Glycosylation of endogenous lipids and proteins by preparations of chicken embryo fibroblasts

Glycosylation of endogenous phosphoisoprenyl lipids and membrane-associated proteins was shown to occur in preparations of chicken embryo fibroblasts incubated with GDP[¹⁴C]mannose and UDP-N-acetylglucosamine. The two preparations used were cells released from the culture dishes by buffered saline containing EDTA and crude membranes from those cells. Both β-mannosyl-phosphoryldolichol and oligosaccharide-phosphoryl lipids with five to eight sugar residues were labelled under the conditions employed. The oligosaccharide isolated from the octasaccharide-lipid fraction was shown to be heterogeneous after an analysis of the products formed by treatment of the oligosaccharide with glycosidases. Some of the oligosaccharides appeared to contain N-acetylglucosamine at positions external to that of [¹⁴C]mannose. Lipids with oligosaccharide moieties of different structures were made by the two preparations. The results of pulse-chase experiments were consistent with the glycosylated lipids being intermediates in glycoprotein biosynthesis.     

Effect of dexamethasone on the synthesis of dolichol-linked saccharides and glycoproteins in hepatocytes prepared from control and inflamed rats.

Hepatocytes were prepared from control and inflamed rats. The incorporation of [14C]mannose into protein was increased in inflamed compared with control hepatocytes. The incorporation of [14C]mannose into protein was also increased when the hepatocytes were cultured in presence of dexamethasone (1 microM), either from control or inflamed rats. At the same time the incorporation of [14C]mannose into dolichol phosphate mannose and dolichol-linked oligosaccharide was increased due to inflammation. The presence of dexamethasone in the hepatocyte culture caused an increased formation of these two products; in particular its effect on oligosaccharide lipid formation was very pronounced. The ratios of activities of formation of [14C]mannose-labelled oligosaccharide lipid in inflamed over control hepatocytes gradually decrease when increasing amounts of exogenous dolichol phosphate was added in cell homogenate assay mixture. These results suggest that the increase of oligosaccharide lipid formation in inflammation could be due to a higher concentration of endogenous dolichol phosphate, as was shown for dolichol phosphate mannose formation in inflammation [Sarkar & Mookerjea (1984) Biochem. J. 219, 429-436]. In contrast, the ratio of activities of [14C]mannose-labelled oligosaccharide lipid between dexamethasone-treated and untreated hepatocytes shows only a slight increase when increasing concentrations of exogenous dolichol phosphate were added to the assays. This suggests that the stimulation of dolichol pyrophosphate oligosaccharide synthesis observed in dexamethasone treatment is probably due to the higher level of enzymes involved in oligosaccharide synthesis rather than higher level of endogenous dolichol phosphate in these cells.     

The participation of lipid-linked oligosaccharide in synthesis of membrane glycoproteins.

Membrane preparations from hen oviduct catalyze the transfer of mannose from GDP-mannose into three components: mannosyl phosphoryl polyisoprenol, oligosaccharide-lipid, and glycoprotein. Eivence that mannosyl phosphoryl polyisoprenol serves as a mannosyl donor for synthesis of both oligosaccharide-lipid and glycoproteins was previously reported (Waechter, C.J., Lucas, J.J., and Lennarz, W.J. (1973) J. Biol. Chem. 248, 7570-7579). In this study the oligosaccharide-lipid has been isolated, and the oligosaccharide has been partially characterized. Based on paper chromatography the oligosaccharide chain contains 7 to 9 glycose units. The glycose at the reducing terminus is N-acetylglucosamine, whereas mannose is found at the nonreducing end. When UDP-N-acetyl[14C]glucosamine is incubated with oviduct membranes in the absence of GDP-mannose, a 14C-labeled chitobiosyl lipid, but little oligosaccharide-lipid is synthesized. When GDP-mannose is also present in the incubation mixture an oligosaccharide-lipid is formed containing N-acetyl[14C]glucosaminyl residues. This oligosaccharide-lipid is chromatographically identical with the [14C]mannose-containing oligosaccharide-lipid isolated in the earlier study cited above. When the N-acetyl[14C]glucosamine-oligosaccharide released from the oligosaccharide-lipid by mild acid is treated with partially purified alpha-mannosidase the major radioactive product is [14C]chitobiose. Evidence that the [14C]mannose-containing oligosaccharide-lipid serves as an oligosaccharide donor for glycoprotein synthesis was obtained by incubation of partially purified oligosaccharide-lipid with the membranes. The products of this incubation were shown to be glycoproteins on the basis of their sensitivity to pronase, as determined by both gel filtration and paper electrophoresis. Similar experiments, using oligosaccharide-lipid doubly labeled with [14C]mannose and N-acetyl[3H]glucosamine, provided evidence that the oligosaccharide chain of the oligosaccharide-lipid is transferred en bloc to glycoprotein s.     

