Transfer of mannose from GDP-mannose to lipid-linked oligosaccharide by soluble mannosyl transferase.

Abstract

A solubilized mannosyl transferase(s) was obtained by treatment of the pig aorta particulate enzyme with the nonionic detergent Nonidet P-40 followed by centrifugation at 100,000 .times. g for 60 min. This enzyme preparation catalyzed the transfer of mannose from GDP-[14C]mannose (but not from [14C]mannosylphosphoryldolichol) to form a heptasaccharide-lipid. The synthesis of this heptasaccharide-lipid required the addition of an acceptor lipid that was isolated from pig liver. The oligosaccharide portion of the acceptor lipid appeared to be a mixture of trisaccharide and pentasaccharide. The formation of hepatasaccharide-lipid did not require divalent cation, was not inhibited by EDTA, and was not inhibited by the anti-biotic amphomycin. The heptasaccharide portion of the heptasaccharide-lipid had the same migration properties on paper chromatograms in 2 different solvent systems as a previously characterized Man5GlcNAc2 oligosaccharide. It also had the same migration properties as the oligosaccharide that accumulates in the presence of amphomycin. All 3 of these oligosaccharides emerged from a Bio-Gel P-4 column in the same position. Radioactive mannose was released from the heptasaccharide by digestion with .alpha.-mannosidase. At least some of the .alpha.-linked mannose residues in the heptasaccharide-lipid are donated directly from GDP-mannose.