Cleavage with phospholipase of the lipid anchor in the cell adhesion molecule, csA, from Dictyostelium discoideum

A cell adhesion molecule, 80-kDa csA, is involved in EDTA-resistant cell contact at the aggregation stage of Dictyostelium discoideum. A 31-kDa csA was isolated from the 80-kDa csA by treatment with Achromobacter protease I. Results from thin-layer chromatography and MALDI-TOF MS analysis indicated that the 31-kDa csA contains ceramide as a component of glycosylphosphatidyl-inositol (GPI). Comparison between the 80-kDa csA and the 31-kDa csA treated with phosphatidylinositol-specific phospholipase C (PI-PLC) or GPI-specific phospholipase D (GPI-PLD) was carried out. Our results indicated that the GPI-anchor of the 31-kDa csA was more sensitive to PI-PLC treatment than that of the 80-kDa csA, and that the anchor in both was easily cleaved by GPI-PLD treatment. They suggested that the resistance of 80-kDa csA to PI-PLC treatment was due to steric hindrance and myo-inositol modification. The results of the 80-kDa csA and the 31-kDa csA treated with sphingomyelinase were similar to those with PI-PLC treatment. In the presence of 1,10-phenanthroline, a GPI-PLD inhibitor, development of Dictyostelium was markedly inhibited, suggesting that GPI-PLD is functional in developmental regulation through cell adhesion.     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

Mutants of Dictyostelium discoideum with altered carbohydrate moieties of contact site A.

Mutants of Dictyostelium discoideum were isolated and found to be defective in the epitope recognized by the monoclonal antibody 120 against the carbohydrate moieties of an integral membrane glycoprotein, contact site A, with the apparent molecular mass of 80 x 10(3). One mutant, HG764, did not express any contact site A and had lost cell contact resistant to EDTA. The others, including HG794, expressed a 68-kDa form of contact site A. In comparison with the parental strain HG592, HG794 showed weaker EDTA-resistant cell contact and the same degree of EDTA-sensitive cell contact. This suggested that the moieties which HG794 lacked were involved in EDTA-resistant cell contact. The 68-kDa contact site A in HG794 could be labeled with wheat germ agglutinin and incorporated [35S] sulfate. The modB mutant HL220 also expresses 68-kDa contact site A, although it cannot be labeled with wheat germ agglutinin. Therefore, the mutants HG794 and HL220 were compared by a complementation test. The diploid strain DG701 expressed 80-kDa contact site A and showed the same degree of EDTA-resistant cell contact as strain HG592. In its EDTA-resistant cell contact, HG794 was stronger than HL220. These results suggest that HG794 is a new mutant, and that there might be at least two processes in the glycosylation of 68-kDa contact site A to the 80-kDa form. The carbohydrate moieties recognized by monoclonal antibody 120 and by wheat germ agglutinin might be involved in EDTA-resistant cell contact.     

Identification of carbohydrate moieties involved in EDTA-stable or EDTA-sensitive cell contact of Dictyostelium discoideum

Antisera against purified contact site A glycoprotein, with an apparent molecular weight of 80 X 10(3) (80 kDa), from Dictyostelium discoideum were raised by using Freund's adjuvant (antiserum-A) and by using Alu-Gel-S (antiserum-B) as immunoadjuvants. They were converted into Fab fragments for the cell agglutination assay. Fab fragments of antiserum-B inhibited only EDTA-stable cell contact, whereas Fab fragments of antiserum-A (Fab-A) inhibited EDTA-sensitive cell contact as well as EDTA-stable cell contact. We prepared several cell types in order to identify target antigens for the adhesion-blocking Fab-A in EDTA-sensitive cell contact or EDTA-stable cell contact. One of these cell types produced contact site A without N-glycosidically-linked carbohydrate chains. It is known that contact site A contains two kinds of N-glycosidically-linked carbohydrate chains (carbohydrates I and II, Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2653-2670). When growth-phase cells were treated with tunicamycin (TM) at a final concentration of 2 micrograms/ml in nutrient medium (TM-pretreated cells), the cells produced contact site A without N-glycosidically-linked carbohydrate chains (53 kDa) at the normal developmental stage. These cells lacked EDTA-sensitive cell contact as well as EDTA-stable cell contact. The neutralization of the adhesion-blocking Fab-A was done by using particulate fractions from each cell type. The blocking activity in EDTA-stable cell contact was neutralized by the cell type with carbohydrate II. Taking these results into consideration, EDTA-stable cell contact may be formed by the interaction between protein moieties of contact site A and carbohydrate II. Concerning EDTA-sensitive cell contact, the blocking activity was neutralized by each cell type irrespective of TM treatment. This suggests that O-glycosidically-linked carbohydrate chains play a role in EDTA-sensitive cell contact. Moreover, the biological activity in EDTA-sensitive cell contact of TM-pretreated cells suggests that N-glycosidically-linked carbohydrate chains may also be involved in this contact.     

