Biosynthesis of Mannose-Containing Lipid-Linked Oligosaccharides by Solubilized Enzyme Preparation from Cultured Soybean Cells

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Abstract
Glycosyl transferases that participate in the assembly of the lipid-linked oligosaccharide intermediates were solubilized from cultured soybean cells using 0.3% Nonidet P-40 (NP-40) in the presence of 10% glycerol. The solubilized enzyme preparation was reasonably stable and 50% of the activity still remained after storage at -10.degree. C for 1 mo. The solubilized enzyme synthesized [14C]Man3GlcNAc2-pyrophosphoryl-polyprenol and [14C]Man5GlcNAc2-pyrophosphoryl-polyprenol when incubated with GDP-[14C]mannose plus a partially purified acceptor lipid isolated from calf liver. The formation of these lipid-linked oligosaccharides did not require the addition of dolichyl-phosphate or metal ions. The addition of 5-10 mm EDT stimulated the incorporation of mannose into lipid-linked oligosaccharides 2- to 3-fold. Since little or no dolichyl-phosphoryl-mannose is formed in the presence of EDT the mannosyl residues added to form Man3GlcNAc2-lipid and Man5GlcNAc2-lipid comes directly from GDP-mannose without the participation of dolichyl-phosphoryl-mannose. The formation of significant amounts of Man6GlcNAc2-lipid, Man7-GlcNAc2-lipid and Man8GlcNAc2-lipid occurred when the above incubations were supplemented with dolichyl phosphate and metal ions. Based on various time course studies and supplementation studies with various additions, the first 5 mannose residues to form Man5GlcNAc2-lipid apparently come directly from GDP-mannose, whereas other mannose units to form larger oligosaccharide-lipids come from dolichyl-phosphoryl-mannose.
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