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.     

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.     

Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.     

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.     

Glycosylation causes an apparent block in translation of immunoglobulin heavy chain

Analysis of nascent heavy chains isolated from MPC11 (gamma 2b heavy chains) and MOPC 21 (gamma 1 heavy chains) mouse myeloma cells demonstrates an accumulation of nascent heavy chains which are slightly smaller in mass (approximately 35,000 daltons) than nascent heavy chains which have just been glycosylated (approximately 38,000 daltons). The accumulation of 35,000-dalton nascent heavy chain appears to be a consequence of the glycosylation process since tunicamycin, an inhibitor of glycosylation, abolishes the apparent translational block manifested by the accumulation of 35,000-dalton nascent chains. Tunicamycin also causes a 15 to 25% increase n the relative rate of synthesis of heavy chain compared to the corresponding rate of synthesis of the nonglycosylated light chain synthesized by the same cell. These results suggest that the translation block, caused by the glycosylation process, of heavy chain synthesis contributes to the imbalance of heavy chain and light chain biosynthesis observed in malignant and normal lymphoid cells.     

Density comparisons of heavy chains of membrane and secreted immunoglobulins of mouse.

In order to explore structural differences between membrane and secreted immunoglobulins the buoyant densities of mouse immunoglobulin (Ig) heavy (H) chains were compared by isopycnic centrifugation in CsCl containing guanidine hydrochloride. The buoyant densities, under denaturing conditions, of mouse myeloma protein MOPC 21 IgG, MOPC 315 IgA and MOPC 104E IgM H chains were consistent with their carbohydrate contents. Mouse membrane IgM and MOPC 104E-secreted IgM H chains were of equal density. The buoyant densities of MOPC 104E-secreted IgM and spleen-cell-secreted IgM H chains were indistinguishable. The IgD-like membrane H chain was denser than membrane IgM H chain, and its carbohydrate content was calculated to be 15.5%. The resolution of the technique was sufficient to conclude that the apparent 1500 mol.wt. difference, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, between membrane and secreted IgM H chains was due to peptide rather than to carbohydrate. The results also imply that intact membrane IgM and IgD bind detergent and are thus integral membrane proteins.     

Glycoprotein synthesis and inhibition of glycosylation by tunicamycin in preimplantation mouse embryos: compaction and trophoblast adhesion

The synthesis of glycoproteins and inhibition of protein glycosylation by tunicamycin were examined during development of preimplantation mouse embryos and trophoblast adhesion. Tunicamycin specifically inhibits glycosylation of asparaginyl residues of glycoproteins. Tunicamycin, 0.25-5.0 microgram/ml, had no effect on early cleavage or aggregation between embryos, but the embryos remained irreversibly uncompacted when control embryos developed to the blastocyst stage. Trophoblast adhesion and giant cell outgrowth were reversibly inhibited and the binding of Con A was also reduced. Incorporation of 3H-mannose into blastocysts was inhibited by 80%, but that of 3H-glucosamine and 3H-leucine by only 28 and 18%, respectively, in the presence of 1.0 microgram/ml tunicamycin. Qualitative analysis showed that the incorporation of the sugars was markedly reduced in the majority of the fractions, but the synthesis of these carbohydrate-deficient glycopeptides was essentially normal. However, protein-polysaccharide fractions with nearly 40% of the incorporated glucosamine and only 5% mannose and 1% leucine were insensitive to inhibition by tunicamycin. Membrane-bound N-glycosidically linked glycoproteins therefore evidently play an important role during compaction and in trophoblast adhesion of mouse embryos.     

Post-translational fate of variant MOPC 315 lambda chains in Xenopus oocytes and mouse myeloma cells

The post-translational fates of three immunoglobulin lambda chain variants of MOPC 315 were investigated in mouse plasmacytoma cell lines and in mRNA-microinjected Xenopus oocytes. Quite unexpectedly we found that one non-secretory variant chain (lambda-43) underwent extensive post-translational N-glycosylation: however the presence of the oligosaccharide moiety did not account for the nonsecretory phenotype nor did it affect the rate of degradation of this lambda chain. Another variant chain (lambda-47) at first believed to be non-secretory, was found to be secreted from oocytes at a very low level, but mostly as a lambda-lambda dimer. In myeloma cells a low level of lambda-47 chain was secreted and again lambda-lambda dimers were the favoured secretory form. The secretory lambda-48 chain also formed lambda-lambda dimers, whereas lambda-43, which was never secreted, was only found as a monomeric lambda chain in both oocytes and myeloma cells. A similar relationship between assembly and secretion was found when oocytes were coinjected with MOPC 21 heavy (gamma 1) chain mRNA and MOPC 315 lambda chain mRNAs. The wild type lambda chain (lambda-48) was able to assemble with the gamma chain in a covalently bound tetramer (gamma gamma lambda lambda). The variant lambda-47 chain was also able to form gamma gamma lambda lambda tetramers, whereas the lambda-43 was not, even when glycosylation was prevented by tunicamycin. Both types of tetramer were secreted. These data reinforce the idea that conformational changes play a major role in the routing of secretory proteins and that the cellular mechanisms by which these changes are recognized are not cell-type specific.     