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.     

Lipid-mediated glycosylation of a white matter membrane glycoprotein with an apparent molecular weight of 24,000.

White matter membrane preparations from pig brain catalyze the transfer of [¹⁴C]mannose from exogenous [¹⁴C]mannosylphosphoryldolichol into an endogenous oligosaccharide lipid. Under the same incubation conditions label is also incorporated into endogenous membrane glycoproteins. The enzymatic labeling of both classes of endogenous acceptors is stimulated by the addition of Ca²⁺. Several enzymatic properties of the mannosyltransferase activity responsible for the transfer of mannose from mannosylphosphoryldolichol into the oligosaccharide lipid intermediate have been examined. The [Man-¹⁴C] oligosaccharide lipid synthesized by this in vitro system has the solubility, hydrolytic and chromatographic characteristics of a pyrophosphate-linked oligosaccharide derivative of dolichol. The free [Man-¹⁴C]oligosaccharide liberated from the carrier lipid by mild acid treatment is estimated to contain 8 glycose units. All of the [¹⁴C]mannosyl units in the [Man-¹⁴C]oligosaccharide derived from exogenous [¹⁴C]mannosylphosphoryldolichol are released as free [¹⁴C]mannose by an α-mannosi-dase. No [¹⁴C]mannose is released during incubation with a β-mannosidase. The presence of an N,N′-diacetylchitobiose unit at the reducing end of the lipid-bound [Man-¹⁴C]oligosaccharide is indicated by its susceptibility to digestion by endo-β-N-acetylglucosaminidase H. Pronase digestion of the enzymatically labeled [Man-¹⁴C]glycoprotein yields a single [Man-¹⁴C]gly-copeptide fraction on Bio-Gel P-6 that appears to be slightly larger than the free [Man-¹⁴C]oligosac-charide released from the carrier lipid by mild acid hydrolysis. The [Man-¹⁴C]glycopeptide is cleaved by endo-β-N-acetylglucosaminidase H, and the neutral [Man-¹⁴C]oligosaccharide product appears to be identical to the product formed when the lipid-bound [Man-¹⁴C]oligosaccharide is degraded by the endoglycosidase. The glycopeptide linkage in the [Man-¹⁴C]glycoprotein is stable to mild alkali treatment. These results are consistent with the dolichol-linked [Man-¹⁴C]oligosaccharide, mannosy-lated via exogenous [¹⁴C]mannosylphosphoryldoiichol, being subsequently transferred en bloc from dolichyl pyrophosphate to asparagine residues in endogenous membrane polypeptide acceptors. SDS-polyacrylamide gel electrophoresis of the [Man-¹⁴C]glycoprotein, labeled when white matter membranes are incubated with [¹⁴C]mannosylphosphoryldolichol. revealed a major labeled polypeptide with an apparent mol wt of 24,000. A minor labeled membrane glycoprotein is also seen, having an apparent mol wt of 105,000.     

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.     

