography on Blue Sepharose and DEAE-Sephacel chromatography in a yield of 33%. Neither endogenous incorporation nor sugar nucleotide degrading enzymes were found in the purified preparation. The transferase had a pH optimum of pH 7.5 and a requirement for Mn 2+ but not for detergents. The K m for UDP-GalNAc was 66 × 10 −6 m, using fetuin as an acceptor. Like β-GalNAc-transferase from other sources the urinary enzyme had a strict requirement for sialylated acceptors. On the basis of enzymatic and chemical treatment of the product obtained by the transfer of [ 3H]GalNAc to 3′-sialyllactose, we propose that the enzyme attaches GalNAc in β-anomeric configuration to 0–4 of the galactose residue that is substituted at O-3 by sialic acid. A preparation of Tamm-Horsfall glycoprotein from a Sd(a−) donor lacking β-Gal-NAc was found to be the best acceptor among the glycoproteins tested. Studies on the transferase activity toward fetuin, human chorionic gonadotropin, and glycophorin A indicated that the enzyme preferentially adds the sugar to the sialylated terminal end of N-linked oligosaccharides. Unlike the β-GalNAc-transferase bound to human kidney microsomes (F. Piller et al. (1986) Carbohydr. Res. 149, 171–184) the urinary transferase is able to transfer β-GalNAc to the NeuAcα-3Galβ-3(NeuAcα-6)GalNAc chains bound to the native glycophorin.