Synthesis of abnormal immunoglobulins by hybridomas from autoimmune "viable motheaten" mutant mice.

Secretory defects in abnormal plasma cells, called Mott cells, that appear in lymphoid tissues of spontaneously autoimmune, "viable motheaten" (mev/mev) mice lead to deposition of immunoglobulin in RER-bound vesicles. Such vesicles have been termed Russel bodies. Cells with Russel bodies can also be observed rarely in normal animals, usually as a result of extreme antigenic loads or pathologic states. To understand why these abnormal cells appear commonly in mev/mev mice, we have established a panel of hybridomas that contain Russell bodies. Using immunochemical analysis and immunoelectron microscopy, we have characterized the secretory defects. Although these hybridoma cells synthesize a normal size heavy chain and it associates with light chain, the Russell bodies have many characteristics of inclusion bodies, which commonly appear in cells synthesizing mutant proteins and often are associated with incompletely or abnormally folded proteins. Pulse-chase experiments showed that immunoglobulins synthesized by these hybridomas accumulate rapidly into insoluble complexes and have an intracellular half life approximately 10 time greater than normal immunoglobulins. The defect affected only the immunoglobulin derived from the mev/mev mice and did not affect the secretion of normal immunoglobulin produced by an IgG1-secreting fusion partner. In addition to accumulating intracellular immunoglobulins, many mutant cell lines also secreted immunoglobulin. Endoglycosidase H digestion was used to determine the state of processing of the N-linked carbohydrates on the immunoglobulin molecules. This analysis demonstrated that the N-linked carbohydrates on the secreted immunoglobulin were resistant to endoglycosidase H digestion, indicating that they were processed normally. The insoluble IgM molecules were sensitive to endoglycosidase H, which is consistent with their localization to the RER. We propose several models by which these abnormal immunoglobulin-secreting cells commonly appear in this autoimmune mutant mouse.     

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.     

Enzymic mechanism of starch breakdown in germinating rice seeds : 12. Biosynthesis of alpha-amylase in relation to protein glycosylation

The biosynthetic mechanism of α-amylase synthesis in germinating rice (Oryza sativa L. cv. Kimmazé) seeds has been studied both in vitro and in vivo. Special attention has been focused on the glycosylation of the enzyme molecule. Tunicamycin was found to inhibit glycosylation of α-amylase by 98% without significant inhibition of enzyme secretion. The inhibitory effect exerted by the antibiotic on glycosylation did not significantly alter enzyme activity.

In an in vitro system using poly-(A) RNA isolated from rice scutellum and the reticulocyte lysate translation system, a precursor form of α-amylase (precursor I) is formed. Inhibition of glycosylation by Tunicamycin allowed detection of a nonglycosylated precursor (II) of α-amylase. The molecular weight of the nonglycosylated precursor II produced in the presence of Tunicamycin was 2,900 daltons less than that of the mature form of α-amylase (44,000) produced in the absence of Tunicamycin, and 1,800 daltons less than the in vitro synthesized molecule.

The inhibition of glycosylation by Tunicamycin as well as in vitro translation helped clarify the heterogeneity of α-amylase isozymes. Isoelectrofocusing (pH 4-6) of the products, zymograms, and fluorography were employed on the separated isozyme components. The mature and Tunicamycin-treated nonglycosylated forms of α-amylase were found to consist of three isozymes. The in vitro translated precursor forms of α-amylase consisted of four multiple components. These results indicate that heterogeneity of α-amylase isozymes is not due to glycosylation of the enzyme protein but likely to differences in the primary structure of the protein moiety, which altogether support that rice α-amylase isozymes are encoded by multiple genes.

     

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.     

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.     

Modification of Epstein-Barr virus replication by tunicamycin.

The effect of tunicamycin, which inhibits N-linked glycosylation, on the replication of Epstein-Barr virus was examined. Tunicamycin markedly reduced the yield of virus from producing cells. At concentrations of 1 to 2 micrograms of tunicamycin per ml, there was a buildup of intracellular virus in P3HR1-Cl13 cells but not in MCUV5 cells; at a concentration of 5 micrograms of tunicamycin per ml in P3HR1-Cl13 cells, viral DNA synthesis was inhibited as well. Viral glycoproteins lacking N-linked sugars were apparently inserted into the cell membrane, and the small amount of virus made in the presence of drug was able to bind specifically to its receptor on B cells. However, the ability of the virus to induce immunoglobulin secretion by fresh human lymphocytes was impaired. This implies a role for viral glycoproteins in the penetration as well as the attachment of virus.     

