title: Characterization of mammalian UDP-GalNAc:glucuronide alpha 1-4-N-acetylgalactosaminyltransferase.
abstract: We previously reported that cultured cells incubated with beta-xylosides synthesized alpha-GalNAc-capped GAG-related xylosides, GalNAc alpha GlcA beta Gal beta Gal beta Xyl beta-R and GalNAc alpha GlcA beta GalNAc beta GlcA beta Gal beta Gal beta Xyl beta-R, where R is 4-methylumbelliferyl or p-nitrophenyl (Manzi et al., 1995; Miura and Freeze, 1998). In this study, we characterized an alpha-N-acetylgalactosaminyltransferase (alpha-GalNAc-T) that probably adds the alpha-GalNAc residue to the above xylosides. Microsomes from several animal cells and mouse brain contained the enzyme activity which requires divalent cations, and has a relatively broad pH optimal range around neutral. The apparent K(m) values were in the submillimolar range for the acceptors tested, and 19 microM for UDP-GalNAc. 1H-NMR analysis of the GlcA-beta-MU acceptor product showed the GalNAc residue is transferred in alpha 1,4-linkage to the glucuronide, which is consistent with previous results reported on alpha-GalNAc-capped Xyl-MU (Manzi et al., 1995). Various artificial glucuronides were tested as acceptors to assess the influence of the aglycone. Glucuronides with a bicyclic aromatic ring, such as 4-methylumbelliferyl beta-D-glucuronide (GlcA-beta-MU) and alpha-naphthyl beta-D-glucuronide, were the best acceptors. Interestingly, a synthetic acceptor that resembles the HNK-1 carbohydrate epitope but lacking the sulfate group, GlcA beta 1,3Gal beta 1,4GlcNAc beta-O-octyl (delta SHNK-C8), was a better acceptor for alpha-GalNAc-T than the glycosaminoglycan-protein linkage region tetrasaccharyl xyloside, GlcA beta 1,3Gal beta 1,3Gal beta 1,4Xyl beta-MU. GlcA-beta-MU and delta SHNK-C8 competed for the alpha-GalNAc-T activity, suggesting that the same activity catalyzes the transfer of the GalNAc residue to both acceptors. Taken together, the results show that the alpha-GalNAc-T described here is not restricted to GAG-type oligosaccharide acceptors, but rather is a UDP-GalNAc:glucuronide alpha 1-4-N-acetylgalactosaminyltransferase.
authors.affiliation: Burnham Institute, La Jolla, CA 92037, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Miura
authors.forename: Y
authors.initials: Y
authors.lastname: Ding
authors.forename: A
authors.initials: A
authors.lastname: Manzi
authors.forename: O
authors.initials: O
authors.lastname: Hindsgaul
authors.forename: H H
authors.initials: HH
authors.lastname: Freeze
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D006027:Glycosides; C010807:xylosides; D017350:N-Acetylgalactosaminyltransferases