Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. Studies on transferrin, very low density lipoprotein, and serum albumin.

Using rat or chick hepatocyte monolayers, we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the synthesis and secretion of serum proteins. Tunicamycin inhibited glucosamine incorporation into rat liver transferrin and the apoprotein B chain of chick liver very low density lipoprotein (VLDL) by 75 to 90%. In contrasts, amino acid incorporation into these two glycoproteins, as well as into the normally unglycosylated proteins, rat serum albumin and apoprotein A of chick liver VLDL, was decreased by only 10 to 25% in the presence of the antibiotic. Despite the inhibitory effect of tunicamycin on glycosylation, secretion of all four proteins was virtually unimpaired. Thus, the carbohydrate moieties of rat liver transferrin or apoprotein B of chick liver VLDL do not appear to play an essential role in the secretion process.     

Tunicamycin inhibition of carbohydrate incorporation into thyroglobulin

Carbohydrate chains formation into thyroglobulin (Tg) is a prerequisite for thyroid hormones formation and completeness of carbohydrates chains is necessary for secretion of Tg into the follicles. Tg biosynthesis has been investigated by in vitro experiments, incubating rat thyroid glands with labeled amino-acid and carbohydrate in the presence of tunicamycin, a specific inhibitor of protein glycosylation. Tunicamycin inhibit Tg biosynthesis which is impaired in carbohydrate chains addition but slightly in the polypeptide synthesis, as shown by inhibition of 3H-glucosamine incorporation. Thus tunicamycin inhibits carbohydrate incorporation into Tg without affecting the polypeptide chain growth and decreases its secretion into the follicles.     

Role of carbohydrates in protein secretion and turnover: effects of tunicamycin on the major cell surface glycoprotein of chick embryo fibroblasts

Using tunicamycin, we have investigated the role of glycosylation in the biosynthesis, processing and turnover of CSP, the major cell surface glycoprotein of chick embryo fibroblasts (CEF). This antibiotic specifically inhibits glycosylation mediated by dolichol pyrophosphate and consequently inhibits the glycosylation of asparaginyl residues of glycoproteins. Tunicamycin inhibited the incorporation of 3H-mannose into CSP by 92--98% and 14C-glucosamine by 84--96%, whereas total protein synthesis was decreased by only 15--45%. Tunicamycin treatment decreased total amounts of CSP by approximately 50--65%, with equal decreases in CSP occurring on the cell surface and in culture medium, whereas intracellular pools of CSP were not substantially affected. In contrast to CSP, three other membrane-associated proteins of apparent molecular weights 75,000, 95,000 and 150,000 daltons were found in increased amounts. Procollagen secretion was not inhibited by tunicamycin. Both procollagen and CSP secretion into culture medium were also not increased in AD6, a glycosylation-deficient, mutant mouse 3T3 cell line compared to wild-type cells. We examined the mechanism of the decrease in CSP after tunicamycin treatment. The rate of CSP biosynthesis as measured by pulse-labeling with 14C-leucine was not altered. Tunicamycin had only a slight effect on the initial times and rates of CSP appearance on the cell surface; some apparent intracellular redistribution of CSP was detected by immunofluorescence. The major effect of tunicamycin treatment was to accelerate the rate of degradation of CSP 2--3 fold. This increase is sufficient to account for the observed decreases after tunicamycin treatment. Our results suggest that carbohydrates may not be essential for CSP or procollagen synthesis, intracellular processing and secretion, but that carbohydrates may help stabilize CSP against proteolytic degradation.     

Functional role of laminin carbohydrate.

Previous work showed that tunicamycin suppresses glycosylation of laminin. In the present work, the role of glycosylation in the secretion of laminin and in the disulfide bonding of laminin subunits was studied, using tunicamycin to inhibit glycosylation. Tunicamycin inhibited extensively the secretion of laminin into culture medium and extracellular matrix even though the treated cells contained higher concentrations of laminin than the control cells. The laminin subunits synthesized in the presence of tunicamycin were disulfide bonded. Thus, suppression of glycosylation did not adversely affect disulfide bonding of the subunits, but did decrease the secretion of laminin. Glycosidases were also used to remove the carbohydrate of laminin to study the role of carbohydrate in the stability of laminin and in its interaction with another extracellular matrix component, heparin. The glycosidases removed about 73% of [3H]glucosamine. Both glycosidase-treated and untreated laminin were stable when incubated with cell lysate or culture medium. The glycosidase-treated laminin bound as efficiently as the untreated laminin to heparin. These results suggest that the presence of a carbohydrate moiety, at least at the level found in untreated laminin, is not essential in binding to heparin or in protecting laminin from proteolytic degradation in the cell or culture medium.     

