Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo.

Influenza-virus-infected cells were labelled with radioactive sugars and extracted to give fractions containing lipid-linked oligosaccharides and glycoproteins. The oligosaccharides linked to lipid were of the 'high-mannose' type and contained glucose. In the glycoprotein fraction, radioactivity was associated with virus proteins and found to occur predominantly in the 'high-mannose' type of glycopeptides. In the presence of the inhibitors 2-deoxy-D-glucose, 2-deoxy-2-amino-D-glucose (glucosamine), 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose incorporation of radiolabelled sugars into lipid- and protein-linked oligosaccharides was decreased. Kinetic analysis showed that the inhibitors affected first the assembly of lipid-linked oligosaccharides and then protein glycosylation after a lag period. During inhibition by deoxyglucose and the fluoro sugars lipid-linked oligosaccharides were formed that contained oligosaccharides of decreased molecular weight. No such aberrant forms were found during inhibition by glucosamine. In the case of inhibition by deoxyglucose it was shown that the aberrant oligosaccharides were not transferred to protein. Inhibition of formation of lipid-linked oligosaccharides by deoxyglucose and fluoro sugars was antagonized by mannose, in which case oligosaccharides of normal molecular weight were formed. The inhibition by glucosamine was reversed by its removal from the medium. The reversible effects of these inhibitors exemplify their usefulness as tools in the study of glycosylation processes.     

CHO cells selected for phytohemagglutinin and con A resistance are defective in both early and late stages of protein glycosylation

The lipid-linked and asparagine-linked oligosaccharides of two lectin-resistant and one parental Chinese hamster ovary (CHO) cell line have been compared by glycosidase digestion and gel filtration analysis of radiolabeled glycopeptides and oligosaccharides. The additional glycosylation defect in a double mutant cell line (CHO-Pha^RConA^R) selected from a phytohemagglutinin-resistant single mutant cell line (CHO-Pha^R) for resistance to concanavalin A has been identified as a block in the synthesis of the lipid-linked oligosaccharide precursor, resulting in a structure with seven instead of the normal nine mannose units. Both the CHO-Pha^RConA^R and CHO-Pha^R cells were completely blocked in the synthesis of complex, acidic type oligosaccharides because of a previously demonstrated deficiency in a particular N-acetylglucosamine transferase activity. In addition, an altered collection of neutral type oligosaccharides (Man_4–7GIcNAc₂) accumulated in the glycoproteins of the double mutant.     

Deoxyglucose inhibition of protein glycosylation: Effects of nucleotide deoxysugars on the formation of glucosylated lipid intermediates

The UDP-derivative of deoxyglucose (UDP-deoxyglucose) inhibits the formation of dolichyl monophosphate glucose (Dol-P-glucose) in chick embryo cell membranes but has no effect on Dol-PP di-N-acetylchitobiose [Dol-PP-(GlcNAc)₂]formation. The effects of UDP-deoxyglucose are not reversed by Dol-P, nor is Dol-P-deoxyglucose formed from this derivative. GDP-deoxyglucose inhibits formation of both Dol-P-glucose and Dol-PP-(GlcNAc)₂. It is shown that GDP-deoxyglucose inhibits in these cases by competition with physiological nucleotide sugars for Dol-P. GDP-deoxyglucose and UDP-deoxyglucose also prevent the attachment of the peripheral glucose residues in Dol-PP-(GlcNAc)₂-Mans_yGlc₃, the immediate precursor of protein-bound oligosaccharides. The inhibition by GDP-deoxyglucose is only in part reversed by Dol-P, probably because deoxyglucose is incorporated into the lipid-linked oligosaccharide instead of glucose.     

Fluoroglucose-inhibition of protein glycosylation in vivo. Inhibition of mannose and glucose incorporation into lipid-linked oligosaccharides

The effects of the glycosylation inhibitor 2-deoxy-2-fluoro-D-glucose on the formation of the lipid-linked oligosaccharides and monosaccharides that are involved in protein glycosylation were investigated. In chick embryo cells treated with fluoroglucose the formation of lipid-linked oligosaccharides cannot go to completion and oligosaccharides with decreased amounts of glucose and mannose can be detected. These oligosaccharides are probably biosynthetic intermediates and serve as acceptors of sugar residues while reversing fluoroglucose-inhibition by the addition of mannose and glucose to the culture medium. In contrast to deoxyglucose, fluoroglucose was not incorporated into lipid-linked oligosaccharides. Fluoroglucose inhibits the formation in vivo of dolichyl phosphate glucose and dolichyl phosphate mannose, but not the transfer of those sugar residues from the lipid monophosphate derivative to the lipid-linked oligosaccharides. The pool size of UDP-glucose, but not of GDP-mannose and UDP-N-acetylglucosamine, was decreased. Also, the formation of lipid-linked N-acetylglucosamine was not affected by fluoroglucose. Fluoroglucose was applied to deplete cellular membranes of endogenous lipid-linked mannose and glucose, and can possibly be used to discern different pathways of glycosylation.     

