title: Characterization of a rat liver Golgi sulphotransferase responsible for the 6-O-sulphation of N-acetylglucosamine residues in beta-linkage to mannose: role in assembly of sialyl-galactosyl-N-acetylglucosamine 6-sulphate sequence of N-linked oligosaccharides.
abstract: Rat liver Golgi membranes were found to contain an enzyme that can transfer sulphate from 3'-phosphoadenosine 5'-phosphosulphate (PAPS) to C-6 of the terminal GlcNAc in beta-linkage to mannose and has properties indicating that it is involved in the synthesis of the NeuAc alpha 2-3(6)Gal beta 1-4GlcNAc(6-SO4) sequences observed in the N-linked carbohydrate units of various glycoproteins. Assays performed with [35S]PAPS (Km 0.67 microM) and GlcNAc beta 1-6Man alpha 1-O-Me (GnMaMe) acceptor (Km 0.71 mM) indicated that the sulphotransferase had a pH optimum of approx. 7.0 and is markedly stimulated by Mn2+ ions (maximum approx. 15 mM) and Triton X-100 (0.05-0.1%). Hydrazine/nitrous acid/NaBH4 treatment of the 35S-labelled product yielded radiolabelled 2,5-anhydromannitol(6-SO4). The sulphated GnMaMc product of the GlcNAc-6-O-sulphotransferase could be galactosylated by a rat liver Golgi enzyme that was shown to have the same properties as the UDP-Gal:GlcNAc beta-1,4-galactosyltransferase from bovine milk. Competition studies performed with GlcNAc and GlcNAc-6-SO4 furthermore indicated that the same liver enzyme acted on both acceptors to produce Gal beta 1-4GlcNAc and Gal beta 1-4GlcNAc(6-SO4) with Km values of 1.04 and 1.68 mM respectively. Because the sulphated N-acetyl-lactosaminc could in turn serve as an acceptor for rat liver sialyltransferase, it seems that this enzyme, together with the Golgi galactosyltransferase and the GlcNAc-6-O-sulphotransferase, could act in concert in assembling the NeuAc alpha 2-3(6)Gal beta 1-4GlcNAc(6-SO4) branches of complex N-linked oligosaccharides.
authors.affiliation: Department of Biological Chemistry, Harvard Medical School, Joslin Diabetes Center, Boston, MA 02215, USA.
authors.forename: R G
authors.initials: RG
authors.lastname: Spiro
authors.forename: Y
authors.initials: Y
authors.lastname: Yasumoto
authors.forename: V
authors.initials: V
authors.lastname: Bhoyroo
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002240:Carbohydrate Sequence; D002417:Cattle; D002855:Chromatography, Thin Layer; D005700:Galactosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008358:Mannose / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; D008345:Manganese; D010724:Phosphoadenosine Phosphosulfate; C022321:PAPS sulfotransferase; D005700:Galactosyltransferases; D015238:Sulfotransferases; D019158:N-Acetylneuraminic Acid; D008274:Magnesium; D008358:Mannose; D000117:Acetylglucosamine