Studies of the mechanism of tunicamycin in hibition of IgA and IgE secretion by plasma cells.

Tunicamycin, an antibiotic which blocks the formation of N-acetylglucosamine-lipid intermediates, thereby preventing glycosylation of glycoproteins, inhibits the secretion of IgA and IgE by MOPC 315 mouse plasma cells and IR162 rat plasma cells, respectively. At 0.5 microng of tunicamycin per ml, D-[14C]glucosamine incorporation into newly synthesized immunoglobulin was inhibited greater than 90% while the overall rate of protein synthesized was much less inhibited (40% in the case of MOPC 315 cells and 13% in the case of IR162 cells). This dose of tunicamycin produced an 85% inhibition of IgA secretion by the MOPC 315 cells and a complete inhibition of intact IgE secretion by the IR162 plasma cells. In contrast, tunicamycin had little effect on the secretion of normally nonglycosylated lambda light chains or on cell-free protein synthesis, demonstrating that tunicamycin is not a general inhibitor of protein synthesis or a non-specific inhibitor of protein secretion. No enhancement of intracellular degradation of nonglycosylated immunoglobulin could be demonstrated. Electron microscopy of tunicamycin-treated MOPC 315 cells revealed marked dilatations of the rough endoplasmic reticulum, and direct immunofluorescence indicated that the dilated rought endoplasmic reticulum contained IgA. These data indicate that glycosylation of newly synthesized IgA and IgE may be necessary for normal secretion to occur.     

Inhibition of overall protein and RNA synthesis as a mechanism for the tunicamycin induced decrease in cytochrome P-450 in rat hepatocytes

In rat hepatocytes maintained in culture, cytochrome P-450 and NADPH cytochrome c reductase activities were decreased by tunicamycin in a dose and time dependent fashion. The effect of tunicamycin was mainly due to inhibition of protein synthesis. Tunicamycin decreased L-[35S] methionine incorporation into many proteins, including a 52 kDa cytochrome P-450 isozyme. Tunicamycin also reduced RNA synthesis. These results indicate that tunicamycin decreased cytochrome P-450 levels in hepatocytes by inhibiting protein and RNA synthesis.     

Biological activities of the two major components of tunicamycin.

Tunicamycin, an antibiotic that inhibits the transfer of N-acetyglucosamine-1-phosphate from UDP-N-acetylglucosamine to dolichol monophosphate and thereby blocks the formation of protein-carbohydrate linkages of the N-glycosidic type, is not a single compound but a mixture of homologous antibiotics. Two major and eight minor homologs have been identified, all of which possess the ability to inhibit protein glycosylation. The biological activities of the two major components of tunicamycin were investigated and found to differ in their ability to inhibit protein glycosylation and in their effectiveness to inhibit protein synthesis. When completely blocking mannose incorporation into protein, one homolog inhibited protein synthesis by 50% while the other had only a negligible effect. The results demonstrate that differences in biological activity can be discriminated among tunicamycin homologs.     

Effect of tunicamycin on IgM, IgA, and IgG secretion by mouse plasmacytoma cells

Tunicamycin, an antibiotic that prevents glycosylation of glycoproteins by blocking the formation of N-acetylglucosamine-lipid intermediates, was used to study the importance of glycosylation for the secretion of immunoglobulins by mouse plasmacytoma lines that produce immunoglobulins of different classes. Biosynthetically labeled secreted and intracellular immunoglobulins were measured by immunoprecipitation assays. Tunicamycin, at a concentration of 0.5 mug/ml produced an 81% inhibition of IgM secretion by MOPC 104E plasma cells without significantly affecting the initial rate of synthesis of intracellular IgM. No increase in the intracellular degradation of nonglycosylated IgM could be demonstrated. Tunicamycin also produced a 64% average inhibition of IgA secretion by several mouse IgA-secreting plasmacytoma lines. In contrast, despite inhibiting the incorporation of D-[14C] glucosamine into newly synthesized IgG, tunicamycin only produced a 28% average inhibition of IgG secretion, which was only slightly more than the nonspecific inhibition of secretion of the normally nonglycosylated lambda2 light chains by variant MOPC 315 plasmacytomas. These data indicate that the extent of inhibition of immunoglobulin secretion produced by tunicamycin depends on the immunoglobulin class produced by the plasma cell.     