Inhibition of beta-1,4-Glucan Biosynthesis by Deoxyglucose : The Effect on the Glucosylation of Lipid Intermediates

GDP- and UDP-deoxyglucose inhibit the incorporation of glucose from UDP-glucose into dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides. GDP-deoxyglucose inhibits by competing with the physiological nucleotide sugars for dolichyl phosphate, and dolichyl phosphate deoxyglucose is formed. This inhibition is reversed by excess of dolichyl phosphate. UDP-deoxyglucose does not give rise to a lipid-linked derivative, and inhibition by this analog is not reversed by dolichyl phosphate. The UDP- and GDP-derivatives of deoxyglucose inhibit the incorporation of glucose into glucose-containing glycoproteins. This effect seems to be the result of the inhibition of lipid intermediates glucosylation and is comparable to the effect produced by coumarin. Cellulose synthetase activity is not affected by UDP- or GDP-deoxyglucose. On the other hand, deoxyglucose inhibits the formation of β-1,4-glucans in vivo.     

Inhibition of protein synthesis also inhibits synthesis of lipid-linked oligosaccharides.

Studies were initiated to determine whether the formation of lipid-linked oligosaccharides was coupled to the synthesis of protein. Canine kidney cells were grown with [2-3H]mannose or [3H]leucine in the presence of cycloheximide or puromycin and the effect of these inhibitors on the synthesis of proteins and lipid-linked oligosaccharides was measured. In all cases, the inhibition of protein synthesis resulted in a substantial inhibition in the incorporation of mannose into the lipid-linked oligosaccharides, although the synthesis of mannosyl-phosphoryl-dolichol was only slightly inhibited. Cycloheximide had no effect on the in vitro incorporation of mannose into lipid-linked oligosaccharides when GDP-[14C]mannose was incubated with aorta microsomal preparations. The inhibition of lipid-linked oligosaccharides was apparently not due to a decrease in the amount of glycosyltransferases as a result of protein degradation in the absence of protein synthesis, nor was it the result of a more rapid degradation of lipid-linked oligosaccharides. The inhibition also did not appear to be due to limitations in the available dolichyl-phosphate. The results suggest that the formation of lipid-linked oligosaccharides may be regulated by end product inhibition.     

Inhibition of lipid-linked saccharide synthesis by bacitracin.

The antibiotic bacitracin was found to inhibit the incorporation of mannose and GlcNAc from their respective sugar nucleotides into lipid-linked saccharides. The inhibition of both systems was apparent in the aorta particulate enzyme system but it was much more pronounced with the solubilized enzyme system. In both cases, GlcNAc incorporation into Dol-P-P-GlcNAc was more sensitive than mannose incorporation into Dol-P-Man, with 50% inhibition being seen at about 0.1–0.2 mm antibiotic. Bacitracin inhibition of mannose incorporation appeared to be overcome at high concentrations of dolichyl phosphate but, in these cases, an unexplained stimulation was observed. However, GlcNAc inhibition could not be overcome by high concentrations of dolichol phosphate, metal ion, or both together. Thus, the mechanism of inhibition by bacitracin is not clear. Bacitracin also inhibited the transfer of mannose from GDP-mannose to lipid-linked oligosaccharides and to glycoprotein in the particulate enzyme, as well as the transfer of radioactivity from Dol-P-Man or from lipid-linked oligosaccharides to glycoprotein. Thus, bacitracin apparently blocks each of the steps in the lipid-linked pathway. In yeast spheroplasts, bacitracin inhibited the incorporation of [¹⁴C]mannose into Dol-P-Man, into lipid-linked oligosaccharides, and into glycoprotein. However, in this case, the antibiotic also blocked the incorporation of leucine into protein. Bacitracin also inhibited the cell-free synthesis of mannosyl-phosphoryl-decaprenol in Mycobacterium smegmatis with 50% inhibition being observed at a concentration of about 0.5 mm.     