Two-step glycosylation of the contact site A protein of Dictyostelium discoideum and transport of an incompletely glycosylated form to the cell surface

Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cells of the 53-kDa precursor with no detectable exposure of this protein on the cell surface. It is concluded that type 1 carbohydrate, which is cotranslationally added in N-glycosidic linkages, is neither required for transport of the protein to the Golgi apparatus nor for type 2 glycosylation or protection of the protein against proteolytic degradation. Incapability of tunicamycin-treated cells of forming EDTA-stable cell contacts suggests a role for type 1 carbohydrate in cell adhesion. Type 2 carbohydrate is added posttranslationally. It is required in the absence of type 1 glycosylation for transport of the protein to the cell surface.     

Mediation of cell-cell adhesion by the altered contact site A glycoprotein expressed in modB mutants of Dictyostelium discoideum

In Dictyostelium discoideum, a surface glycoprotein with Mr 80,000 (gp80) has been found to mediate the EDTA-resistant contact sites A at the aggregation stage of development. To evaluate the role of the carbohydrate moiety in cell-cell adhesion, we have examined the accumulation and activity of an altered gp80 molecule in two glycosylation (modB) mutants. Both mutants synthesize an altered gp80 of lower molecular size. This modB-gp80 can be detected by the monoclonal antibody 80L5C4, which is capable of blocking cell-cell adhesion (C. -H. Siu, T. Y. Lam, and A. Choi, (1985) J. Biol. Chem. 260, 16,030-16,036). The mutant cells exhibit both EDTA-sensitive and EDTA-resistant types of cell-cell binding, though to a lesser extent than that of the parental strain, and the EDTA-resistant binding sites are blocked in the presence of 80L5C4 Fab. Mutant cells can also bind Covaspheres conjugated with gp80. These results suggest that the modB-gp80 protein still retains the domain essential for its cell binding activity and the carbohydrate moiety affected by the modB mutation is not directly involved in cell-cell adhesion.     

Carbohydrate and other epitopes of the contact site A glycoprotein of Dictyostelium discoideum as characterized by monoclonal antibodies

