IN-VITRO BIOSYNTHESIS OF A DECASACCHARIDE PROTOTYPE OF MULTIPLY BRANCHED POLYLACTOSAMINOGLYCAN BACKBONES

Multiply branched polylactosaminoglycans are expressed in glycoprotein s and glycolipids of many cells. Interest in their biology stems from their abundant expression in early embryonal cells and from their abil ity to carry multiple lectin-binding determinants, which makes them pr ominent ligands and antagonists of cell adhesion proteins. A prototype of their backbones is represented by the decasaccharide LacNAc beta 1 -3'(LacNAc beta 1-6')LacNAc beta 1-3'(LacNAc beta 1-6')LacNAc (5), whe re LacNAc is the disaccharide Gal beta 1-4GlcNAc. Here, we describe in vitro biosynthesis of glycan 5. Incubation of the linear hexasacchari de LacNAc beta 1-3'LacNAc beta 1-3'LacNAc (1) with UDP-GlcNAc and a mi dchain beta 1,6-GlcNAc transferase activity (GlcNAc to Gal), present i n rat serum [Gu, J., Nishikawa, A., Fujii, S., Gasa, S., & Taniguchi, N. (1992) J. Biol. Chem. 267, 2994-2999], gave the doubly branched oct asaccharide LacNAc beta 1-3'(GlcNAc beta 1-6')LacNAc beta 1-3'(GlcNAc beta 1-6')LacNAc (4). The latter was converted to 5 by enzymatic beta 1,4-galactosylation. In the initial branching reaction of 1, two isome ric heptasaccharide intermediates, LacNAc beta 1-3'LacNAc beta 1-3'(Gl cNAc beta 1-6')LacNAc (2) and LacNAc beta 1-3'(GlcNAc beta 1-6')LacNAc beta 1-3'LacNAc (3), were formed first at comparable rates. Later, bo th intermtdiates were converted to 4, revealing two distinct pathways of the reaction: 1 --> 2 --> 4 and 1 --> 3 --> 4. These data suggest t hat, regardless of their chain length, linear polylactosamines similar to 1 contain potential branching sites at each of the internal galact oses. The enzyme-binding epitope of 1 is probably LacNAc beta 1-3'LacN Ac, because the trisaccharides GlcNAc beta 1-3'LacNAc and LacNAc beta 1-3Gal as well as the tetrasaccharide GlcNAc beta 1-3'LacNAc beta 1-3G al were poor accepters, while LacNAc beta 1-3'LacNAc was a good one. M idchain beta 1,6-GlcNAc transferase activities present in serum of sev eral mammalian species, including man, resembled closely the rat serum activity in their mode of action and in their acceptor specificity. W e suggest that analogous membrane-bound Golgi enzymes are involved in the biosynthesis of multiply branched polylactosamines in vivo.