Biosynthesis of prolyl hydroxylase: evidence for two separate dolichol-media pathways of glycosylation

Prolyl hydroxylase is a glycoprotein containing two nonidentical subunits, alpha and beta. The alpha subunit of prolyl hydroxylase isolated from 13-day-old chick embryos contains a single high mannose oligosaccharide having seven mannosyl residues. Two forms of alpha subunit have been shown to exist in enzyme purified from tendon cells of 17-day-old chick embryos, one of which (alpha) appears to be identical in molecular weight and carbohydrate content with the single alpha of enzyme from 13-day-old chick embryos, as well as another form (alpha') that contains two oligosaccharides, each containing eight mannosyl units [see Kedersha, N. L., Tkacz, J. S., & Berg, R. A. (1985) Biochemistry (preceding paper in this issue)]. Biosynthetic labeling studies were performed with chick tendon cells using [2-3H]mannose, [6-3H]glucosamine, [14C(U)]mannose, and [14C(U)]glucose. Analysis of the labeled products using polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that only the oligosaccharides on alpha' incorporated measurable mannose or glucosamine isotopes; however, both alpha subunits incorporated 14C amino acid mix and [14C(U)]glucose [metabolically converted to [14C(U)]mannose] under similar conditions. Pulse-chase labeling studies using 14C amino acid mix demonstrated that both glycosylated polypeptide chains alpha and alpha' were synthesized simultaneously and that no precursor product relationship between alpha and alpha' was apparent. In the presence of tunicamycin, neither alpha nor alpha' was detected; a single polypeptide of greater mobility appeared instead. Incubation of the cells with inhibitory concentrations of glucosamine partially depressed the glycosylation of alpha' but allowed the glycosylation of alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Glycoprotein biosynthesis in intestinal epithelial cells during differentiation. Incorporation of [14C]mannose from GDP-[14C]mannose into dolichol derivatives

Epithelial cells of the rat small intestine were collected as a gradient of villus to crypt cells. Homogenates of these cells incubated with GDP-D-[14C]mannose in the presence of MnCl2 incorporated radioactivity into dolichyl mannosyl phosphate and a mixutre of dolichyl pyrophosphate oligosaccharides varying in the size of their oligosaccharide moiety. The labeled oligosaccharides formed in villus cell homogenates appeared shorter than those formed in crypt cell homogenates. The addition of dolichyl phosphate greatly stimulated the synthesis of dolichyl mannosyl phosphate. The initial rate of synthesis of dolichyl mannosyl phosphate from GDP-D-[14C]mannose and exogenous dolichyl phosphate was highest in an intermediate cell fraction having a low specific activity of sucrase and alkaline phosphatase and an intermediate specific activity of thymidine kinase. To compare the rates of dolichyl mannosyl phosphate synthesis in the different cell fractions, it was essential to control degradation of GDP-D-[14]mannose by the addition of AMP to the incubation, since villus cells degraded GDP-D-[14C]mannose much faster than crypt cells.     

Mannosyltransferase activity in calf pancreas microsomes. Formation of 14C-labeled lipid-linked oligosaccharides from GDP-D-[14C]mannose and pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate.