A series of monoclonal antibodies against a developmentally regulated protein of Dictyostelium discoideum, the contact site A glycoprotein, were used in immunoblots to label proteins of cells harvested at three stages of development: during the growth phase, at the aggregation competent stage, and at the slug stage. The antibodies fell into two groups according to their reactivity with partially or fully deglycosylated forms of the 80 kDa glycoprotein. Group A antibodies reacted not only with a 66 kDa, but also with a 53 kDa product of tunicamycin-treated wild-type cells, and they reacted with a 68 kDa component produced by HL220, a mutant that carries a specific defect in glycosylation. The 68 kDa product of the mutant was not completely unglycosylated. Like the 80 kDa glycoprotein of the wild type, which carried sulfate at carbohydrate residues, the mutant product was sulfated. In the presence of tunicamycin, the mutant produced a 53 kDa component indistinguishable from that of the wild type, which represents, most likely, the non-N-glycosylated protein portion of the contact site A glycoprotein. The group A antibodies showed almost no cross-reactivity with other proteins of the developmental stages tested, in accord with their postulated specificity for the protein moiety of the contact site A molecule. Group B antibodies did not react with the 53 kDa product of tunicamycin-treated cells, nor with the 68 kDa component of mutant HL220. These antibodies were of varying specificity. Some of them were almost as specific as group A antibodies, others cross-reacted with many proteins, particularly of the slug stage. Competition or non-competition between various group B antibodies for binding to the contact site A glycoprotein allowed sub-classification of these antibodies. According to two criteria, group B antibodies were characterized as anti-carbohydrate antibodies: (1) some of these antibodies were blocked by N-acetylglucosamine; (2) none of them reacted with the 68 kDa product or any other protein of mutant HL220. These results indicate that the 80 kDa glycoprotein carries two types of carbohydrate: type 1 carbohydrate that is sulfated and present on the 68 kDa product of mutant HL220, and type 2 carbohydrate that reacts with group B antibodies and is present on the 66 kDa product of tunicamycin-treated wild-type cells. Type 2 carbohydrate moieties are also present on many glycoproteins that are enriched in the prespore area of the slugs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Post-translational glycosylation of the contact site A protein of Dictyostelium discoideum is important for stability but not for its function in cell adhesion

The functions of type 1 and 2 carbohydrates of the contact site A (csA) glycoprotein of Dictyostelium discoideum have been investigated using mutants lacking type 2 carbohydrate. In two mutant strains, HG220 and HG701, a 68-kd glycoprotein was synthesized as the final product of csA biosynthesis. This glycoprotein accumulated to a much lower extent on the surfaces of mutant cells than the mature 80-kd glycoprotein did in wild-type cells. There was also no accumulation of the 68-kd glycoprotein observed within the mutant cells nor was a precursor of lower molecular mass detected, in accordance with previous findings that indicated cotranslational linkage of type 1 carbohydrate by N-glycosylation. Pulse-chase labelling showed that a 50-kd glycopeptide was cleaved off from the mutant 68-kd glycoprotein and released into the medium, while the fully glycosylated 80-kd glycoprotein of the wild type was stable. These results assign a function to type 2 carbohydrate in protecting the cell-surface-exposed csA glycoprotein against proteolytic cleavage. HG220 cells were still capable of forming EDTA-stable contacts to a reduced extent, consistent with the low amounts of the 68-kd glycoprotein present on their surfaces. Thus type 1 rather than type 2 carbohydrate appears to be directly involved in intercellular adhesion that is mediated by the csA glycoprotein. Tunicamycin-treated wild-type and mutant cells produce a 53-kd protein that lacks both type 1 and 2 carbohydrates. While this protein is stable and not transported to the cell surface in the wild type, it is cleaved in the mutants and fragments of it are released into the extracellular medium. These results suggest that the primary defect in the two mutants studied is relief from a restriction in protein transport to the cell surface, and that the defect in type 2 glycosylation is secondary.     

Replacement of the phospholipid-anchor in the contact site A glycoprotein of D. discoideum by a transmembrane region does not impede cell adhesion but reduces residence time on the cell surface

The contact site A (csA) glycoprotein of Dictyostelium discoideum, a cell adhesion molecule expressed in aggregating cells, is inserted into the plasma membrane by a ceramide-based phospholipid (PL) anchor. A carboxyterminal sequence of 25 amino acids of the primary csA translation product proved to contain the signal required for PL modification. CsA is known to be responsible for rapid, EDTA-resistant cohesion of cells in agitated suspensions. To investigate the role of the PL modification of this protein, the anchor was replaced by the transmembrane region and short cytoplasmic tail of another plasma membrane protein of D. discoideum. In cells transformed with appropriate vectors, PL-anchored or transmembrane csA was expressed under the control of an actin promoter during growth and development. The transmembrane form enabled the cells to agglutinate in the presence of shear forces, similar to the PL-anchored wild-type form. However, the transmembrane form was much more rapidly internalized and degraded. In comparison to other cell-surface glycoproteins of D. discoideum the internalization rate of the PL-anchored csA was extremely slow, most likely because of its exclusion from the clathrin-mediated pathway of pinocytosis. Thus, our results indicate that the phospholipid modification is not essential for the csA-mediated fast type of cell adhesion but guarantees long persistence of the protein on the cell surface.     

