Modulation of nucleotide pyrophosphatase in plasmacytoma cells.

The effect of glucocorticoid hormones on the protein responsible for both nucleotide pyrophosphatase (EC 3.6.1.9) and alkaline phosphodiesterase I (EC 3.1.4.1) activities was examined in murine MOPC 315 plasmacytoma cells. Incubation of these cells with dexamethasone resulted in parallel increases in pyrophosphatase and phosphodiesterase specific activities. The incorporation of [3H]mannose into N-linked oligosaccharide precursors was also analyzed in cells following hormone modulation. In cells treated for 36 hours or cultured continuously with dexamethasone, the resulting increase in enzyme specific activities was accompanied by a decrease in [3H]mannose incorporation, consistent with the hypothesis that in some cell types, nucleotide pyrophosphatase activity is involved in the regulation of glycoprotein synthesis.     

Glycoprotein biosynthesis in animal cells grown in suspension culture. Assembly of lipid-linked saccharides and formation of protein-bound ''high-mannose'' oligosaccharides.

Glycoprotein biosynthesis was studied with mouse L-cells grown in suspension culture. Glucose-deprived cells incorporated [3H]mannose into 'high-mannose' protein-bound oligosaccharides and a few relatively high-molecular-weight lipid-linked oligosaccharides. The latter were retained by DEAE-cellulose and turned over quite slowly during pulse--chase experiments. Increased heterogeneity in size of lipid-linked oligosaccharides developed during prolonged glucose deprivation. Sequential elongation of lipid-linked oligosaccharides was also observed, and conditions that prevented the assembly of the higher lipid-linked oligosaccharides also prevented the formation of the larger protein-bound 'high-mannose' oligosaccharides. In parallel experiments, [3H]mannose was incorporated into a total polyribosome fraction, suggesting that mannose residues were transferred co-translationally to nascent protein. Membrane preparations from these cells catalysed the assembly from UDP-N-acetyl-D-[6-3H]glucosamine and GDP-D-[U-14C]mannose of polyisoprenyl diphosphate derivatives whose oligosaccharide moieties were heterogeneous in size. Elongation of the N-acetyl-D-[6-3H]glucosamine-initiated glycolipids with mannose residues produced several higher lipid-linked oligosaccharides similar to those seen during glucose deprivation in vivo. Glucosylation of these mannose-containing oligosaccharides from UDP-D-[6-3H]glucose was restricted to those of a relatively high molecular weight. Protein-bound saccharides formed in vitro were mainly smaller in size than those assembled on the lipid acceptors. These results support the involvement of lipid-linked saccharides in the synthesis of asparagine-linked glycoproteins, but show both in vivo and in vitro that protein-bound 'high-mannose' oligosaccharide formation can occur independently of higher lipid-linked oligosaccharide synthesis.     

Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway

Two complementing mutations in lipid-linked oligosaccharide biosynthesis have been isolated following a [3H]mannose suicide enrichment. Rather than making the wild type precursor oligosaccharide, Glc3man9Glc-NA2-P-P-dolichol, the mutants, alg5-1 and alg6-1, accumulate Man9GlcNAc2-P-P-dolichol as their largest lipid-linked oligosaccharide in vivo and in vitro. When UDP-[3H]Glc was added to microsomal membranes of each mutant, neither could elongate Man9GlcNAc2-P-P-dolichol and only alg6-1 could synthesize dolichol-phosphoglucose. When dolicholphospho[3H]glucose was added to microsomes from alg5-1, alg6-1, or the parental strain, only alg5-1 and the parental strain made glucosylated lipid-linked oligosaccharides. These results indicate that alg5-1 cells are unable to synthesize dolichol phosphoglucose while alg6-1 cells are unable to transfer glucose from dolichol phosphoglucose to the unglucosylated lipid-linked oligosaccharide. We also present evidence that both mutants transfer Man9GlcNAc2 to protein.     

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.     

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.     

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.     

Glycoprotein biosynthesis in plants. Formation of lipid-linked oligosaccharides of mannose and N-acetylglucosamine by mung bean seedlings.