Effects of Several Tunicamycin-like Antibiotics on Glycoprotein Biosynthesis in Mung Beans and Suspension-cultured Soybean Cells

The antibiotics Streptovirudin and 24010 were tested to determine their effects on the formation of lipid-linked saccharide intermediates associated with glycoprotein biosynthesis in mung bean (Vigna radiata) and suspension-cultured soybean cells (Glycine max cv. Mandarin). In vitro both compounds strongly inhibited the transfer of N-acetyl[³H]glucosamine from UDP-N-[³H]acetylglucosamine to N-acetylglucosaminyl-pyrophosphoryl-polyisoprenol and lipid-linked oligosaccharides, although they had no apparent effect on the incorporation of [¹⁴C]mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol with a small inhibition into lipid-linked oligosaccharides. In vivo, Streptovirudin and tunicamycin dramatically inhibited the incorporation of N-[¹⁴C]acetylglucosamine and [³H]mannose into Pronase-released material (glycoproteins), whereas there was no effect on [³H]leucine incorporation into Pronase-released material (protein). Because the action of Streptovirudin and antibiotic 24010 in plants and other systems is similar to that for tunicamycin, these antibiotics are believed to be closely related. The use of tunicamycin is discussed with respect to its importance in studying glycoprotein biosynthesis and function in animal and plant systems.     

Structure of the lipid-linked oligosaccharides that accumulate in class E Thy-1-negative mutant lymphomas.

Studies reported in the preceding paper (Trowbridge and Hyman, 1979) have demonstrated that Thy-1^? mutant lymphoma cells of the class E complementation group lack the normal high molecular weight lipid-linked oligosaccharide, but instead accumulate two smaller species termed I and II. This paper reports studies which elucidate the structures of lipid-linked oligosaccharides I and II. By subjecting oligosaccharides radiolabeled with ³H-mannose, ³H-glucose or ³H-glucosamine to methylation, acetolysis, periodate oxidation and exoglycosidase digestion, the structures were shown to be: where R = GlcNac B1,4(3) GlcNAc. A comparison of I and II with lipid-linked oligosaccharides from normal Chinese hamster ovary cells indicates that both I and II are normal biosynthetic intermediates. On the basis of these data we suggest that the defect in the class E mutant cells is the lack of an α1,3 mannosyltransferase involved in the conversion of the Man₅GlcNAc₂ lipid-linked oligosaccharide to the Man₆GlcNAc₂ intermediate. It is also impossible that the same enzyme is involved in conversion of the Glc₃Man₅GlcNAc₂ lipid-linked oligosaccharide to Glc₃Man₆GlcNAc₂. The latter reaction, however, has not yet been demonstrated in normal cells.     

Isolation of a temperature-sensitive FM3A mutant deficient in asparagine-linked glycosylation by selecting for resistance to tritiated mannose suicide

Protein glycosylation mutants in the mouse mammary carcinoma cell line FM3A were selected for ability to withstand exposure to [2-3H]mannose at 39 degrees C. G258 , one of the mutant cells isolated, has been characterized. G258 cells were temperature-sensitive for cell growth. Moreover, G258 cells showed temperature sensitivity for [3H]mannose incorporation into the TCA-insoluble fraction. To study the biochemical basis of the defect in glycoprotein biosynthesis, the formation of lipid-linked saccharides was examined. The results showed that the formation of lipid-linked oligosaccharides was severely inhibited in G258 cells at 39 degrees C. At 33 degrees C, G258 cells synthesized Glc3Man9GlcNAc2-PP-Dol, the fully assembled lipid-linked oligosaccharides, but at 39 degrees C, G258 cells were able to synthesize merely the smaller lipid-linked oligosaccharides (approximately up to Man3GlcNAc2 -PP-Dol), but were unable to synthesize the larger lipid-linked oligosaccharides.     

Tunicamycin inhibits protein glycosylation in suspension cultured soybean cells

Soybean cells in suspension culture incorporate [³H]mannose into dolichyl-phosphoryl-mannose and into lipid-linked oligosaccharides as well as into extracellular and cell wall macromolecules. Tunicamycin completely inhibited the formation of lipid-linked oligosaccharides at a concentration of 5 to 10 micrograms per milliliter, but it had no effect on the formation of dolichyl-phosphoryl-mannose. Tunicamycin did inhibit the incorporation of [³H]mannose into cell wall components and extracellular macromolecules, but even at 20 micrograms per milliliter of antibiotic there was still about 30% incorporation of mannose. The radioactivity in these macromolecules was localized in mannose (70%), rhamnose (20%), galactose (8%), and fucose (2%) in the absence of antibiotic. But when tunicamycin was added, very little radioactive mannose was found in cell wall or extracellular components. The incorporation of [³H]leucine into membrane components and [¹⁴C]proline into cell wall components by these suspension cultures was unaffected by tunicamycin. However, tunicamycin did inhibit the appearance of leucine-labeled extracellular macromolecules, probably because it prevented their secretion.     

