Steps in the biosynthesis of mosquito cell membrane glycoproteins and the effects of tunicamycin

A cultured cell line of the mosquito, Aedes aegypti, is sensitive to tunicamycin as expected from the ability of crude membrane preparations to catalyse the formation of N-acetylglucosamine-linked dolichyl pyrophosphate. Formation of dolichylphosphomannose was also detected and this reaction was totally insensitive to tunicamycin. Incorporation of radioactive mannose into total acid-precipitable glycoproteins was inhibited greater than 90% in whole cells by tunicamycin, while the incorporation of leucine and glucosamine was less affected. Separation of the radioactive hexosamines from acid hydrolysates of cells incubated with [¹⁴C]glucosamine and tunicamycin showed predominant labelling of galactosamine, whereas in control cells not treated with the drug both glucosamine and galactosamine were labelled equally. Evidently, mosquito cells synthesise N-glycosidically linked carbohydrate chains assembled through tunicamycin-sensitive steps involving dolichyl pyrophospho-oligosaccharides, and O-glycosidically linked chains rich in N-acetylgalactosamine, the assembly of which is unaffected by tunicamycin. These results support structural evidence (Butters, T.D. and Hughes, R.C. (1981) Biochim. Biophys. Acta 640, 655–671) for the presence of high mannose N-glycans and N-acetylgalactosamine-richO-glycans in mosquito cell glycoproteins. The absence of complex N-glycans was confirmed by the demonstration of negligible activities of N-acetylglucosaminyl-, galactosyl- and sialyltransferases responsible for assembly of the terminal sequences of N-glycans of mature mammalian glycoproteins.     

Incorporation of [14C]glucosamine into synaptosomes in vitro

Abstract— Synaptosomes isolated from rat cerebral cortex by zonal centrifugation in-corporated radioactive glucosamine into macromolecules in vitro as glucosamine, galactosamine, N-acetylneuraminic acid, and glucuronic acid. The largest percentage of incorporated radioactivity was recovered in the particulate fraction in which radioactive carbohydrates were bound in covalent linkage requiring acid hydrolysis or enzymatic digestion for release. Less than 20 per cent of the particulate radioactivity represented incorporation into gangliosides. Some 20 per cent of the radioactivity was incorporated into proteins as glucosamine, identified in hydrolysates by paper chromatography and by the amino acid analyser. After incubation, radioactivity was demonstrable in the proteins as sialic acid by paper chromatography and specific enzymic digestion; and as glucuronic acid by chromatography, electrophoresis, and digestion with hyaluronidase. Incorporation of carbohydrate was stimulated by sodium and potassium at concentrations demonstrated to enhance incorporation of amino acids, and involved the macro-molecules of all subsynaptosomal fractions. Significant incorporation of radioactivity was found in the synaptic plasma membrane. The synthesis of glycoproteins was suggested by simultaneous incorporation of [¹⁴C]glucosamine and [³H]leucine into glycopeptides subsequently hydrolysed and subjected to polyacrylamide gel electrophoresis and two-dimensional paper chromatography and electrophoresis. Such studies demonstrated that amino acids and carbohydrates may be incorporated into glycoproteins of the synaptic membrane and suggest the possibility of local synthesis as well as modification of material brought to the nerve ending by axoplasmic flow.     

THE SECRETORY PATHWAYS OF RAT SERUM GLYCOPROTEINS AND ALBUMIN : Localization of Newly Formed Proteins within the Endoplasmic Reticulum

These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein. N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membrane-attached polysomes and not into proteins from free polysomes. Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes. Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae. Albumin was mostly recovered (98%) in the cisternae, with negligible amounts in the membranes. To determine whether the radio-active sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the ¹⁴C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins. The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae.     

Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [¹⁴C]glucosamine and [³H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated ceils by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [¹⁴C]glucosamine and [³H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [³H]glucosamine and from lectin stimulated cells labeled with [¹⁴C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

Biosynthesis ofSaccharomyces cerevisiae glycoproteins: Nature of some participating glycolipids