Calf pancreas microsomes incorporated radioactive D-mannose from GDP-D-[14C]mannose into lipid-bound oligosaccharides extracted with chloroform/methanol/water (10/10/2.5, v/v). Several products, which probably differed in the size of the oligosaccharide moiety, were labeled. These could be partially resolved by thin layer chromatography and DEAE-cellulose chromatography. The labeled lipid-bound oligosaccharides were retained on DEAE-cellulose more strongly than synthetic dolichyl alpha-D-[14C]mannopyranosyl phosphate. They were stable to mild alkali, but labile to acid and hot alkali. Acid treatment yielded a neutral 14C-labeled oligosaccharide fraction which was estimated by gel filtration to have a minimum of 8 monosaccharide residues. Hot alkali treatment yielded a mixture of neutral and acidic 14C-labeled oligosaccharides which could be transformed into neutral products by alkaline phosphatase. The D-[14C]mannose residues were alpha-linked at the nonreducing terminus of the oligosaccharides since they could be removed completely with alpha-mannosidase. Most of the D-[14C]mannose-labeled oligosaccharides were retained on concanavalin A Sepharose and eluted with methyl alpha-D-mannopyranoside. Pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate incubated with calf pancreas microsomes in the presence of sodium taurocholate was efficiently utilized as donor of alpha-D-mannosyl residues in lipid-bound oligosaccharides. The products formed from dolichyl beta-D-[14C]mannopyranosyl phosphate were identical with those formed from GDP-D-[14C]mannose, and evidence was obtained to show that the dolichyl beta-D-[14C]mannopyranosyl phosphate was serving as donor without prior conversion to GDP-D-[14C]mannose. Transfer of mannose from dolichyl beta-D-[14C]mannopyranosyl phosphate to lipid-bound oligosaccharides took place at a pH optimum of 7.3, whereas transfer to the precipitate containing glycoproteins was greatest at pH 6.0 in Tris/maleate buffer. The addition of divalent cation was not required, but low concentrations of EDTA were extremely inhibitory. The carbohydrate composition of the lipid-bound oligosaccharides of microsomal membranes was investigated by gas-liquid chromatography and by reduction with sodium borotritide. A heterogeneous mixture of oligosaccharides containing N-acetyl-D-glucosamine, D-mannose, and D-glucose varying in proportions from approximately 1/2.5/0.5 to 1/5/1.5 was obtained with glucosamine at the reducing end. Acid treatment of the lipid-bound oligosaccharide fraction yielded dolichyl pyrophosphate, suggesting that at least some of the oligosaccharides were linked to dolichol through a pyrophosphate group.     

Evidence for mannolipids as intermediates in mannose transfer to thyroid rough microsomal glycoproteins.

Thyroid rough microsomes catalyzed the transfer of mannose from GDP-mannose to endogeneous glycoprotein(s) and to glycolipids comprising a recently described dolichol phosphomannose extractable with usual organic solvents and a material tentatively identified as an oligosaccharide lipid. The labeling of the two lipids was consistent with a role in mannose transfer to glycoprotein(s). When partially purified dolichol phospho[14C] mannose was incubated with rough microsomes, a part of the label appeared in the second lipid, suggesting a role as intermediate, and less rapidly in glycoprotein(s). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis did not allow to ascertain whether or not the glycoproteins receiving label from these sugar lipids comprised thyroglobulin precursors.     

A lipid-linked oligosaccharide intermediate in glycoprotein synthesis. Characterization of [Man-14C]glycoproteins labeled from [Man-14C]oligosaccharide-lipid and GDP-[14C]Man.

Endogenous proteins of cell-free preparations of hen oviduct labeled from GDP-[14C]Man or from [Man-14C]oligosaccharide-lipid have been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Under the conditions tested, a polypeptide chain of molecular weight about 25,000 was the principle acceptor for the oligosaccharide moiety of exogenous [Man-14C]oligosaccharide-lipid. The product labeled by [Man-14C]oligosaccharide-lipid appeared identical with one of three glycoproteins formed when GDP-[14C]Man was incubated with a crude membrane fraction. These three proteins (apparent molecular weight of 75,000, 55,000, and 25,000) accounted for nearly two-thirds of the [14C]mannose-labeled glycoprotein products using GDP-[14C]Man and either the crude membrane fraction or a total oviduct homogenate. Thus, all of the mannose acceptor proteins present in the oviduct homogenate appear to be membrane-bound. Analyses of the [Man-14C]glycoproteins labeled from GDP-[14C]Man in membrane fractions from hen kidney, liver, brain, and oviduct indicated that a labeled polypeptide of apparent molecular weight 25,000 was the only major protein product common to the four preparations.     