Cell-free enzyme particles from mung bean seedlings catalyze the incorporation of mannose from GDP-[¹⁴C]mannose and GlcNAc from UDP-[³H]GlcNAc into glycolipids and into glycoprotein. The most rapidly labeled product from GDP-mannose was characterized as a mannosyl-phosphoryl-polyisoprenol, whereas that from UDP-GlcNAc was a mixture of GlcNAc-(pyro)phosphoryl-polyisoprenol and a disaccharide composed of two N-acetylglucosamine residues attached to the polyisoprenol by a phosphoryl or pyrophosphoryl linkage. Radioactivity from GDP-mannose and UDP-GlcNAc was also incorporated into more polar lipids which have been partially characterized as a series of oligosaccharide-(pyro)phosphoryl-lipids. The mannose-labeled oligosaccharides released from these lipids by mild acid hydrolysis were found to contain GlcNAc at their reducing end indicating that these oligosaccharides contain both GlcNAc and mannose. Both the GlcNAc-labeled and the mannose-labeled oligosaccharides gave multiple radioactive peaks upon paper chromatography indicating that they are composed of a series of different sized oligosaccharides. Finally, radioactivity from GDP-[¹⁴C]mannose and UDP-[³H]GlcNAc is incorporated into an insoluble component. Ten percent of the mannose label and all of the GlcNAc label in this insoluble material could be solubilized by digestion with Pronase. The glycopeptides released by Pronase digestion appeared to be approximately the same size as the oligosaccharides from the lipid-linked oligosaccharides based on gel filtration chromatography on Sephadex G-50. The results are consistent with a mechanism for glycoprotein synthesis involving lipid-linked oligosaccharide intermediates.     

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.     

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.     

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.     

Studies of the biosynthesis of the mannose 6-phosphate receptor in receptor-positive and -deficient cell lines

Phosphomannosyl residues present on lysosomal enzymes are specifically recognized by the mannose 6-phosphate receptor protein. This interaction results in the selective targeting of lysosomal enzymes to lysosomes. While this pathway is operative in many cell types, we have found four cultured cell lines that are deficient in the ability to bind lysosomal enzymes containing phosphomannosyl residues to their intracellular or surface membranes (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). These cells appear to segregate lysosomal enzymes by an alternate intracellular pathway. To determine the basis for the lack of mannose 6-phosphate receptor activity in these cell lines, we studied the biosynthesis of the receptor in receptor-positive (BW5147) and receptor-deficient (P388D1 and MOPC 315) cells. The cells were labeled with [2-3H]mannose or [35S]methionine and the receptor was immunoprecipitated with an antireceptor antiserum. BW5147 cells synthesize a receptor protein whose size increases after translation/glycosylation. MOPC 315 cells produce an apparently normal receptor and degrade it rapidly. P388D1 cells fail to synthesize any detectable receptor. The receptor from BW5147 and MOPC 315 cells is a glycoprotein with both high mannose and complex asparagine-linked oligosaccharides. The complex-type units become fully sialylated and remain so during long periods of chase.     

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 $\text{Mycobacterium smegmatis}$.     

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.     

Metabolism of 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose in yeast and chick-embryo cells.

2-Deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose have been prepared by tritiation of the corresponding unlabeled 2-fluoro sugars. The tritiated 2-fluoro sugars are phosphorylated and activated by UTP and by GTP to yield UDP-2-deoxy-2-fluoro-D-[3H]glucose, UDP-2-deoxy-2-fluoro-D-[3H]mannose, GDP-2-deoxy-2-fluoro-D-[3H]glucose and GDP-2-deoxy-2-fluoro-D-[3H]mannose in both cell types. The nucleotide derivatives could also be labeled in the nucleotide moiety by feeding the cells with [14C]uridine or [14C]guanosine in the presence of unlabeled 2-fluoro sugar. No evidence was obtained for metabolic steps in which the six-carbon chain of 2-fluoro sugars was not preserved. No epimerisation of the label to 2-deoxy-2-fluoro-D-[3H]galactose could be observed by radioactive gas-liquid chromatography of the enzymatic cleavage products of the different 2-fluoro sugar metabolites isolated from either cell type. Yeast and chick embryo cells both incorporate 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose specifically into glycoproteins, although this incorporation is very low when compared to the incorporation of 2-deoxy-D-[3H]glucose.     

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.     

