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

Abstract

Glycosyl transferases that participate in the assembly of the lipidlinked oligosaccharide intermediates were solubilized from cultured soybean cells using 0.3% Nonidet P40 (NP40) in the presence of 10% glycerol. The solubilized enzyme preparation was reasonably stable and 50% of the activity still remained after storage at -10°C for I month.The solubilized enzyme synthesized ["C]Man3GlcNAc2-pyrophosphorylpolyprenol and I'4CjMansGlcNAc2-pyrophosphoryl-polyprenol when incubated with GDP-'4Cjmannose 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. In fact, the addition of 5 to 10 millimolar ethylenediaminetetraacetate 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 ethylenediaminetetraacetate, the results suggest that the mannosyl residues added to form Man3GlcNAcrlipid and MansGlcNAc2-lipid come directly from GDP-mannose without the participation of dolichyl-phosphoryl-mannose. On the other hand, the formation of significant amounts of Man6GlcNAc2-lipid, Man7GlcNAc2-lipid, and MansGlcNAc2-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, it appears likely that the first five mannose residues to form MansGlcNAcrlipid come directly from GDP-mannose, whereas other mannose units to form larger oligosaccharide-lipids come from dolichylphosphoryl-mannose.In plants as well as animal cells, lipid-linked saccharide intermediates are involved in the formation of the N-linked oligosaccharide chains of membrane and secretory proteins (6,22,23,26 phoma cell line was isolated that had lost the ability to synthesize dolichyl-phosphoryl-mannose, but this mutant could still produce the Man5GlcNAc2-lipid (4). However, when cell-free extracts of this mutant were supplemented with dolichyl-phosphoryl-mannose, they could elongate the Man5GlcNAc2-lipid to larger oligosaccharides. Finally, a number ofthe mannosyl transferases that participate in the formation of the Man5GlcNAc2-lipid have been solubilized (1 1, 16, 17, 24, 25) and the a-1,2 and a-1,3 mannosyl transferases have been partially purified (17,24). These enzymes required GDP-mannose, rather than dolichylphosphoryl-mannose, as the mannosyl donors (16,25).In plants, the lipid-linked saccharide pathway has not been studied in nearly as much detail. The mung bean particulate enzyme catalyzes the incorporation of mannose from GDPmannose into Man1GlcNAc2-lipid, Man2GlcNAc2-lipid, Man3-GlcNAc2-lipid, Man5GlcNAc2-lipid, and Man7GlcNAc2-lipid (13). These oligosaccharides were partially characterized and a pathway of mannose addition was postulated. However, it was not determined whether the various mannose units were donated via GDP-mannose or dolichyl-phosphoryl-mannose. In the present repor...

Structure, biosynthesis, and function of asparagine‐linked glycans on plant glycoproteins

“…In plant cells, as in mammalian cells (reviewed in Hirschberg and Snider 1987), several studies indicate that the Glc3Man9GlcNAc2 oligosaccharide precursor is assembled on a dolicholpyrophosphate carrier. Of greatest interest is the finding that the 5 mannose residues required for the formation of a Man^GlcNAcj-lipid come directly from GDP-mannose, while the other mannose residues are transferred across the membrane attached to a dolichol molecule (Hori et al 1985). Thus plants and animals appears to use the same strategy to solve a difficult topographical problem.…”

Purification and Properties of UDP-GlcNAc:Dolichyl-Pyrophosphoryl-GlcNAc GlcNAc Transferase from Mung Bean Seedling

“…The oligosaccharides were partially characterized as Man(GlcNAc)2-PP-lipid, Man2(GlcNAc)2-PP-lipid, Man3(GlcNAc)2-PP-lipid, Man5(GlcNAc)2-PP-lipid, and Man7-(GlcNAc)2-PP-lipid. Recently, the mannQsyl transferases from suspension-cultured soybean cells were solubilized (17). The solubilized enzyime preparation synthesized Man3- ' Supported by grants from the National Institutes of Health (AM 21800) and the Robert A. Welch Foundation.…”

“…However, when the incubations were done in the presence of EDTA and amphomycin, both of which inhibit the formation of dolichyl-phosphoryl-mannose, only the Man3(GlcNAc)2-lipid and the Man5(GlcNAc)2-lipid were formed ( 17). That study suggested that the first five mannose residues in the lipid-linked oligosaccharides come directly from GDP-mannose, whereas the next four mannose units come from dolichylphosphoryl-mannose.…”

Properties of Solubilized UDP-GlcNAc: Dolichyl Phosphate-GlcNAc-1-P-Transferase from Soybean Cultured Cells

“…The study of individual enzymes is necessary in order to know the regulation and control points of this pathway. Previously, we solubilized several mannosyl transferases from soybean cultured cells and identified the products formed by transfer of mannose from GDP-mannose to the acceptor lipids ( 18). The present study describes the solubilization and properties of GlcNAc-1 -P-transferase from soybean cultured cells.…”