A particulate membrane preparation fromSaccharomyces cerevisiae catalyzed the incorporation of mannose from GDP-mannose into lipids that were extractable in chloroform-methanol. One lipid has been previously characterized as dolichyl phosphomannose. Another one was purified by chromatography on silicic acid, DEAE-cellulose and Sephadex LH-20 and found to be alkali unstable. The lipid moiety was shown to be dolichol and the glycosydic part contained mannose, glucose and glucosamine.Radioactive mannose was also incorporated at a slower rate into more polar compounds. They were soluble in chloroform-methanol-water and were seen to liberate neutral oligosaccharides after alkaline hydrolysis.Radioactive mannose was also incorporated into substances which behave chemically as glycoproteins since they were insoluble in organic solvents, water and trichloroactic acid. Pronase treatment of the trichloroacetic acidinsoluble material released water-soluble oligosaccharides.When the particulate preparation which had been extracted with chloroform-methanol at –20 C, was incubated with GDP-(U-¹⁴C)mannose, radioactivity was incorporated into glycolipids that were soluble in chloroform-methanol-water and into glycoproteins. This result suggests that at least part of the mannose was transferred to endogenous acceptors independent of dolichyl phosphomannose.     

Effects of 2-deoxyglucose, glucosamine, and mannose on cell fusion and the glycoproteins of herpes simplex virus.

2-Deoxyglucose and glucosamine were found to inhibit cell fusion caused by a syncytial mutant of herpes simplex virus and to inhibit the glycosylation of viral glycoproteins in the infected cells. The inhibition of fusion and the inhibition of glycosylation caused by 2-deoxyglucose were substantially prevented when mannose was also present during infection. When glycosylation was inhibited, three new bands were found in major glycoprotein region on sodium dodecyl sulfate-polyacrylamide gels. These bands may be precursors to the normal glycoproteins. The correlation between fusion and glycosylation in the presence of 2-deoxyglucose, glucosamine, and mannose suggests that the cells cannot fuse if their glycoproteins have a considerably reduced carbohydrate content.     

Synthesis of sulfated glycoproteins by glial precursor cells

Populations of enriched glial precursor cells and astrocytes isolated from primary cultures of newborn rat brain were used to study the synthesis of sulfated glycoproteins. Both cell types incorporated [³H]glucosamine and [³⁵S]sulfate into carbohydrate side chains of proteoglycans and glycoproteins. The rate of incorporation of [³H]glucosamine into the oligosaccharides and the pattern of distribution of the label into high mannose and complex glycopeptides recovered from the glycoproteins appeared to be similar for the two glial cell types. However, clear differences were noted in the rate of oligosaccharide sulfation activities. Thus the cultures of precursor glia were about four times more active than cultures enriched in astroglia in their ability to incorporate [³⁵S]sulfate into glycoproteins.     

Increased degradation of carbohydrate deficient arterial basement membrane-like material from 2-deoxyglucose modified myomedial cell cultures

The effect of 1.6 mM 2-deoxyglucose on the incorporation of [3H]leucine, [3H]glucosamine and [35S]sulphate into arterial basement membrane-like (BM) material was evaluated. BM-like material was isolated from the cell-matrix layer of cultured arterial smooth muscle cells by a sonication-differential centrifugation technique. 1.6 mM 2-deoxyglucose inhibited the 24 hr incorporation of [3H]glucosamine into BM-like material by 46% with a reduction in both [3H]glucosamine labelled glycopeptides and glycosaminoglycans. A marked decrease in [35S]sulphate incorporation (reduced by 80%) was demonstrated suggesting that 2-deoxyglucose may affect sulphatation of glycosaminoglycans. At 1.6 mM 2-deoxyglucose no effect on [3H]leucine incorporation was found. By gel filtration on Bio-Gel P6 a heterogeneous mixture of shortened glycopeptides was found after 2-deoxyglucose. The electrophoretic mobility of fibronectin and other glycoprotein components of BM-like material was increased. The stability of carbohydrate deficient BM-like material against removal/degradation was evaluated. A significantly increased removal of [3H]leucine from insufficiently glycosylated BM-like material was observed after a 24 hr chase period. The increased removal/degradation of BM-like material formed in the presence of 2-deoxyglucose was found to be a cellular dependent event.     

Partial Purification and Some Properties of Aggregation Factor from Pronase-Susceptible Floe Forming Flavobacterium Strain B

Globomycin inhibited the incorporation of [¹⁴C]diaminopimeric acid (Dap) into the cold 5% TCA insoluble fraction of Escherichia coli H2143 at higher concentrations than the minimum inhibitory concentration (MIC).

One-sixth or -seventh molecules of the lipoprotein were found per one molecule of N-acetyl glucosamine (GlucNAc) or Dap in globomycin-treated cells as compared with one-twelfth or -thirteenth molecules in normal cells. Among globomycin-resistant cells isolated, one-tenth were lipoprotein-less mutants and they showed a slightly swollen form and leaked RNase into the medium. It was interesting that spheroplast formation of the mutants in the presence of the antibiotic was not observed even at a high concentration.     