Effects of cycloheximide and tunicamycin on cell surface expression of pancreatic muscarinic acetylcholine receptors

The importance of glycosylation in cell surface expression of muscarinic receptors in cultured guinea pig pancreatic acini was investigated. Recovery of the muscarinic receptor population after carbachol-induced down regulation was blocked by cycloheximide but not by tunicamycin, although tunicamycin reduced [3H]mannose incorporation into acinar macromolecules by up to 90%. Tunicamycin treatment also failed to alter carbachol stimulation of amylase secretion from cultured acini. These results indicate that glycosylation of the glandular subtype of muscarinic receptor in the pancreatic acinar cell is not necessary for its insertion in the plasma membrane or for its functional activity.     

Involvement of Glycoproteins in the Development of Early Mouse Embryos: Effect of Tunicamycin and α, α' Dipyridyl in vitro

Early mouse embryos grown in tissue culture were treated with tunicamycin, an inhibitor of protein glycosylation or with αα' dipyridyl, an inhibitor of collagen secretion. Neither treatment blocked development of cleavage stage embryos nor did either interfere with blastocyst formation, hatching, or adhesion to the substratum at low concentrations. However, both treatments caused marked and specific changes in the morphology of the blastocyst outgrowth. Treatment of embryos with tunicamycin caused severe deterioration of the trophoblast layer and subsequent disintegration of the inner cell mass. Tunicamycin completely inhibited the incorporation of mannose into proteins. Treatment with αα' dipyridyl caused dose dependent retardation of the inner cell mass while the trophoblast cells were virtually unaffected. These alterations in morphogenesis occurred only in embryos treated at the blastocyst stage or later in development. Changes caused by α,α' dipyridyl could be partially reversed by addition of collagen to the culture. These findings might indicate the involvement of extracellular matrix macromolecules in embryonic organization.     

Tunicamycin inhibits the differentiation of ST 13 fibroblasts to adipocytes with suppression of the insulin binding activity

ST 13 cells are a clonal line of murine fibroblasts that are capable of differentiating into adipocyte-like cells $\text{in}\text{vitro}$. When the cells were maintained as a confluent monolayer, they began to accumulate lipid droplets and to exhibit a rapid increase of insulin binding activity. Tunicamycin, a specific inhibitor of dolichol-mediated protein glycosylation, blocked this adipose conversion without affecting cell growth and total protein synthesis. The inhibitory effect of tunicamycin was dose-dependent and reversible. Enhancement of the incorporation of [¹⁴C]acetate into triglyceride fraction accompanying the adipose conversion was completely inhibited by tunicamycin, whereas the incorporation into phospholipid fraction was only partially affected. The insulin binding activity increased about 10-fold during differentiation, but was completely suppressed in tunicamycin-treated cells.     

The role of glycosylation in storage-protein synthesis in developing pea seeds

Intact pea (Pisum sativum L.) cotyledons were incubated with [¹⁴C]glucosamine at several stages of seed development and the resultant radioactive proteins were analysed by gel electrophoresis combined with immunoaffinity chromatography and sucrose gradient fractionation. Glucosamine was incorporated into at least five vicilin polypeptides (approx. molecular weight 70,000; 50,000, two components; 14,000, two components). No incorporation was detected into the subunits of legumin. Tunicamycin at 50 g/ml largely inhibited glucosamine incorporation but had little effect on the incorporation of ¹⁴C-labelled amino acids into cotyledon proteins, including vicilin. The assembly of vicilin polypeptides into full-sized protein oligomers (7–9 S) was also unaffected by tunicamycin. Chromatography on concanavalin A confirmed that glycosylation of cotyledon proteins was inhibited by tunicamycin. It is concluded that glycosylation of most cotyledonary proteins involves lipid-linked sugar intermediates, but that glycosylation itself is not an essential step in the synthesis of vicilin polypeptides nor in their assembly into oligomers.Abbreviations IgG immunoglobulin G - M W_t approximate molecular weight based on electrophoretic mobility relative to that of protein standards - SDS-PAGE Na-dodecyl sulfate-polyacrylamide gel electrophoresis     

Re-expression of nonglycosylated surface IgA in trypsin-treated MOPC 315 plasmacytoma cells.