Abnormal lipid-linked oligosaccharides in class E Thy-1-negative mutant lymphomas.

The glycosylation defect of Thy-1-mutant lymphomas of the class E complementation group has been identified as a block in the synthesis of the lipid-linked oligosaccharide precursor of the asparagine-linked oligosaccharides of glycoproteins. Two major lipid-linked oligosaccharides were isolated from the mutant cells. Both oligosaccharides were smaller than the lipid-linkid oligosaccharides of wild-type lymphomas and, in contrast to the lipid-linked oligosaccharides isolated from wild-type cells, both were resistant to digestion with endoglycosidase H. The oligosaccharides of newly synthesized polypeptides in class E Thy-1-cells were also resistant to endoglycosidase H digestion, providing strong evidence that they are derived from the abnormal lipid-linked oligosaccharides.     

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 $\text{Mycobacterium smegmatis}$.     

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.     

Inhibition of the formation of lipid-linked intermediates in normal and transformed cells by a purified tunicamycin homologue

Summary The deffects of a purified homologue of tunicamycin (B₂-tunicamycin) on the biosynthesis of lipid-linked intermediates participating in protein glycosylation in normal embryonic fibroblasts, 3T3 and virally transformed (simian virus 40 and polyoma virus) mouse fibroblasts grown in culture were investigated. Long incubations (20 h) with the antibiotic caused a higher degree of inhibition of sugar incorporation into glycoproteins in transformed cells. However, the formation of lipid-linked intermediates was inhibited to a similar level in both cell types. When time dependent inhibition experiments were carried out using transformed cells, an earlier and stronger inhibition of the formation of lipid-oligosaccharides occurred (70% inhibition at 30 min). In 3T3 cells, prolonged incubation (6–8 h) was necessary in order to reach a similar degree of inhibition. Formation of lipid-sugar was also inhibited to a greater extent by B₂-tunicamycin in transformed cells. This inhibition was not clearly time dependent. Analysis of the newly synthesized glycolipids in 3T3 and in transformed cells after B₂-tunicamycin treatment have shown reduction in dolichyl-P-P-sugars as well as in other glycolipids. Dimethylsulfoxide (10%) and linoleic acid (0.5 mg/ml) markedly increased the level of tunicamycin activity in 3T3 cells while phosphatidylcholine (2 mg/ml) partially reversed it. The stronger and faster inhibition of the formation of lipid intermediates of the dolichyl-phosphate cycle caused by B₂-tunicamycin in transformed cells, described here for the first time, may therefore be due to differences in penetration of the antibiotic into these cells.Abbreviations DMEM Dulbecco's modified Eagle's medium - DMSO dimethylsulfoxide - MF mouse fibroblasts from Balb/c mouse embryos - 3T3 Balb/3T3 mouse fibroblastic line - SV40 Simian virus 40 - PY polyoma virus - TLC thin layer chromatography     

Biosynthesis of mannose-containing lipid-linked oligosaccharides by solubilized enzyme preparation from cultured soybean cells

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°C for 1 month. 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. 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 Man₃GlcNAc₂-lipid and Man₅GlcNAc₂-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 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, it appears likely that the first five mannose residues to form Man₅GlcNAc₂-lipid come directly from GDP-mannose, whereas other mannose units to form larger oligosaccharide-lipids come from dolichyl-phosphoryl-mannose.     