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.     

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.     

FUCOSE INCORPORATION INTO GLYCOPROTEINS OF MOUSE BRAIN

—Radioactive fucose was incorporated into glycoproteins of brain in vivo. After intracerebral administration of this precursor, radioactive glycoproteins were the sole detectable product. The glycoproteins formed appeared to have a slow turnover but this was due, at least in part, to re-utilization of fucose released from degraded glycoproteins. Incorporation of fucose into glycoproteins differed from that of glucosamine, since a much smaller proportion of the radioactive fucose was incorporated into soluble glycoproteins. Fucose was rapidly incorporated into glycoproteins of nerve endings, although there was relatively little incorporation into glycoproteins associated with the soluble component of the nerve-ending fraction. As found in previous studies with glucosamine, soluble glycoproteins of nerve endings turned over relatively rapidly. Pretreatment with acetoxycycloheximide markedly inhibited incorporation of fucose into glycoproteins of brain. In contrast to the results with glucosamine, comparable inhibition was observed for fucose in all subcellular fractions of brain including the particulate and soluble components derived from the nerve-ending fraction.     

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.     

Biosynthesis of the carbohydrate portion of immunoglobins. Kinetics of synthesis and secretion of [3H]leucine-, [3H]galactose- and [3H]mannose-labelled myeloma protein by two plasma-cell tumours

The kinetics of incorporation of leucine, galactose and mannose into intracellular and secreted myeloma protein, MOPC 21 IgG(1) and MOPC 46 kappa-type light chain, by cell suspensions of two myeloma plasma-cell tumours, MOPC 21 and MOPC 46, were similar. Radioactive galactose was incorporated to over 90% into galactose residues of intracellular and secreted protein, mannose to over 90% into glucosamine and mannose residues of intracellular protein and to over 90% into glucosamine, mannose and fucose residues of secreted protein, but not into galactose residues. The results show that specific residues in the carbohydrate portion of myeloma proteins can be labelled by specific radioactive monosaccharides, and suggest that fucose residues are added, while myeloma protein is in its final stage of secretion from the plasma cell. The kinetics of incorporation indicate at least three sequential precursor-product relationships between different intracellular forms and the secreted form of myeloma protein.     

The effect of tunicamycin on development of the mammalian embryonic pancreas

The role of cell-surface glycoproteins in histogenesis of the embryonic rat pancreas was investigated by studying the effect of tunicamycin (TM) on in vitro development. TM has been shown to block glycosylation of asparagine residues in glycoproteins by inhibiting formation of dolichol oligosaccharide intermediates. Exposure of Day 15 pancreatic rudiments to 1.0 μg TM/ml for 15 or 24 hr inhibited [³H]mannose, [³H]glucosamine, and [³H]fucose incorporation by 95, 85, and 90%, respectively, while [³H]leucine incorporation was reduced by 35%. Similar results were obtained with Day 17 rudiments. These trends were confirmed using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Inhibition of [³H]monosaccharide incorporation correlated with reduced binding of RCA I-ferritin conjugates to the cell surface and both effects of TM were reversed by reculturing rudiments in medium lacking the antibiotic. Morphologically, TM treatment resulted in a delay in pancreatic histogenesis and this delay correlated with an inhibition of the normal increase in specific activity of amylase, an acinar cell secretory protein. These effects were not mimicked by treatment with cycloheximide at a concentration which inhibited [³H]leucine incorporation to the same degree observed with TM. The percentage of delayed rudiments decreased as reculturing in the absence of TM was extended.     

A modified microassay for human macrophage activation: I. Activation by antigen-stimulated lymphocytes and correlation with delayed hypersensitivity

The degree of activation of glass-adherent human blood monocyte-macrophages cultured with autologous lymphocytes was assessed by measurement of [¹⁴C]glucosamine uptake. In the absence of streptokinase-streptodornase (SK-SD) or purified protein derivative of tuberculin (PPD) minimal incorporation of the labeled compound occurred. Enhanced glucosamine uptake in the presence of antigen was positively correlated with cell donor-delayed skin hypersensitivity (PPD) and in vitro lymphoproliferative response (PPD, SK-SD). Increasing antigen or mononuclear cell concentrations resulted in increasing macrophage glucosamine uptake. Lymphoblasts, cell clumping, and macrophages with prominent pseudopodia were seen on stained monolayers of stimulated cells. Radioautography of such monolayers showed that the radiolabel was present only in mononuclear phagocytes. Adherent cell protein also generally increased in stimulated monolayers but did not account for the enhanced glucosamine uptake. Measurement of radioactive glucosamine incorporation into human macrophages is a useful tool to assess their degree of activation by lymphocytes stimulated by specific antigen.     

