Metabolism of 3-deoxy-3-fluoro-D-mannose and 4-deoxy-4-fluoro-D-mannose by Saccharomyces cerevisiae S288C.

Incubation of Saccharomyces cerevisiae S288C with 4-deoxy-4-fluoro-D-[1-14C]-mannose resulted in the formation of three metabolites that were characterized as 4-deoxy-4-fluoro-D-[1-14C]mannose 1,6-bisphosphate, 4-deoxy-4-fluoro-D-[1-14C]-mannose 6-phosphate and GDP-4-deoxy-4-fluoro-D-[1-14C]mannose. In addition, radioactive material was incorporated into a particulate fraction composed primarily of cell-wall polysaccharides. Compared with the 4-fluoro sugar, 3-deoxy-3-fluoro-D-[1-14C]mannose was not transported into yeast cells as well, and its conversion into sugar nucleotide was much less efficient. Metabolites that were isolated after incubation with the 3-fluoro analogue were identified as 3-deoxy-3-fluoro-D-[1-14C]mannose 1,6-bisphosphate, 3-deoxy-3-fluoro-D-[1-14C]mannose 6-phosphate and GDP-3-deoxy-3-fluoro-D-[1-14C]mannose. Little radioactivity was transferred into the cell-wall fraction.     

Tridecaptin stimulates the formation of lipid-linked saccharides in aorta.

The peptide antibiotic tridecaptin caused a 2--4-fold stimulation in the incorporation of mannose from GDP-[14C]mannose and glucose from UDP-[3H]glucose into lipid-linked monosaccharides by both the particulate and the soluble enzyme fractions from pig aorta. In both cases, the major products and the ones stimulated by antibiotic were dolichyl phosphate mannose and dolichyl phosphate glucose. The stimulation in activity was unaffected by increasing concentrations of dolichyl phosphate, GDP-mannose, UdP-glucose, Mn2+ or the detergent Nonidet P40. Tridecaptin stimulation was apparently not due to protection of sugar nucleotide substrate, since addition of various concentrations of sugar nucleotides did not alter the stimulation. Nor did the addition of tridecaptin result in any increase in the amount of radioactive sugar nucleotide recovered from incubation mixtures. Tridecaptin bound to the particulate enzyme and could not be removed by centrifugation of the particles.     

In vivo and in vitro inhibition of lipid-linked saccharide and glycoprotein synthesis in plants by amphomycin

The effect of the polypeptide antibiotic, amphomycin, on the in vitro and in vivo synthesis of polyprenyl-linked sugars and glycoproteins in plants was examined. This antibiotic blocked the transfer of mannose from GDP-[¹⁴C]mannose into mannosyl-phos-phoryl-dolichol by a particulate enzyme preparation from mung beans and also inhibited the transfer of GlcNAc from UDP-[³H]GlcNAc to GlcNAc-pyrophosphoryl-polyisoprenol. The in vitro incorporation of these sugars into trichloroacetic acid-insoluble material was also markedly inhibited by this antibiotic. Since most of the radioactivity incorporated into this insoluble material is rendered water-soluble by treatment with pronase, it seems likely that these sugars are incorporated into glycoproteins whose synthesis is sensitive to amphomycin. Amphomycin also inhibited the transfer of glucose from UDP-[¹⁴C]glucose to steryl glucosides, although this system was less sensitive to antibiotic than was synthesis of the polyprenyl-linked sugars. The antibiotic did not block the in vitro transfer of glucose from UDP-[¹⁴C]glucose to β-glucans. In carrot slice cultures, amphomycin also inhibited the incorporation of [¹⁴C]mannose into glycolipid and glycoprotein, but it did not prevent the incorporation of [¹⁴C]lysine into protein.     

Bacitracin Inhibits the Synthesis of Lipid-linked Saccharides and Glycoproteins in Plants

The particulate enzyme fraction from mung bean (Phaseolus aureus) seedlings catalyzes the incorporation of mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol and of N-acetylglucosamine from UDP-[³H]N-acetylglucosamine into N-acetylglucosamine-pyrophosphoryl-polyisoprenol. Bacitracin inhibits the transfer of both of these sugars into the lipid-linked saccharides with 50% inhibition being observed at 5 mm bacitracin. This antibiotic did not inhibit the transfer of glucose from UDP-[¹⁴C]glucose into steryl glucosides or the incorporation of glucose into a cell wall glucan. Bacitracin also inhibited the in vivo incorporation of [¹⁴C]mannose into mannosyl-phosphoryl-dolichol and into glycoprotein by carrot (Daucus carota) slices. While bacitracin also inhibited the incorporation of lysine into proteins by these slices, protein synthesis was less sensitive than glycosylation. Thus at 2 mm bacitracin glycosylation was inhibited 92%, while protein synthesis was inhibited only 50%.     