Deoxyglucose transport of uninfected, murine sarcoma virus-transformed, and murine leukemia virus-infected mouse cells

The initial rates of deoxy-D-glucose transport by cultures of growing and density-inhibited mouse embryo cells and lines of mouse cells transformed spontaneously or after infection by murine leukemia virus or murine sarcoma virus were investigated as a function of the deoxyglucose concentration. The apparent K_m for deoxyglucose transport was about the same for all types of cells (1–2 mM). The V_max of secondary cultures of mouse embryo cells decreased from 6 nmoles/10⁶ cells/minute for sparse cultures to less than 1 nmole/10⁶ cells/minute for density-inhibited cultures. The V_max was about the same whether estimated in monolayer culture or in suspensions of cells dispersed by treatment with trypsin. The V_max for deoxyglucose transport by the established cells, whether transformed spontaneously or by virus infection, was 4 to 25 times higher than that for density-inhibited mouse embryo cells and was independent of the cell density of the cultures. Deoxyglucose transport was competitively inhibited by Cytochalasin B, Persantin, glucose and 3-O-methyl-D-glucose and the apparent K_i values of inhibition were similar for the mouse embryo cells and the various cell lines. Similarly, the sensitivity of the glucose transport systems to inactivation by p-chloromercuribenzoate was about the same for all types of cells. The results suggest that the glucose transport system of the normal mouse embryo cells and the cells of the various established lines is qualitatively the same, but that the number of functional transport sites differs for the various cell lines and decreases markedly in mouse embryo cells with an increase in cell density of the cultures.     

The effect of mannosamine on the formation of lipid-linked oligosaccharides and glycoproteins in canine kidney cells

Madin-Darby canine kidney (MDCK) cells normally form lipid-linked oligosaccharides having mostly the Glc3Man9GlcNAc2 oligosaccharide. However, when MDCK cells are incubated in 1 to 10 mM mannosamine and labeled with [2-3H]mannose, the major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 structures. Since both of these oligosaccharides were susceptible to digestion by endo-beta-N-acetylglucosaminidase H, the Man5GlcNAc2 must be different in structure than the Man5GlcNAc2 usually found as a biosynthetic intermediate in the lipid-linked oligosaccharides. Methylation analysis also indicated that this Man5GlcNAc2 contained 1----3 linked mannose residues. Since pulse chase studies indicated that the lesion was in biosynthesis, it appears that mannosamine inhibits the in vivo formation of lipid-linked oligosaccharides perhaps by inhibiting the alpha-1,2-mannosyl transferases. Although the lipid-linked oligosaccharides produced in the presence of mannosamine were smaller in size than those of control cells and did not contain glucose, the oligosaccharides were still transferred in vivo to protein. Furthermore, the oligosaccharide portions of the glycoproteins were still processed as shown by the fact that the glycopeptides were of the complex and hybrid types and were labeled with [3H]mannose or [3H]galactose. In contrast, control cells produced complex and high-mannose structures but no hybrid oligosaccharides were detected. The inhibition by mannosamine could be overcome by adding high concentrations of glucose to the medium.     

Incorporation of glucose into lipid-linked saccharides in aorta and its inhibition by amphomycin

The particulate enzyme from pig aorta catalyzed the transfer of glucose from UDP-glucose into glucosyl-phosphoryl-dolichol, into lipid-linked oligosaccharides, and into glycoprotein. Radioactive lipid-linked oligosaccharides were prepared by incubating the extracts with GDP-[¹⁴C]mannose and UDP-[³H]glucose. When the labeled oligosaccharides were run on Bio-Gel P-4, the two different labels did not exactly coincide; the ³H peak eluted slightly earlier indicating that it was of higher molecular weight than the ¹⁴C material, but there was considerable overlap. The purified oligosaccharide(s) contained glucose, mannose, and N-acetylglucosamine but the ratios of these sugars varied from one enzyme preparation to another, probably depending on the endogenous oligosaccaride-lipids present in the microsomal preparation. Treatment of the [³H]glucose-labeled oligosaccharide with α-mannosidase gave rise to a ³H-labeled oligosaccharide which moved somewhat faster on Bio-Gel P-4 than the original oligosaccharide, suggesting it had lost one or two sugar residues. These data indicate that mannose and glucose are in the same oligosaccharide. The antibiotic, amphomycin, inhibited the transfer of glucose from UDP-glucose into the lipid-linked saccharides. However the synthesis of glucosyl-phosphoryl-dolichol was much more sensitive then was the synthesis of lipid-linked oligosaccharides. The glucose-labeled oligosaccharide produced in the absence of amphomycin was of high molecular weight based on paper chromatography. But in the presence of partially inhibitory concentrations of antibiotic, the oligosaccharide migrated more rapidly on paper chromatograms. However, amphomycin had no effect on the synthesis of glucosyl-ceramide by the aorta extracts. In fact, the antibiotic may stimulate glucosyl-ceramide by making more of the substrate, UDP-glucose, available for synthesis of this lipid.