Glycoprotein biosynthesis in plants. Demonstration of lipid-linked oligosaccharides of mannose and N-acetylglucosamine.

Previous studies from this laboratory have shown that particulate preparations from maturing cotton fibers catalyze the transfer of mannose from GDP-[14C]mannose into mannosylphosphorylpolyisoprenol (Forsee, W. T., and Elbein,A. D. (1973) J. Biol. Chem. 248, 2858-2867). In this report, we show that these particulate preparations also catalyze the inocoporation of mannose from GDP-[14C]mannose into lipid-linked oligosaccharides and into glycoprotein. The oligosaccharide-lipids were treated with dilute acid to liberate the water-soluble oligosaccharides and these oligosaccharides could then be separated into seven or eight distinct radioactive peaks by paper chromatography in isobutyric acid/NH4OH/H2betaO (57/4/39). The smallest of the oligosaccharides appears to be a trisaccharide with the structure Man leads to GlcNAc-GlcNAc. Thus the oligosaccharides attached to the lipids apparently range in size from those having 3 glycose units to those having approximately 8 to 10 glycose units. The radioactivity in the smaller-sized oligosaccharide-lipids could be chased into the larger oligosaccharide-lipids by a second incubation in the presence of unlabeled GDP-mannose. The sugar at the reducing ends of the oligosaccharides was identified as GlcNAc while some mannose (20 to 30%) was present in alpha linkages at the nonreducing ends...     

The effect of glycoprotein-processing inhibitors on fucosylation of glycoproteins

The influenza viral hemagglutinin contains L-fucose linked alpha 1,6 to some of the innermost GlcNAc residues of the complex oligosaccharides. In order to determine what structural features of the oligosaccharide were required for fucosylation or where in the processing pathway fucosylation occurred, influenza virus-infected MDCK cells were incubated in the presence of various inhibitors of glycoprotein processing to stop trimming at different points. After several hours of incubation with the inhibitors, [5,6-3H]fucose and [1-14C]mannose were added to label the glycoproteins, and cells were incubated in inhibitor and isotope for about 40 h to produce mature virus. Glycopeptides were prepared from the viral and the cellular glycoproteins, and these glycopeptides were isolated by gel filtration on Bio-Gel P-4. The glycopeptides were then digested with endo-beta-N-acetylglucosaminidase H and rechromatographed on the Bio-Gel column. In the presence of castanospermine or 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, both inhibitors of glucosidase I, most of the radioactive mannose was found in Glc3Man7-9GlcNAc structures, and these did not contain radioactive fucose. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, most of the [14C]mannose was in a Man9GlcNAc structure which was also not fucosylated. However, in the presence of swainsonine, an inhibitor of mannosidase II, the [14C]mannose was mostly in hybrid types of oligosaccharides, and these structures also contained radioactive fucose. Treatment of the hybrid structures with endoglucosaminidase H released the [3H]fucose as a small peptide (Fuc-GlcNAc-peptide), whereas the [14C]mannose remained with the oligosaccharide. The data support the conclusion that the addition of fucose linked alpha 1,6 to the asparagine-linked GlcNAc is dependent upon the presence of a beta 1,2-GlcNAc residue on the alpha 1,3-mannose branch of the core structure.     

The formation of lipid-linked sugars as intermediates in glycoprotein synthesis in rabbit mammary gland

