Presence in a plant of a compound similar to the dolichyl diphosphate oligosaccharide of animal tissue

Staneloni, Roberto J.; Tolmasky, Marcelo E.; Petriella, Claudio; Ugalde, Rodolfo A.; Leloir, Luis F.

doi:10.1042/bj1910257

Cited by 39 publications

(19 citation statements)

References 10 publications

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“…A single block, affecting the enzyme responsible for the addition of the eighth mannosyl residue, could account, therefore, for the accumulation in C. fasciculata of the lipidbound oligosaccharide depicted above. When animal, plant, or fungal cells were labeled and the DolPP derivatives were isolated by using procedures comparable to those employed here, the main compound found was Glc3MangGlcNAc2-P-P-Dol (2,5,8). This was one of the principal arguments for concluding that this compound is the saccharide donor of protein glycosylation in those organisms.…”

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“…It has become evident in recent years that glycosylation of asparagine residues in eukaryotic cell proteins involves dolichol pyrophosphate (DolPP)-bound oligosaccharides as intermediates (1). It has been reported that in animal tissues (2,3), yeasts (4)(5)(6), and probably also in insects (7) and plants (8), the oligosaccharide transferred from the lipid derivative to proteins is composed of two N-acetylglucosamine, nine mannose, and three glucose residues. A report by Lehle suggests that in yeast the oligosaccharides containing two or three glucose residues are equally transferred to protein (9).…”

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Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.

Parodi¹,

Allué²,

Cazzulo³

1981

Proc. Natl. Acad. Sci. U.S.A.

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Cells of the insect parasite Crithidia fasciculata incubated with ['4C]glucose were found to possess only one lipidbound oligosaccharide with solubility in chloroform/methanol/ water mixtures and net charge similar to the charges of dolichol pyrophosphate derivatives. The saccharide moiety could be released from lipid by mild acid hydrolysis. Several enzymatic and chemical treatments of the oligosaccharide indicated that the latter had the structure Mana-+Mana---Mana--[Mana-+ Mana-+Man(al--6)]Man--GlcNAc(l1--4)GlcNAc. Two labeled oligosaccharides were liberated from proteins by a sequential treatment with a protease and endo-13-N-acetylglucosaminidase H. One ofthe protein-bound oligosaccharides had the same structure as the lipid-linked compound, whereas in the second oligosaccharide some mannose residues had been replaced by galactose units, but both compounds migrated as did a Man7GlcNAc standard. These were the largest oligosaccharides obtained even after short labeling periods. It is suggested that glycosylation ofproteins in the protozoan Crithidiafasciculata does not involve glucosylated lipid-bound oligosaccharides as intermediates. It has become evident in recent years that glycosylation of asparagine residues in eukaryotic cell proteins involves dolichol pyrophosphate (DolPP)-bound oligosaccharides as intermediates (1). It has been reported that in animal tissues (2, 3), yeasts (4-6), and probably also in insects (7) and plants (8), the oligosaccharide transferred from the lipid derivative to proteins is composed of two N-acetylglucosamine, nine mannose, and three glucose residues. A report by Lehle suggests that in yeast the oligosaccharides containing two or three glucose residues are equally transferred to protein (9). However, no evidence was presented indicating that the oligosaccharides used in the assay had the same specific activity.The protein-bound oligosaccharides are then processed by loss of some of their monosaccharide constituents and addition ofother residues directly from the respective sugar nucleotides. We here report evidence suggesting that in the protozoan Crithidta fasciculata, an insect obligate parasite, the oligosaccharide transferred to protein contains two N-acetylglucosamine and seven mannose residues and that the oligosaccharide may be processed once bound to protein.MATERIALS AND METHODS Materials.[14C]Glucose (284 Ci/mol; 1 Ci = 3.7 X 1010 becquerels) was from New England Nuclear. Jack bean a-mannosidase type III and Streptomyces griseus protease type VI were purchased from Sigma and endo-,B3N-acetylglucosaminidase H (endo H), from Miles. Isolation of Lipid-Bound Oligosaccharides. C. fasciculata cells (Anopheles isolate ATCC 11745) were grown in the medium described by Bacchi et aL (10) without agar at 28°C. Six hundred milliliters of culture containing about 2 g of cells (logarithmic phase) were cooled on ice and centrifuged at 2500 X g for 10 min at 4°C. The cell pellet was resuspended in 30 ml of ice-cold minimal Eagle's medium without glucose but containing 5...

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confidence: 99%

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confidence: 99%

Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.

Parodi¹,

Allué²,

Cazzulo³

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…In plants, although the biosynthesis of lipid-linked oligosaccharides (8) and the formation of the final product Glc3Man9GlcNAc2-P-P-lipid (17,26,33), have been demonstrated, so far no studies have been made on the characterization or the purification of any of the individual enzymes involved in this pathway. The study of individual enzymes is necessary in order to know the regulation and control points of this pathway.…”

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

Kaushal¹,

Elbein²

1986

Plant Physiol.