A concanavalin A-resistant Chinese hamster ovary cell line is deficient in the synthesis of [3H]glucosyl oligosaccharide-lipid.

In this report we present an initial determination of the biochemical defect present in a Chinese hamster ovary cell line selected for resistance to concanavalin A. Membranes of this mutant, B211, incorporated at least 10-fold less mannose from GDP-[14C]mannose into oligosaccharide-lipid than membranes of the wild type. In the presence of dolichol phosphate, membranes of the mutant and wild type exhibited similar rates of synthesis of number of early intermediates, namely, mannosylphosphoryldolichol, N-acetylglucosaminyl- and N,N'-diacetylchitobiosylpyrophosphoryldolichol, glucosylphosphoryldolichol, and mannosyloligosaccharide-lipid. The membranes of B211 did not incorporate glucose from UDP-[3H]glucose into oligosaccharide-lipid or protein. Comparison by gel filtration chromatography of oligosaccharides derived from the oligosaccharide-lipids of B211 and wild type cells labeled with [2-3H]mannose revealed that B211 cells incorporated little if any label into an oligosaccharide corresponding to the most excluded oligosaccharide labeled by wild type cells. This concanavalin A-resistant cell line appears to lack the ability to glucosylate oligosaccharide-lipid.     

Glycosyltransfer by pea membranes from sugar nucleotides to added prenyl phosphates

Pea membranes supplied with GDP-[14C]mannose, UDP-N-[14C]acetylglucosamine or UDP-[14C]glucose catalyze the transfer of 14C-labeled sugars or sugar phosphates to endogenous lipid acceptors as well as to exogenously added dolichyl phosphates. Fully unsaturated polyprenyl phosphates were not used as effective acceptors by this system. Mannosyl-P-dolichol was formed most rapidly in the presence of long-chained dolichyl-P while mannosyl-PP-, glucosyl-PP- and GlcNAc-PP-dolichol were preferentially formed from relatively short-chained dolichyl phosphate acceptors. Glucosyl-PP- and mannosyl-PP-dolichol accumulated in the preparation without further metabolism, but GlcNAc-PP-dolichol was lengthened by addition of a second GlcNAc plus several [14C]mannose units to form an oligosaccharide fraction susceptible to the action of endoglycosidase H. This lipid-linked oligosaccharide could then be glycosylated in the presence of UDP-[14C]glucose to form a longer oligosaccharide. It is concluded that levels of endogenous dolichyl phosphates in pea membranes are rate-limiting for several of the key glycosyltransferases required for oligosaccharide assembly.     

Glycosylation in vitro of Semliki-Forest-virus and influenza-virus glycoproteins and its suppression by nucleotide-2-deoxy-hexose

Cell-free enzyme preparations from cultured fibroblasts infected with Semliki forest virus or fowl plague virus (an influenza A virus) incorporate [14C]-mannose into dolichol-phosphate-mannose, lipid-linked oligosaccharides and into endogenous virus-specific glycoproteins. When GDP-2-deoxy-D-[14C]glucose serves as substrate 2-deoxy-D-[14C]glucose is transferred to dolichol phosphate yielding dolichol-monophosphate-2-deoxy-D-[14C]glucose. UDP-2-deoxy-D-[14C]glucose gives rise also to a lipid which, however, is not a polyprenol derivative. The transfer of [14C]mannose to lipid-extractable fractions and glycoproteins in vitro is blocked by GDP-2-deoxy-D-glucose. It can be restored by exogenous dolichol monophosphate only with regard to the formation of dolichol-monophosphate-[14C]mannose-labelled oligosaccharides into glycoproteins. UDP-2-deoxy-D-glucose has no inhibitory effect on transfer reactions of [14C]mannose from GDP-[14C]mannose into various lipid fractions or into glycoprotein. It is concluded therefore, that the inhibition of glycosylation brought about by 2-deoxyglucose in vivo is caused by an interference of its GDP derivative with the formation of a correct lipid-oligosaccharide.     

Novel mannose carrier in the trypanosomatid Crithidia fasciculata behaving as a short alpha-saturated polyprenyl phosphate.