1. The incorporation of d-[1-(14)C]mannose, d-[2-(3)H]mannose and N-acetyl-d-[1-(14)C]-glucosamine into glycoproteins and lipid-linked intermediates of mammary explants obtained from lactating rabbits was studied. The amount of radioactivity incorporated into lipid-linked intermediates was very low compared with the incorporation into protein. Most of the radioactivity incorporated into the chloroform/methanol-soluble fraction was present as neutral lipid. Radioactivity from d-[2-(3)H]mannose was incorporated mainly into the fatty acid moiety, whereas radioactivity from d-[1-(14)C]mannose and N-acetyl-d-[1-(14)C]glucosamine was present in the glycerol moiety of triacylglycerol. 2. The labelled lipid-linked intermediate that was soluble in chloroform/methanol/water (10:10:3, by vol.) was partially characterized and was found to exhibit properties characteristic of an oligosaccharide linked to lipid via a pyrophosphate bridge. It migrated largely as a single zone of radioactivity on t.l.c. and was eluted from a column of DEAE-cellulose acetate as a single peak by 50mm-ammonium acetate. 3. The oligosaccharide moiety was released from the lipid by mild acid hydrolysis. The size of the oligosaccharide was estimated by paper chromatography to be 10 or 11 monosaccharide units. 4. d-[1-(14)C]Mannose was incorporated largely into glycopeptides with molecular weights in the range 40000-80000, as determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Label from N-acetyl-d-[1-(14)C]glucosamine was incorporated into a glycopeptide with an electrophoretic mobility identical with that of rabbit casein (mol.wt. 32000) as well as into glycopeptides of higher molecular weight. 5. Approx. 50% of the total radioactivity in the protein labelled from N-acetyl-d-[1-(14)C]glucosamine was present as galactosamine, a component of the carbohydrate portion of rabbit casein. No labelled galactosamine was present in the lipid-linked oligosaccharide labelled from N-acetyl-d-[1-(14)C]glucosamine. It thus appears that the lipid-linked oligosaccharide is not involved in the glycosylation of casein.     

A lipid-linked oligosaccharide intermediate in glycoprotein synthesis in oviduct. Structural studies on the oligosaccharide chain.

The structure of the oligosaccharide chain of the lipid-linked oligosaccharide that serves as a donor of oligosaccharide chain to proteins of hen oviduct membranes has been investigated. A [Man-14C]glycopeptide fraction was prepared from membrane glycoproteins labeled with GDP-[14C]mannose. Reductive alkaline cleavage of this glycopeptide yielded a reduced oligosaccharide that, by four criteria, was identical with reduced [Man-14C]oligosaccharide prepared from [Man-14C]oligosaccharide-lipid. The structure of the oligosaccharide chain of the [Man-14C]glycopeptide was investigated by cleavage with a specific endo-beta-N-acetylglucosaminidase, followed by treatment of the released oligosaccharide with purified al alpha-and beta-mannosidases. By this procedure it was possible to establish the structure of the cleavage product as (alpha-Man)n-beta-Man-(1 leads to 4)-GlcNAc. Similar studies were performed on the [GlcNAc-14C]oligosaccharide prepared by hydrolysis of [GlcNAc-14C]oligosaccharide-lipid. The results indicate that the structure of the intact oligosaccharide is (alpha-Man)n-beta-Man-(1 leads 4)-beta-GlcNAc-(1 leads to 4)-GlcNAc. These experiments, coupled with earlier enzymatic studies on synthesis of the glycoproteins from the lipid-linked oligosaccharide, provide strong evidence that the structure of the oligosaccharide intermediate and the oligosaccharide chain of the glycoprotein product contain the same core structure found in many secretory glycoproteins.     

Lipid-linked oligosaccharides containing glucose in lactating rabbit mammary gland.

1. Microsomal fractions of lactating rabbit mammary gland incubated with UDP-glucose formed lipid-linked mono- and oligo-saccharides. The lipid-linked monosaccharide had chromatographic properties similar to those of dolichol phosphate mannose and yielded glucose on acid hydrolysis. 2. Incubation of the microsomal fraction with GDP-[U14C]-mannose yielded an oligosaccharide lipid of approximately seven monosaccharide units. Further incubation with UDP-glucose increased the size of the oligosaccharide by approximately two units. 3. Explants of lactating rabbit mammary gland incorporated [U-14C]glucose into both lipid-linked mono- and oligo-saccharides. The oligosaccharide lipid was of approx. 11 monosaccharide units. 4. Considerable redistribution of radioactive label occurred in the explant system, and radioactively labelled glucosamine and mannose, as well as glucose, were detected on acid hydrolysis of the oligosaccharide lipid. 5. Glucose was also detected in the acid hydrolysate of explant proteins. Radioactive glucosamine, galactosamine, galactose and mannose were also found in this fraction.     