The importance of glycosylation for the re-expression of surface immunoglobulin in trypsin-treated MOPC 315 plasmacytoma cells was examined by using tunicamycin, an antibiotic that prevents glycosylation by inhibiting the formation of N-acetylglucosamine-lipid intermediates. Tunicamycin greatly inhibited the secretion of nonglycosylated MOPC 315 IgA in trypsin-treated cells. Two hours after trypsin treatment, there was an 80% inhibition of secretion as measured by immunoprecipitation assays of biosynthetically labeled immunoglobulin. However, tunicamycin had no effect on the time course of re-expression of surface IgA in these cells as measured by TNP-sheep erythrocyte rosette formation and [125I] TNP-albumin binding to the plasmacytoma cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 125I-labeled cell surface IgA re-expressed in the presence of tunicamycin revealed a protein with an apparent m.w. identical to nonglycosylated MOPC 315 alpha-chains, further suggesting that nonglycosylated surface IgA was being inserted into the plasma membrane. This protein did not bind to concanavalin A-Sepharose. These data suggest that in MOPC 315 plasmacytoma cells, glycosylation is necessary for immunoglobulin secretion but not for immunoglobulin expression at the cell surface.     

Effects of weakly basic amines on proteolytic processing and terminal glycosylation of secretory proteins in cultured rat hepatocytes.

We examined the effects of weakly basic amines on the secretion and post-translational modifications of secretory proteins in cultured rat hepatocytes. Weakly basic amines such as methylamine, chloroquine and NH4Cl strongly inhibited not only protein secretion, but also the proteolytic conversion of a proform of complement C3, allowing the precursor to be released into the medium. The amines, however, had no effect on the proteolytic conversion of prohaptoglobin into its subunits. Since available evidence indicates that the conversion of pro-C3 occurs at the Golgi complex while that of prohaptoglobin takes place in the endoplasmic reticulum, it is most likely that the weak bases specifically affect the proteolytic event occurring at the Golgi complex. Electron microscopic observations confirmed that the amines caused morphological changes of the Golgi complex, consisting of dilated cisternae and swollen vacuoles. When the glycosylation of alpha 1-protease inhibitor and haptoglobin was examined, it was found that the amines caused a marked accumulation in the cells of both glycoproteins corresponding to the mature secreted forms. Neuraminidase digestion demonstrated that the glycoproteins accumulating in response to the amines had acquired terminal sialic acid. The results indicate that the amines do not significantly affect terminal glycosylation, in contrast with their definite effect on proteolytic processing, despite the fact that both modifications take place in the Golgi complex.     

Localization of the lipid intermediate pathway of protein glycosylation in oviduct cell types

Oviduct tissue slices were incubated with [3H]-leucine or [3H]-mannose in the presence and absence of tunicamycin, a specific inhibitor of lipid-mediated protein glycosylation. Conditions were established where tunicamycin had maximal effect on [3H]-mannose incorporation (greater than 90% inhibition) but a minimal effect on [3H]-leucine incorporation (less than 10% inhibition) into total TCA-insoluble products. Analysis of incubated tissues by SDS-polyacrylamide gel electrophoresis revealed that in the absence of tunicamycin, [3H]-mannose was incorporated into only a few proteins, of which ovalbumin represented the major radiolabeled component. Tunicamycin markedly reduced the incorporation of [3H]-mannose into ovalbumin and other oviduct glycoproteins. In contrast, analysis by SDS-polyacrylamide gel electrophoresis showed that [3H]-leucine was incorporated into a variety of proteins in the absence of tunicamycin. The radioactivity profile of some of these proteins was shifted toward lower Mr when oviduct slices were incubated in the presence of tunicamycin, with only a minimal decrease in protein labeling. Light microscopic autoradiograms of tissue incubated with [3H]-leucine in either the presence or absence of tunicamycin exhibited extensive labeling of tubular gland and epithelial cells. In the absence of tunicamycin, these cell types also become markedly labeled with [3H]-mannose; however, incorporation of label in both cell types was substantially reduced in the presence of tunicamycin. Qualitatively, labeling of tubular gland cells appeared greater than that of epithelial cells, largely due to the concentration of silver grains over the dense population of secretory vesicles in the tubular gland cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycosylation of apolipoproteins by cultured rat hepatocytes. Effect of tunicamycin on lipoprotein secretion.

Cultured rat hepatocytes were used to measure hepatic synthesis of rat plasma glycoproteins. [3H]Glucosamine was progressively incorporated into the protein of hepatocyte culture media very-low-density lipoprotein, low-density lipoprotein, high-density lipoprotein and the p greater than 1.21 g/ml fraction after 3.5 and 6.5 h incubation. Apolipoproteins B, E and C, as well as transferrin, were identified as glycoproteins. The association of radioactivity with apolipoprotein C of hepatocyte very-low-density and high-density lipoproteins suggests that apolipoprotein C-III-3, the only C apoglycoprotein in the rat, is synthesized de novo by the hepatocytes. Treatment of hepatocytes with tunicamycin, a specific inhibitor of protein glycosylation, resulted in a substantial decrease in [3H]glucosamine incorporation into hepatocyte very-low-density, low-density and high-density lipoproteins and p greater than 1.21 g/ml protein, but had little or no effect on secretion. In the rat, hepatic secretion of lipoproteins and transferrin does not appear to be dependent on prior protein glycosylation.     