Characterization of the glycosylation of a human IgM produced by a human-mouse hybridoma

We analysed the oligosaccharides of a human IgM produced bya human-human-mouse hybridoma at each of its five conservedheavy chain glycosylation sites. Consistent with previous reports,this IgM possesses sialylated oligosaccharides at Asn171, Asn332and Asn395, and high-mannose-type oligosaccharides at Asn402.In contrast to previous reports for human IgMs, we find thatAsn563 is not occupied by oligosaccharide on perhaps 25% ofIgM heavy chains, while occupied Asn563 sites contain both high-mannose-typeand sialylated oligosaccharides. These latter results are consistentwith the glycosylation at Asn563 previously reported for themouse MOPC 104E IgM. We demonstrate that both the human hybridomaIgM and the mouse MOPC 104E IgM are mixtures of pentamers andhexamers, raising the possibility that the unique findings concerningthe glycosylation at Asn563 in this study and the previous studyof the MOPC 104E IgM could be related, at least in part, tothe different packing requirements of the hexameric geometryand the accessibility of oligosaccharides in the hexameric geometryfor processing to complex type. In addition, we used high-pHanion-exchange (HPAE) chromatography, neutral anion-exchangechromatography, fluorophore-assisted carbohydrate electrophoresisand Western blots to compare the oligosaccharide compositionsof the human hybridoma IgM, pooled human serum IgM and two mousemonoclonal IgMs (MOPC 104E and TEPC 183). Of note is the presenceof N-glycolylneuraminic acid (NeuGc) and N-acetymeuraminic acid(NeuAc) at a 2:1 ratio in the oligosaccharides of the humanhybridoma IgM. The presence of both NeuGc and NeuAc complicatesthe interpretation of HPAE chromato-graphs. glycosylation high-pH anion-exchange chromatography human IgM human—mouse hybridoma oligosaccharide     

Murine plasma cells secreting more than one class of immunoglobulin heavy chain. III. Immunoglobulin production by established cultures and cloned lines of SAMM 368.

SAMM 368, a plasmacytoma which produces IgA-kappa and IgG2b-kappa was established in vitro from primary explants or after animal passage. The 9 lines that were established produced both paraproteins. The production of both immunoglobulins by single cells was demonstrated by immunofluorescent staining and cloning. Continuous culture of 3 parent lines for 18 months and 11 cloned lines for periods from 5 to 7 months demonstrated that double production is a stable characteristic of this plasmacytoma. Two single paraprotein-producing varients (IgG2b or IgA) were derived when cells were cultured in the presence of Fungizone. Chromosomal analysis of SAMM 368 indicates that this double producing tumor has a modal number similar to those observed in myelomas producing a single immunoglobulin class.     

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.     

Glucose-dependent glycosylation of secretory glycoprotein in mouse myeloma cells.

The mouse myeloma tumor, MOPC-46, produces a kappa-type immunoglobulin light chain that may be isolated from the urine of tumor-bearing animals. This protein possesses a single carbohydrate side chain, attached by glycosylamine linkage to asparagine residue 28. When viable single cell suspensions of the tumor are incubated in vitro in minimum essential medium containing sodium pyruvate as a source of carbon and energy, the major protein synthesized and secreted corresponds to a nonglycosylated form of the kappa light chain. However, when glucose or mannose are substituted for sodium pyruvate as a source of carbon, the immunoglobulin light chain is synthesized and secreted in the fully glycosylated, native form. The dependence of normal glycosylation of the protein on the presence of either glucose or mannose in the medium is relatively specific for these compounds since substitution with either fructose, galactose, glycerol, ribose, or N-acetylglucosamine was ineffective. The nonglycosylated protein produced in the presence of sodium pyruvate was characterized as nonglycosylated MOPC-46 light chain by immunoprecipitation and gel electrophoresis. An identical nonglycosylated protein was produced by tumor cells in the presence of glucose when the incubation mixtures contained tunicamycin.     

Complement proteins C2, C4 and factor B. Effect of glycosylation on their secretion and catabolism.

Tunicamycin, an inhibitor of N-acetylglucosaminylpyrophosphopolyisoprenol-dependent glycosylation, was used to study the effect of glycosylation on the synthesis, post-translational modification, secretion and function of the complement proteins that are associated with the major histocompatibility complex in humans, mice and guinea pigs. Tunicamycin blocked glycosylation of pro-C4, C2 and factor B and inhibited secretion of the corresponding native complement proteins synthesized by guinea-pig peritoneal macrophages in tissue culture. In addition, underglycosylated pro-C4 was more rapidly catabolized intracellularly than the corresponding fully glycosylated pro-complement protein. C4 protein secreted by cells incubated with tunicamycin had approximately the same specific biological activity as the protein obtained from control culture media, suggesting that carbohydrate is not required for its activity in immune haemolysis. Direct studies of carbohydrate incorporation and the tunicamycin effect suggested an unequal distribution of sugar among the C4 subunits, with maximal incorporation of carbohydrate into alpha-, and less into the beta-chain of the native protein.     

Incomplete glycosylation of ASN 563 in mouse immunoglobulin M

Mouse immunoglobulin IgM was prepared from MOPC 104E ascites fluid and [3H]-mannose labeled tumor cells. The purified protein was used to prepare Fc fragments which were cleaved by cyanogen bromide. Gel filtration allows complete separation of the C-terminal glycosylation site. Amino acid and carbohydrate analyses show that Asn 563 of murine IgM is glycosylated only about 44% of the time.