Studies of the biosynthesis of the cation-dependent mannose 6-phosphate receptor in murine cell lines

We have studied the biosynthesis of the cation-dependent mannose 6-phosphate receptor in murine BW5147 lymphoma cells and MOPC 315 plasmacytoma cells. The cells were labeled with [35S]methionine or [2-3H]mannose and the receptor immunoprecipitated with an anti-receptor antiserum. The receptor was first detected as a glycoprotein with an apparent molecular mass of 40 kDa. This intermediate was rapidly processed to a mature form which was stable during 22 h of chase. In these cells, the mature receptor has an apparent molecular mass of 43 kDa. The 3-kDa increase occurs as a result of processing of Asn-linked high-mannose oligosaccharides to complex-type units.     

The effect of glycoprotein-processing inhibitors on the secretion of glycoproteins by Madin-Darby canine kidney cells

The effects of various glycoprotein-processing inhibitors on the biosynthesis and secretion of N-linked glycoproteins was examined in cultured Madin-Darby canine kidney (MDCK) cells. Since incorporation of [2-3H]mannose into lipid-linked saccharides and into glycoproteins was much greater in phosphate-buffered saline (PBS) than in serum-supplemented basal medium (BME), most experiments were done in PBS. Castanospermine, an inhibitor of glucosidase I, caused the formation of glycoproteins having mostly Glc3Man7-9(GlcNAc)2 structures; deoxymannojirimycin, an inhibitor of mannosidase I, gave mostly glycoproteins with Man9(GlcNAc)2 structures; swainsonine, an inhibitor of mannosidase II, caused the accumulation of hybrid types of oligosaccharides. Castanospermine and swainsonine, either in PBS or in BME medium, had no effect on the incorporation of [2-3H]mannose or [5,6-3H]leucine into the secreted glycoproteins and, in fact, there was some increase in mannose incorporation in their presence. These inhibitors also did not affect mannose incorporation into cellular glycoproteins nor did they affect the biosynthesis as measured by mannose incorporation into lipid-linked saccharides. On the other hand in PBS medium, deoxymannojirimycin, at 25 micrograms/mL, caused a 75% inhibition in mannose incorporation into secreted glycoproteins, but had no effect on the incorporation of [3H]leucine into the secreted glycoproteins. Since deoxymannojirimycin also strongly inhibited mannose incorporation into lipid-linked oligosaccharides in PBS, the decreased amount of radioactivity in the secreted and cellular glycoproteins may reflect the formation of glycoproteins with fewer than normal numbers of oligosaccharide chains, owing to the low levels of oligosaccharide donor. However, in BME medium, there was only slight inhibition of mannose incorporation into lipid-linked saccharides and into cellular and secreted glycoproteins.     

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.     

Involvement of Lipid-linked Oligosaccharides in Synthesis of Storage Glycoproteins in Soybean Seeds

Membrane preparations from developing soybean (var. Prize) cotyledon tissue, at the time of synthesis of storage glycoproteins, catalyze the sequential assembly of lipid-linked oligosaccharides from uridine-5'-diphospho-N-acetyl-d-[6-(3)H] glucosamine and guanosine-5'diphospho-d-[U-(14)C]mannose. The maximum size of lipid-linked oligosaccharide that accumulates contains the equivalent of 10 saccharide units on the basis of Bio-Gel P-2 gel filtration studies. These lipid-linked oligosaccharides show similar characteristics to polyisoprenyl diphosphate derivatives on diethylaminoethyl-cellulose chromatography and are potential intermediates in glycoprotein biosynthesis in this tissue. These glycolipids do not appear to turn over in pulse-chase experiments and no completed storage glycoproteins were detected among the products of these incubations.Tissue slices from cotyledons at the same stage of development synthesize lipid-linked oligosaccharides from [(3)H]mannose and [(3)H]glucosamine with sizes equivalent to 1, 7, 10, and approximately 15 saccharide units. In pulse-chase experiments, the lipid-linked saccharides with the equivalent of 1 and 10 units rapidly turnover, whereas those with 7 and 15 units do not. Examination of the higher oligosaccharide peaks (10 and 15) by Bio-Gel P-4 gel filtration shows them to comprise 2 distinct subsets of oligosaccharides containing different proportions of glucosamine and mannose units. Tissue slices synthesize products which resemble the completed 7S storage glycoproteins as judged by similarity of molecular weight and precipitation with specific antisera. Analysis of the oligosaccharides obtained by hydrazinolysis of glycoproteins shows the presence of a similar size "high-mannose" type N-linked oligosaccharides as in other glycoproteins from animal and plant cells.     

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.