Crithidia fasciculata cells incubated with [14C]glucose or membranes derived from the same protozoan incubated with GDP-[14C]mannose were found to synthesize a lipid monophosphate mannose. No glucosylated mild acid-labile compound was formed in vivo or in vitro when UDP-[14C]glucose was used instead of GDP-[14C]mannose. The lipid moiety of the mannosyl derivative formed behaved as a polyprenol having 11 isoprene residues as judged by t.l.c. and be gel filtration in sodium deoxycholate-containing buffers. The mannolipid was not broken on treatment with hot phenol, suggesting the existence of an alpha-saturated isoprene unit. This is the first case reported in which a mannosyl phospholipid involved in sugar transfer in a eukaryotic cell behaves as if it was similar to that of bacterial polyprenols, although having its putative alpha-isoprene unit saturated to the same extent as dolichols from higher organisms.     

Amphomycin inhibits the incorporation of mannose and GlcNAc into lipid-linked saccharides by aorta extracts

Amphomycin inhibits the incorporation of mannose from GDP-[¹⁴C]mannose and GlcNac from UDP-[³H]GlcNAc into lipid-linked saccharides by either a particulate or a solubilized enzyme fraction from pig aorta. The solubilized enzyme was much more sensitive to the antibiotic than was the particulate fraction with 50% inhibition being observed at 8–15 μg of amphomycin. Although the antibiotic inhibited mannose transfer from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol, lipid-linked oligosaccharides and glycoprotein, the synthesis of mannosyl-phosphoryl-dolichol was much more sensitive to amphomycin. Amphomycin also inhibited the incorporation of mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryldecaprenol in particulate extracts of $\text{Mycobacterium smegmatis}$.     

Lipid-bound sugars in malignant human breast tumors

Microsomal preparations from malignant human breast tumors catalyzed the transfer of mannose and glucose from GDP-[14C]-Man and UDP-[14C]-Glc into lipid-linked sugars and glycoprotein-like substances. As judged by several criteria the obtained lipid-linked monosaccharides behaved as dolichyl phosphate mannose and dolichyl phosphate glucose whereas lipid-linked oligosaccharides behaved as polyprenyl diphosphate derivatives. The optimum conditions for mannosyl- and glucosyl-transfer reactions and the effect of dolichyl phosphate, detergent and EDTA on incubation mixture were described.     

Schistosoma mansoni: glycosyl transferase activity and the carbohydrate composition of the tegument.

Homogenates of adult Schistosoma mansoni contain enzymes which transferred [¹⁴C]mannose, [¹⁴C]glucose, and [¹⁴C]galactose from GDP-[U-¹⁴C]mannose, UDP-[U-¹⁴C]glucose, and UDP-[U-¹⁴C]galactose respectively to a lipid acceptor; in comparison, free [¹⁴C]mannose, GDP-[U-¹⁴C]fucose, and UDP-[U-¹⁴C]acetyl-glucosamine were poorly transferred. The lipid acceptor is believed to be an intermediate in the glycosylation of the worm's glycoproteins and in the biosynthesis of oligosaccharides and glycolipids. The tegument of adult worms was isolated by the freeze-thaw procedure and sugars associated with macromolecules in this fraction were analyzed; the major monosaccharide components were glucose, galactose, and mannose. These results suggest that the mechanism of glycosylation of the adult schistosome's tegumental macromolecules may occur through the glycosyl transferase system. The schistosome mannosyl transferase (EC 2.4.1), which is membrane bound was solubilized with 0.1% Triton X-100 without loss of activity; after density gradient centrifugation there was a peak of enzymic activity in a region of density 1.08, which could not be associated with any particular organelle.     

Solubilization and properties of mannose and N-acetylglucosamine transferases involved in formation of polyprenyl-sugar intermediates.

A particulate fraction from porcine aorta catalyzed the incorporation of N-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc into both GlcNAc-pyrophosphorylpolyprenol and GlcNAc-GlcNAc-pyrophosphorylpolyprenol. This transfer utilized endogenous lipid and required a divalent cation. Mn2+ was the best metal ion and was optimum at 2.3 mM. This same particulate fraction was previously shown to transfer mannose from GDP-[14C]mannose to endogenous lipid to form mannosylphosphorylpolyprenol (Chambers, J., and Elbein, A.D. (1975) J. Biol. Chem. 250, 6904-6915). Both the GlcNAc activities and the mannose activity were solubilized by treatment of the particulate fraction with the detergent Nonidet P-40. The enzymes were partially purified by chromatography on DEAE-cellulose and on Sephadex G-200. These soluble enzymes required the addition of acceptor lipid for activity. An acidic lipid fraction, isolated from pig liver and having the properties of dolichyl phosphate, was active with either the GlcNAc or the mannose transferase. Chemically synthesized dolichyl phosphate was also active with either of these enzymes. The products formed from either GlcNAc or mannose by the soluble transferases were similar to those formed by the particulate enzyme. Thus the major product formed from UDP-[3H]GlcNAc was GlcNAc-pyrophosphoryldolichol with small amounts of the disaccharide-lipid while the product formed from GDP-[14C]mannose was mannosylphosphoryldolichol.     

