title: In vitro biosynthesis of a decasaccharide prototype of multiply branched polylactosaminoglycan backbones.
abstract: Multiply branched polylactosaminoglycans are expressed in glycoproteins and glycolipids of many cells. Interest in their biology stems from their abundant expression in early embryonal cells and from their ability to carry multiple lectin-binding determinants, which makes them prominent ligands and antagonists of cell adhesion proteins. A prototype of their backbones is represented by the decasaccharide LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc (5), where LacNAc is the disaccharide Gal beta1-4GlcNAc. Here, we describe in vitro biosynthesis of glycan 5. Incubation of the linear hexasaccharide LacNAc beta1-3'LacNAc beta1-3'LacNAc (1) with UDP-GlcNAc and alpha midchain beta1,6-GlcNAc transferase activity (GlcNAc to Gal), present in rat serum [Gu, J., Nishikawa, A., Fujii, S., Gasa, S., & Taniguchi, N. (1992) J. Biol. Chem. 267, 2994-2999], gave the doubly branched octasaccharide LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (4). The latter was converted to 5 by enzymatic beta1,4-galactosylation. In the initial branching reaction of 1, two isomeric heptasaccharide intermediates, LacNAc beta1-3'LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (2) and LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'LacNAc (3), were formed first at comparable rates. Later, both intermediates were converted to 4, revealing two distinct pathways of the reaction: 1 --> 2 --> 4 and 1 --> 3 --> 4. These data suggest that, regardless of their chain length, linear polylactosamines similar to 1 contain potential branching sites at each of the internal galactoses. The enzyme-binding epitope of 1 is probably LacNAc beta1-3'LacNAc, because the trisaccharides GlcNAc beta1-3'LacNAc and LacNAc beta1-3Gal as well as the tetrasaccharide GlcNAc beta1-3'LacNAc beta1-3Gal were poor acceptors, while LacNAc beta1-3'LacNAc was a good one. Midchain beta1,6-GlcNAc transferase activities present in serum of several mammalian species, including man, resembled closely the rat serum activity in their mode of action and in their acceptor specificity. We suggest that analogous membrane-bound Golgi enzymes are involved in the biosynthesis of multiply branched polylactosamines in vivo.
authors.affiliation: Institute of Biotechnology and Department of Biosciences, University of Helsinki, Finland.
authors.forename: A
authors.initials: A
authors.lastname: Leppanen
authors.forename: H
authors.initials: H
authors.lastname: Salminen
authors.forename: Y
authors.initials: Y
authors.lastname: Zhu
authors.forename: H
authors.initials: H
authors.lastname: Maaheimo
authors.forename: J
authors.initials: J
authors.lastname: Helin
authors.forename: C E
authors.initials: CE
authors.lastname: Costello
authors.forename: O
authors.initials: O
authors.lastname: Renkonen
mesh_terms: D000606:Amino Sugars / Q000096:biosynthesis* / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002384:Catalysis; D002417:Cattle; D006056:Golgi Apparatus / Q000201:enzymology; D006736:Horses; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000097:blood / Q000378:metabolism; D009844:Oligosaccharides; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D051381:Rats; D012756:Sheep; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000606:Amino Sugars; D009844:Oligosaccharides; D011134:Polysaccharides; C039448:lactosaminoglycan; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase