Characterization of the Oligosaccharides from Lipid-Linked Oligosaccharides of Mung Bean Seedlings

Hori, Hidetaka; Elbein, Alan D.

doi:10.1104/pp.70.1.12

Cited by 27 publications

(17 citation statements)

References 30 publications

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“…Lima bean lectin and pineapple bromelain also contain asparagine-linked oligosaccharides (13,18), but the oligosaccharide structures of these proteins are quite different from the typical high-mannose oligosaccharides. That is, these oligosaccharides do not contain the typical Mana Since relatively little information is available on the incorporation of xylose or fucose into plant glycoproteins, the present study was undertaken to examine the fucosylation of plant The membrane fraction was suspended in CHC13:CH30H:H20 (1:1:1) to extract the lipid-linked monosaccharides as previously described (3,10). After phase separation and removal of the lower CHC13 layer (which contains the lipid-linked monosaccharides and perhaps other glycolipids), the particulate material was reisolated by centrifugation and extracted with CHC13:CH30H:H20 (10:10:3) to extract the lipid-linked oligosaccharides (10).…”

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“…That is, these oligosaccharides do not contain the typical Mana Since relatively little information is available on the incorporation of xylose or fucose into plant glycoproteins, the present study was undertaken to examine the fucosylation of plant The membrane fraction was suspended in CHC13:CH30H:H20 (1:1:1) to extract the lipid-linked monosaccharides as previously described (3,10). After phase separation and removal of the lower CHC13 layer (which contains the lipid-linked monosaccharides and perhaps other glycolipids), the particulate material was reisolated by centrifugation and extracted with CHC13:CH30H:H20 (10:10:3) to extract the lipid-linked oligosaccharides (10). After this extraction, the particulate material was reisolated by centrifugation, washed several times with methanol and then water, and digested exhaustively with pronase to release soluble glycopeptides.…”

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Fucosylation of Membrane Proteins in Soybean Cultured Cells

Hori¹,

Kaushal²,

Elbein³

1985

Plant Physiol.

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Cultures of soybean cells incorporate 15,6-3H1-L-fucose into various cellular components including lipids and proteins. The membrane glycoproteins were digested with pronase to produce glycopeptides, and the glycopeptides were isolated on columns of Biogel P4. The major fucoselabeled glycopeptide sized as a Hexose,s1rN-acetylglucosamine2 (GlcNAc2) on columns of Biogel P4. Fucose incorporation was also examined in the presence of the processing inhibitor swainsonine, and the glycosylation inhibitor tunicamycin. In the presence of swainsonine, the incorporation of fucose was not reduced but the glycopeptides were smaller in size and migrated like Hexosel2 13-GicNAc2 structures. On the other hand, tunicamycin inhibited the incorporation of fucose into the glycopeptides by 70 to 80%, indicating that the L-fucose was present in N-linked oligosaccharides.The membrane glycoproteins were doubly-labeled by incubating soy- In animal cells, once the oligosaccharide is transferred to protein, it is rapidly processed by the removal of all three glucose residues (27) and a number of mannose residues (21,22) via a number ofspecific processing glycosidases. In addition, a number of sugars such as GIcNAc, galactose, sialic acid, and fucose may be added to give rise to the high-mannose, hybrid and complex types of oligosaccharides that are typically found in these cells (8). For example, the addition ofL-fucose to animal glycoproteins occurs after the removal of three glucoses and four mannoses and the addition of one GlcNAc (17).The reactions involved in processing of plant glycoproteins have not been studied to any great extent, but in these systems also, the initial reactions involve the removal of all three glucose residues (11). However

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Fucosylation of Membrane Proteins in Soybean Cultured Cells

Hori¹,

Kaushal²,

Elbein³

1985

Plant Physiol.

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“…Thus, a number oflipid-linked oligosaccharides, containing mannose and GlcNAc, have been isolated from various plant systems (2,8,13,22) and many of these oligosaccharides have structures that closely resemble or are identical to those of animal systems (2,13,22,27). The final lipid-linked oligosaccharide intermediate, the Glc3Man9GlcNAc2-pyrophosphoryl-dolichol, has also been isolated from several plant systems (20,27) and its structure partially characterized (14).…”

Section: Discussionmentioning

confidence: 99%

“…The pellet from this centrifugation was suspended in Tris-HCI buffer containing 10% glycerol at the rate of 2 ml of buffer/ tained as described above (10)(11)(12)(13)(14)(15) Preparation of Acceptor Lipid. The acceptor-lipid that was used in these studies as a mannosyl acceptor was prepared from pig liver as previously described (13). Briefly After phase separation, the lower phase containing lipids was discarded and the upper phase was concentrated to dryness, dissolved in a small volume of water and analyzed by chromatography on Biogel P-4.…”

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

Hori

Kaushal²,

Elbein³

1985

Plant Physiol.

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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...

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Glucomannan synthesis in pea epicotyls: The mannose and glucose transferases

Piro

Zuppa

Dalessandro

et al. 1993

Planta

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Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a beta-1,4-[14C]mannan from GDP-D-[U-14C]-mannose, a mixed beta-1,3- and beta-1,4-[14C]glucan from GDP-D-[U-14C]-glucose and a beta-1,4-[14C]-glucomannan from both GDP-D-[U-14C]mannose and GDP-D-[U-14C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The beta-glucan synthase had different properties from other preparations which bring about the synthesis of beta-1,3-glucans (callose) and mixed beta-1,3- and beta-1,4- glucans and which use UDP-D-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-D-xylose in addition to GDP-D-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-D-glucose acted competitively in the presence of GDP-D-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-D-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-D-glucose and GDP-D-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-D-mannose and GDP-D-glucose to bring about the synthesis of the heteropolysaccharide.

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Characterization of the Oligosaccharides from Lipid-Linked Oligosaccharides of Mung Bean Seedlings

Cited by 27 publications

References 30 publications

Fucosylation of Membrane Proteins in Soybean Cultured Cells

Fucosylation of Membrane Proteins in Soybean Cultured Cells

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

Glucomannan synthesis in pea epicotyls: The mannose and glucose transferases

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