In Vitro Biosynthesis of a Decasaccharide Prototype of Multiply Branched Polylactosaminoglycan Backbones

• Published in: Biochemistry (Easton) 1997-06, Vol.36 (23), p.7026-7036
• Main Authors: Leppänen, Anne, Salminen, Heidi, Zhu, Ying, Maaheimo, Hannu, Helin, Jari, Costello, Catherine E, Renkonen, Ossi
• Format: Article
• Language: English
• Subjects: Amino Sugars - biosynthesis; Amino Sugars - chemistry; Animals; Binding Sites; Carbohydrate Sequence; Catalysis; Cattle; Golgi Apparatus - enzymology; Horses; In Vitro Techniques; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Sequence Data; N-Acetylglucosaminyltransferases - blood; N-Acetylglucosaminyltransferases - metabolism; Oligosaccharides; Polysaccharides - biosynthesis; Polysaccharides - chemistry; Rats; Sheep; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9627673

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 LacNAcβ1−3‘(LacNAcβ1−6‘)LacNAcβ1−3‘(LacNAcβ1−6‘)LacNAc (5), where LacNAc is the disaccharide Galβ1−4GlcNAc. Here, we describe in vitro biosynthesis of glycan 5. Incubation of the linear hexasaccharide LacNAcβ1−3‘LacNAcβ1−3‘LacNAc (1) with UDP-GlcNAc and a midchain β1,6-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 LacNAcβ1−3‘(GlcNAcβ1−6‘)LacNAcβ1−3‘(GlcNAcβ1−6‘)LacNAc (4). The latter was converted to 5 by enzymatic β1,4-galactosylation. In the initial branching reaction of 1, two isomeric heptasaccharide intermediates, LacNAcβ1−3‘LacNAcβ1−3‘(GlcNAcβ1−6‘)LacNAc (2) and LacNAcβ1−3‘(GlcNAcβ1−6‘)LacNAcβ1−3‘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 LacNAcβ1−3‘LacNAc, because the trisaccharides GlcNAcβ1−3‘LacNAc and LacNAcβ1−3Gal as well as the tetrasaccharide GlcNAcβ1−3‘LacNAcβ1−3Gal were poor acceptors, while LacNAcβ1−3‘LacNAc was a good one. Midchain β1,6-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.