The role of the carbohydrate moiety in the biologic properties of fibrinogen

The carbohydrate moiety of some glycoproteins influences their secretion and functional properties. We have examined the importance of the oligosaccharide chains of fibrinogen in this regard. Fibrinogen was labeled de novo by the addition to rabbit hepatocyte monolayer cultures of either 3H-amino-acids or [2-3H] mannose, in the presence or absence of tunicamycin, a potent inhibitor of glycosylation. Inhibition of glycosylation, which ranged from 75 to 80%, was determined by incorporation of [2-3H]mannose as quantitated by gel filtration. Synthesis and secretion of fibrinogen were quantitated by 3H-amino-acid incorporation, using anti-fibrinogen immunoaffinity column chromatography of medium and cell homogenates. Tunicamycin did not appreciably inhibit fibrinogen synthesis, as compared to a 30-40% inhibition of overall protein synthesis, determined by incorporation of 3H-amino-acids into trichloroacetic acid-precipitable material. There was no evidence that secretion of fibrinogen was impaired. Fibrinogen from medium was copurified by adding cold plasma fibrinogen as carrier. Nonglycosylated fibrinogen was found to be functional as demonstrated by incorporation of radioactivity into clots of the copurified material at a rate identical to that of glycosylated fibrinogen. When clotted in the presence of Ca2+ and Factor XIII, cross-linking of glycosylated and nonglycosylated fibrin was demonstrable on fluorography of sodium dodecyl sulfate-polyacrylamide gels, showing disappearance of gamma-chain and appearance of gamma-gamma-dimers.     

Effect of tunicamycin on in vitro ripening of tomato pericarp tissue

Ripening of pericarp tissue from mature green, early breaker and late breaker stages of tomato ( Lycopersicon esculentum Mill. cv. Dombito) fruit development was inhibitied by tunicamycin. Ripening was evaluated by lycopene accumulation, chlorophyll degradation, rate of ethylene production and cell wall-bound polygalacturonase (EC 3.2.1.15) activity. Maximum inhibition of these ripening parameters occurred at a treatment of 240 μ M tunicamycin for 2 h except for cell wall-bound polygalacturonase activity, which was greatly inhibited by concentrations of 12 μ tunicamycin or higher. Tunicamycin treatment at 120 μ M for 2 h inhibited the incorporation of [³H]-mannose into macromolecules (about 70%) and pronase-sensitive material (about 65%) and the incorporation of [³H]-leucine into proteins (about 20%). Our results indicate that protein glycosylation plays an important role in the ripening of tomato pericarp tissue.     

Secretion of lysyl oxidase by cultured human skin fibroblasts and effects of monensin, nigericin, tunicamycin and colchicine

Lysyl oxidase is an extracellular enzyme that initiates crosslink formation in the major connective tissue proteins, the collagens and elastin. This enzyme activity accumulated in a fresh medium of cultured human skin fibroblasts for at least 24 h, but the accumulation was distinctly non-linear after the first 12 h. Most of the total enzyme activity was present in the medium, the activity found in the cell layer representing about 30% of the total activity at 4 h, and about 10-15% at 24 h. The bulk of the cell-layer-associated activity appeared to be extracellular, as more than half was lost upon trypsinization. Culturing of the cells for 8 h in the presence of either monensin or nigericin, ionophores known to inhibit the secretion of many proteins at the level of the Golgi complex, markedly reduced the accumulation of lysyl oxidase activity in the medium. Monensin was particularly effective, as it produced a distinct inhibition even at a 10 nM concentration, reaching 50% at 30 nM. Both ionophores also reduced enzyme activity in the cell layer, whereas no definite decrease was seen in the activity of the trypsinized cells. The effect of monensin was evidently not due to any general toxicity on the part of the drug, since even a 500 nM concentration gave no inhibition of the incorporation of [3H]leucine into total protein. Tunicamycin also reduced lysyl oxidase activity in the medium and to a lesser extent in the cell layer, but the effective dose, 1-10 micrograms/ml, also inhibited the incorporation of [3H]leucine into total protein. The reduced enzyme activity may therefore not be due to a direct effect of tunicamycin on the glycosylation of the lysyl oxidase protein itself but may be mediated through other actions of the drug. Colchicine caused no inhibition in lysyl oxidase activity secretion even at a 10 microM concentration, although it has been reported to inhibit collagen secretion at doses more than one order of magnitude lower.