Incorporation of mannose and glucose into prenylphosphate sugars in isolated human platelet membranes.

Isolated platelet membranes synthesize mannosylretinyl phosphate and dolichylmannosyl phosphate from GDP-[14C]mannose, but only dolichylglucosyl-phosphate is synthesized from UDP-[14C]glucose. Addition of exogenous retinylphosphate specifically stimulates the biosynthesis of mannosylretinylphosphate.     

Glycoproten biosynthesis in Trypanosoma brucei. The glycosylation of Glycoproteins located in and attached to the plasma membrane

1. Glycosyltransferase activity incorporating N-[14C]acetylglucosamine ([14C]GlcNAc) from uridine diphosphate N-[14C]acetylglucosamine (UDP-[14C]GlcNAc) into endogenous proitein acceptors was localized primarily in the plasma membrane of Trypanosoma brucei. 2. The acceptor site for the nucleotide sugar was further localized exclusively to the cytoplasmic face of the plasma membrane. 3. The glycosyltransferase produced elongation of the growing oligosaccharide chains while they were attached to their peptide acceptors. 4. This glycosyltransferase activity was incapable of initiating sugar attachment directly to amino acid residues within peptide acceptors. 5. The dolichyl-phosphate-sugar pathway for glycoprotein biosynthesis was either absent of only present at a very low level in T. brucei when compared to rat liver. 6. All oligosaccharide chains accepting GlcNAc were of the same or very similar lengths. 7. Both O-glycosidic (26%) and N-glycosidic (74%) linkages (exclusive of hydroxylysine attachment) were found. 8. Glycosyltransferase activity required either Mn2+ or Mg2+, had a pH optimum of 6.5 and was temperature-dependent. 9. The kinetics of incorporation were complex, probably a result of multiple acceptors or glycosyltransferases whose activities were characterized by a Km of 30 microM for UDP-GlcNAc with a V of 40 pmol x mg protein -1 x min-1 for the highest affinity system and a Km of approximately 2 mM for UDP-GlcNAc with a V of approximately 400 pmol x mg protein-1 x min-1 for the lowest affinity system. 10. Glycosyltransferases using UDP-GlcNAc, uridine diphosphate glucose, uridine diphosphate galactose and guanidine diphosphate mannose as glycosyl donors were observed. Each peptide acceptor was specific for a singloe labelled sugar in the absence of other unlabelled nucleotide sugars. 11. The final extent of incorporation of GlcNAc was due primarily to exhaustion of peptide acceptor. 12. An inhibitor of UDP-[14C]GlcNAc incorporation into plasma membranes was found in the cytoplasmic fraction.     

Evidence that the lipid carrier for N-acetylglucosamine is different from that for mannose in mung beans and cotton fibers

Cell-free enzyme particles from mung beans (Phaseolus aureus) or cotton (Gossypium hirsutum L.) fibers catalyze the incorporation of mannose from GDP-[¹⁴C]mannose and N-acetylglucosamine from UDP-[³H]-N-acetylglucosamine into polyprenyl-type lipids. These lipids have been synthesized and purified and the lipid moieties compared to each other as well as to dolichyl phosphate and to lipids isolated from similar mannoseand N-acetylglucosamine-containing lipids from liver and aorta.

The following lines of evidence indicate that in plants, the lipid carrier for N-acetylglucosamine is different from the lipid carrier for mannose: [List: see text]

We propose that the apparent difference in the lipid carrier for these two sugars may be a point of control of glycoprotein synthesis.

     

Arrangement of glucose residues in the lipid-linked oligosaccharide precursor of asparaginyl oligosaccharides.

The lipid-linked oligosaccharide synthesized in vitro, in the presence of 1.0 microM UDP-[3H]Glc, GDP-[14C]Man, and UDP-GlcNAc has been isolated and the structure of the oligosaccharide has been analyzed. The oligosaccharide contains 2 N-acetylglucosamine, 9 mannose, and 3 glucose residues. The N-acetylglucosamine residues are located at the reducing terminus. The 3 glucose residues are arranged in a linear order at one of the nonreducing termini in the sequence Glc 1,2--Glc 1,3--Glc--(Man)9 (GlcNAc)2. The structural analysis was made possible largely by the availability of glucosidase preparations of fungal anad microsomal origin which remove glucose residues from the oligosaccharide without releasing mannose residues.     