Retinoids increase the incorporation of D-[3H]galactose into epidermal glycoproteins

All-trans retinoic acid increased the incorporation of D-[³H]galactose into particulate and soluble glycoproteins in the epidermis of cultured pig skin slices nearly two-fold. Increased incorporation of D-[³H]galactose was not blocked by tunicamycin. This effect was specific for D-[³H]galactose since the incorporation of D-[³H]glucosamine and L-[¹⁴C]leucine into epidermal glycoproteins was unaffected by all-trans retinoic acid. All-trans retinoic acid and 13-cis retinoic acid had quantitatively similar effects on D-[³H]galactose incorporation. All-trans retinyl acetate and an aromatic retinoic acid analogue (‘Etretinate’) were less effective. SDS polyacrylamide gel electrophoresis and fluorography showed increased incorporation of D-[³H]galactose into all epidermal glycoproteins in the presence of all-trans retinoic acid. There was no evidence for synthesis of new glycoproteins such as mucins.     

Inhibitory effects of tunicamycin on procollagen biosynthesis and secretion

Chick embryo cells were briefly exposed to the antibiotic, tunicamycin. Pre-exposed cells, compared to control cultures, showed a severe, progressive inhibition of the incorporation of glucosamine and mannose into total cellular macromolecules. Inhibition of the incorporation of glycine, leucine and proline was also progressive but not as marked as for the carbohydrates. Cellular secretion of all macromolecules was severely impaired. while comparison of the procollagens showed no difference in their subunit size or in their degree of glycosylation; the intracellular content of procollagen polypeptides was similar for both types of cells. In vitro studies showed that tunicamycin selectively inhibited glucosamine, but not mannose, incorporation into macromolecules. The composite results indicate that tunicamycin effectively inhibits protein synthesis, protein glycosylation and protein secretion in chick embryo cells.     

Inhibitory effects of tunacamycin on procollagen biosynthesis and secretion

Chick embryo cells were briefly exposed to the antibiotic, tunicamycin. Pre-exposed cells, compared to control cultures, showed a severe, progressive inhibition of the incorporation of glucosamine and mannose into total cellular macromolecules. Inhibition of the incorporation of glycine, leucine and proline was also progressive but not as marked as for the carbohydrates. Cellular secretion of all macromolecules was severely impaired, while comparison of the procollagens showed no difference in their subunit size or in their degree of glycosylation; the intracellular content of procollagen polypeptides was similar for both types of cells. In vitro studies showed that tunicamycin selectively inhibited glucosamine, but not mannose, incorporation into macromolecules. The composite results indicate that tunicamycin effectively inhibits protein synthesis, protein glycosylation and protein secretion in chick embryo cells.     

Synthesis in vitro of glycoprotein in regenerating rat liver

The metabolism of glucosamine in regenerating rat liver was studied in liver slices. [1-¹⁴C]Glucosamine was incorporated into acid-soluble fraction, rapidly converted to UDP-N-acetylhexosamine and transferred to acid-insoluble fraction. Electrophoretic analysis revealed that most of the radioactive macromolecules released from the slices to the incubation medium were plasma glycoproteins.The incorporation of [1-¹⁴c]glucosamine into UDP-N-acetylhexosamine significantly increased from 6 h to 48 h after partial hepatectomy. On the contrary, the incorporation into acid-insoluble fractions of slice and medium decreased to about 50% of the control values. The rate of transfer of N-acetylhexosamine from UDP-N-acetylhexosamine to acid-insoluble fractions also decreased at 12 h and 48 h respectively. This indicates that the transfer of N-acetylhexosamine to glycoproteins decreases during 48 h of liver regeneration.The enhancement of [1-¹⁴C]glucosamine incorporation into UDP-N-acetylhexosamine is due to an accumulation of the label in the larger pool of this compound. Evidently, some control mechanism may operate on the transfer of N-acetylhexosamine from UDP-N-acetylhexosamine to glycoproteins in regenerating rat liver.