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The GJcNAc-l-P-transferase was solubilized from microsomal preparations of soybean cultured cells by treatment with 1% Triton X-100. The solubilized enzyme catalyzed the formation of dolichyl pyrophosphoryl-GlcNAc when incubated with UDP-GlcNAc and dolichyl phosphate. The GlcNAc-l-P-transferase activity was stimulated by the addition of phosphatidylglycerol and phosphatidylinositol, but was inhibited by phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The K. value for dolichyl-phosphate was 6.2 micromolar and that determined for UDP-GlcNAc was 0.42 micromolar. The pH optimum for the GIcNAc-l-P reaction was between 7.2 and 7.6; maximum activity occurred at about 10 millimolar Mg2". The addition of unlabeled GDPmannose or UDP-glucose considerably inhibited enzyme activity which could be restored to nearly the original value by addition of more dolichyl phosphate to the incubation mixture. On the other hand, the addition of unlabeled ADP-glucose and GDP-glucose enhanced the enzyme activity.This stimulation by these sugar nucleotides was found to be due to the protection of the substrate UDP-1H1-GlcNAc from pyrophosphatase degradation. The GlcNAc-l-P-transferase reaction was very sensitive to tunicamycin and 50% inhibition required less than 1 microgram of antibiotic per milliliter. Amphomycin, showdomycin, and diumycin also inhibited this reaction but at higher concentrations.

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“…Thus, few of the oligosaccharides have been isolated or characterized, nor are the steps in the synthesis known. Re-cently, however, an oligosaccharide-lipid with the composition Glc3Man9GlcNAc2 was identified in several plant tissues (18,27).…”

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

Hori

Elbein²

1982

Plant Physiol.

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Lipid-linked oligosaccharides were synthesized with the particulate enzyme preparation from mung bean (Phaseols aureus) seedlngs in the presence of GDP-1J4C1 mannose. The oligosaccharides were released from the lipids by mild acid hydrolysis and purified by several passages on Biogel P4 columns. Five different oUgosacchardes were purified in this way.Based on their relative elution constants (Kd) compared to a variety of standard oligosaccharides, they were sized as (mannose-acetylglucosamine) Man7GlcNAc2, Man5GIcNAc2, Man3GlcNAc2, Man2GlcNAc2, and ManGlcNAc2. These oUgosaccharides were treated with endoglucosaminidase H and a-and 8i-mannosidase, and the products were examined on Biogel P4 columns. They also were subjected to a number of chemical treatments including analysis of the reducing sugar by NaB3H4 reduction, methylation analysis, and in some cases acetolysis. From these data, the likely structures of these oligosaccharides are as foUows: E, Man,BGlcNAc-GlcNAc; D, Mana1-*3Manfi-GlcNAc-GlcNAc; C, Manal-+ 2Mana-1 3Man#-GlcNAc-GlcNAc; B, Manal-2Mana1-*2Mana1--* 3(ManaI-.6)Man,8-GlcNAc-GlcNAc; and A, Manal-2Mana1--+ 2Manal-3(Manal-3lManal-6I Manal--.6)Man8-GkcNAc-GlcNAc.The synthesis of the Man7GlcNAc2 was greatly diminished when tunicamycin (10 pg/ml) was added to the incubation mixtures.Many plant and animal glycoproteins have an oligosaccharide composed of mannose, N-acetylglucosamine (GlcNAc) and possibly other sugars, that is attached to the protein in a GlcNAcasparagine bond (10,17,32). The biosynthetic pathway for the formation of this oligosaccharide involves a series of membranebound glycosyl transferases that utilize dolichol-linked saccharide intermediates (10, 32). In the animal systems, it is now wellestablished that these enzymes catalyze the transfer of GlcNAc, mannose, and glucose to dolichyl-phosphate to form a dolichylpyrophosphoryl-oligosaccharide having the composition, Glc3Man9GlcNAc2 (5,20,22). This oligosaccharide is then transferred to the protein (6,7,24,28). Several of the intermediate lipid-linked oligosaccharides have been isolated from animal tissues and the oligosaccharides have been characterized (4,15,32,33). Plant cells also contain glycoproteins having oligosaccharides attached in GlcNAc--asparagine bonds (10), and the structure of the oligosaccharide of soybean lectin has been shown to be a highmannose type (21).Although the general pathway of assembly of the lipid-linked saccharides of plant cells is known (1-3, 8, 13, 19) The reactions were terminated by the addition of 2 ml of CHC13:CH30H (1:1) and 0.5 ml of H20. The mixture was stirred vigorously, and the layers were separated by centrifugation. The lower CHC13 layer was removed and saved, and the aqueous phase and interface were extracted with another 1 ml of CHC13. The combined CHC13 phases, which contained lipid-linked monosaccharides and lower mol wt lipid-linked saccharides, were extracted with CHC13:CH30H:H20 (3:48:47) before being placed in scintil-12 www.plantphysiol.org on May 9, 2018 -Published by Dow...

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Presence in a plant of a compound similar to the dolichyl diphosphate oligosaccharide of animal tissue

Cited by 39 publications

References 10 publications

Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.

Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.

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

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

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