Dietary effects on the formation of dolichyl monophosphate mannose by microsomal preparations of rat adipose tissue

Microsomal preparations from rat adipose tissue catalyse the transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to an endogenous acceptor forming a [¹⁴C]mannosyl lipid. The mannosyl lipid co-chromatographs with hen oviduct dolichyl monophosphate β-mannose on three solvent systems. It is stable to mild alkaline hydrolysis, but strong alkaline treatment yields a compound that co-migrates with mannose 1-phosphate. The mannosyl lipid is labile to mild acid hydrolysis, yielding [¹⁴C]mannose. Formation of the compound is reversible by GDP, but not GMP, and is stimulated by exogenous dolichyl phosphate.

The kinetics of transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to form dolichyl monophosphate mannose were studied by using preparations derived from rats fed on one of four diets: G (high glucose), L (high lard), F (fructose) or GC (high glucose, 0.9% cholesterol). The K_m and V_max. values for transfer from GDP-mannose were virtually indistinguishable in the four preparations.

In the absence of exogenous dolichyl phosphate, the largest amount of transfer of [¹⁴C]mannose into the mannosyl lipid was observed with preparations from fructose-fed animals. Preparations from glucose-fed animals showed about 60% as much transfer, whereas membranes from rats fed the other diets showed intermediate values between the fructose- and glucose-fed animals. The inclusion of cholesterol in the glucose diet elicited an increase in transfer of mannose.

Under conditions of saturating exogenous dolichyl phosphate, preparations from lard-fed animals have 1.5 times as much enzyme activity as do preparations from animals fed the other three diets.

     

Biosynthesis of yeast glycoproteins. Processing of the oligosaccharides transferred from dolichol derivatives.

The oligosaccharides previously bound to dolichol diphosphate were isolated from Saccharomyces cerevisiae cells incubated with [U-14C]glucose. Five compounds were obtained that migrated with RGlucose of 0.100, 0.120, 0.145, 0.180, and 0.215 on paper chromatography. All of them contained mannose and 2 N-acetylhexosamine residues. The substances that migrated with the three lower RGlucose values had, in addition, glucose units. The structure of the oligosacchardies was very similar if not identical with that of the oligosaccharides isolated from the dolichol diphosphate derivatives synthesized "in vitro" by yeast or rat liver particulate preparations or "in vivo" by dog thyroid or rat liver slices as judged by their migration on paper chromatography, monosaccharide composition, and degradation compounds produced by alpha-mannosidase treatment or acetolysis. The oligosaccharides previously bound to asparagine residues in proteins were isolated from yeast cells which had been pulsed with [U-14C]glucose and chased with medium containing the unlabeled monosaccharide. The samples taken after very short pulses contained four oligosaccharides that migrated with RGlucose of 0.100, 0.120, 0.145, and 0.180 on paper chromatography. The first three compounds contained glucose, mannose, and 2 N-acetylhexosamine residues whereas the one that migrated with a RGlucose of 0.180 was devoid of the former monosaccharide. Samples taken after short chase periods revealed that the compounds that migrated with the lower RGlucose values gradually disappeared and were converted to the oligosaccharide with the higher RGlucose value was they lost their glucose residues. Similar analysis as those mentioned above showed that the structures of these compounds were similar to those of the dolichol diphosphate-bound oligosaccharides. Samples taken after longer chase periods revealed that the oligosaccharide that migrated with a RGlucose of 0.180 was subsequently either enlarged by the addition of more mannose residues or trimmed to smaller sizes.