Biosynthesis of mannan and mannolipids from GDP-Man by membrane fractions of sycamore cell cultures

Abstract

Membrane fractions have been prepared from sycamore (Acer pseudoplatanus) cell suspensions grown in liquid medium. These fractions catalyzed the transfer of mannoxyl-units from GDP-(14C) Man into a polymannoside, a mannolipid and oligosaccharide-lipids. The polymannoside was partially solubilized by proteolytic digestion or maceration in sodium dodecyl sulfate-urea mixtures. However no evidence is available of a covalent linkage between the biosynthesized glycan and peptides. The structural analysis of the (14C)mannan showed that the polysaccharide was a homopolymer of beta-(1 leads to 4) linked mannose with a few branches. During the incubation of the membranes with the substrate, the polymer chains elongated with a large number of sugar units and contained 25 to 40 hexose residues per non-reducing end monomer. GDP-Glc was a competitive inhibitor of the GDP-Man: beta-mannan mannosyl-transferase, whereas GDP-Man activated a GDP-Gle: beta-glucan glucosyl-transferase present in the same membrane preparation. Two kinds of glycolipids were synthesized in the presence of GDP-(14C)Man. The first (I) contained a polar moiety characterized as a mannose-phosphate and was very similar to polyprenyl-phosphate-mannose identified in plants by Alam and Hemming. The other mannolipid (II) was hydrolyzed by mild acid into labeled oligosaccharides of high molecular weight. This material was separated into two oligosaccharide fractions, the first (II A) of MW over 5000, the second (II B) of MW around 1700. II B contained at most two labeled mannose residues per chain, linked to the non-reducing end of unlabeled units which probably contained neutral sugars and N-acetyl-osamine(s) near the reducing end. Oligosaccharide II A seemed to contain one or several II B chains.