The assembly of lipid-linked oligosaccharides in plant and animal membranes

Membrane preparations from growing regions of pea stems and activelydividing mouse L-cells form lipid-linked saccharides from GDP-mannose and UDP-N-acetylglucosamine. These lipids have properties which are consistent with those of mono-and di-phosphoryl polyisoprenyl derivatives. In experiments using plant membranes, the monophosphoryl derivative labeled with GDP-(¹⁴C) mannose contains mannose only, while the diphosphoryl derivative labeled with the same nucleotide sugar is heterogeneous, containing oligosaccharides corresponding to mannosaccharides of 5, 7, and 9-12 residues. Only the diphosphoryl polyisoprenyl derivatives are labeled with UDP-(¹⁴C)glucosamine and these contain predominantly chitobiose and N-acetylglucosamine itself. Unlabeled GDP-mannose added after UDP-N-acetyl (¹⁴C)glucosamine results in the formation of higher lipid-linked oligosaccharides which are apparently the same as those which are labeled with GDP-(¹⁴C)mannose alone. Incubation of the membranes with GDP-(¹⁴C)mannose in the presence of Mn²⁺, unlabeled UDP-glucose or unlabeled UDP-N-acetylglucosamine results in marked changes in the accumulation of both the polyisoprenyl monophosphoryl mannose and polyisoprenyl diphosphoryl oligosaccharides. Animal cell membranes synthesise lipid-linked oligosaccharides when incubated with UDP-N-acetylglucosamine and GDP-mannose. These oligosaccharides are similar in size to those synthesised by the plant membranes but their formation is more efficient. The potential roles of these compounds in glycoprotein biosynthesis in both plant and animal tissues is discussed.     

Synthesis and rapid purification of UDP-[6-3H]galactose

UDP[6-3H]galactose of high specificity can be obtained by oxidation of the C-6 hydroxymethyl group of UDP-galactose by galactose oxidase and subsequent reduction by sodium borotritide. One-step purification of the nucleotide sugar involves anion-exchange chromatography on a Pharmacia Mono Q column. Radiolabeled UDP-N-acetylgalactosamine can also be synthesized and purified by this procedure. Both nucleotide sugars can be used for sugar incorporation studies using the appropriate glycosyltransferase.     

Formation of lipid-linked oligosaccharides by MOPC 315 plasmacytoma cells. Decreased synthesis by a nonsecretory variant

MOPC 315 is a BALB/c plasmacytoma which secretes a trinitrophenol-binding IgA lambda 2 paraprotein. We have investigated the incorporation of [3H]mannose into lipid-linked oligosaccharide precursors in wild-type MOPC 315/J and variant nonsecretory 315/P cells. In pulse labeling experiments, no differences could be detected in the ability of the two cell types to incorporate [3H]mannose into lipid-linked oligosaccharides containing 5 or less mannose residues. In contrast, quantitation of the incorporation of [3H]mannose into larger lipid-linked oligosaccharides and proteins revealed a 49 and 40% decrease, respectively, in the 315/P cells compared to wild-type cells. Further characterization of the lipid-linked structures documented a marked decrease in glucosylated oligosaccharides isolated from 315/P cells. When membranes from the two cell lines were analyzed for their ability to transfer [3H]glucose from UDP-[3H]glucose to [3H]glucosylphosphoryldolichol, an apparent deficiency was noted in the 315/P preparations. However, if assay conditions were adjusted to include AMP in the reaction mixtures, no differences in the in vitro synthesis of [3H]glucosylphosphoryldolichol or [3H]glucose-labeled oligosaccharide-lipid could be detected. In these reactions AMP was found to prevent hydrolysis of UDP-[3H]glucose by inhibiting nucleotide pyrophosphatase (EC 3.6.1.9), the specific activity of which was determined to be more than 100 times greater in variant 315/P compared to wild-type MOPC 315/J cells. This large difference in specific activity was not accompanied by similar differences in the activity of several other enzymes analyzed. A decrease in whole cell UDP-glucose pool size was not detected in 315/P cells. Therefore, if nucleotide pyrophosphatase is important for the control of substrates for glycosylation, it must regulate nucleotide sugar levels at a site other than the cytoplasm of cells, perhaps at the location of synthesis of the larger lipid-linked oligosaccharides.     

Incorporation of mannose and glucose into prenylphosphate sugars in isolated human platelet membranes

Isolated platelet membranes synthesize mannosylretinyl phosphate and dolichylmannosyl phosphate from GDP-[¹⁴C]mannose, but only dolichylglucosyl-phosphate is synthesized from UDP-[¹⁴C]glucose.Addition of exogenous retinylphosphate specifically stimulates the biosynthesis of mannosylretinylphosphate.