Inhibition of cell-cell binding at the aggregation stage of Dictyostelium discoideum development by monoclonal antibodies directed against an 80,000-dalton surface glycoprotein

Monoclonal antibodies were prepared against a putative cell-cell adhesion molecule, a surface glycoprotein with an apparent Mr of 80,000 (gp80), from Dictyostelium discoideum. Seven monoclonal antibodies directed against gp80 were characterized and found to fall into three distinct classes. Class I consisted of one monoclonal antibody, is monospecific for gp80, and probably recognizes the peptide portion of the molecule. This class was capable of blocking the EDTA-resistant contact sites effectively. Class II recognized the carbohydrate moiety of gp80 and cross-reacted with a large number of glycoproteins. These monoclonal antibodies partially inhibited cell reassociation. Class III recognized gp80 and one other glycoprotein of Mr 95,000. This class had no effect on cell-cell binding. The class I monoclonal antibody was most potent in inhibiting cell reassociation at the aggregation stage of development. Its effect decreased drastically as development progressed and became negligible by the culmination stage. These observations are consistent with a direct role of gp80 in cell-cell binding and suggest a transient function for gp80 at the aggregation stage.     

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.     

Changes in glycosylation alter the affinity of the human transferrin receptor for its ligand

When transferrin receptors of human erythroleukemic cells were pulse-labeled with [35S]methionine and then chased in the absence of radioactive precursor, the first detectable immunoprecipitable form of the receptor had a molecular mass of 85 kDa. This form of the receptor was converted to the mature form of 93 kDa with a half-time of about 40-60 min. Both the immature (85 kDa) and mature (93 kDa) receptors associated as dimers, the native form of the receptor. The 85-kDa, as well as the 93-kDa, receptors bound to a monoclonal antibody raised against the transferrin receptor or to transferrin-Sepharose. In order to determine whether glycosylation was necessary for ligand binding, purified receptors were isolated from cells grown in the presence of tunicamycin. When K562 cells were grown in the presence of tunicamycin, an 80-kDa nonglycosylated form of the receptor was synthesized. This nonglycosylated receptor was also capable of dimer formation; however, much less of it reached the cell surface than the fully glycosylated form, although both untreated and tunicamycin-grown cells appeared to synthesize transferrin receptors at similar rates. Although the number of receptor molecules/cell was similar in control and tunicamycin-treated cells, the nonglycosylated receptors exhibited a much lower affinity for transferrin than those of untreated cells; in contrast, when receptors were purified by immunoprecipitation and digested with bacterial alkaline phosphatase, no difference was observed between the affinity of these receptors and undigested immunoprecipitated receptors. These results suggest that glycosylation is not necessary for specific binding of transferrin to its receptor, but the affinity of this binding can be influenced greatly by the presence or absence of carbohydrate residues.     

Characterization and Distribution of O-Glycosylated Carbohydrates in the Cell Adhesion Molecule, Contact Site A, fromDictyostelium discoideum

This paper presents further investigation of the properties of carbohydrate II in the cell adhesion molecule, contact site A, fromDictyostelium discoideum.A purified contact site A was digested withAchromobacterprotease I to produce a 31-kDa fragment to which carbohydrate II was mainly bound and a 21-kDa fragment containing the NH₂terminus of contact site A, which was identified as Ala-Pro-Thr-Ile-Thr-Ala. The NH₂terminus of the 31-kDa fragment was Thr-Glu-Ala-Thr-Thr-Ser. It was estimated from the cDNA sequence data of contact site A that more than 20 Ser/Thr residues exist as target sites for the O-linked oligosaccharides in the 31-kDa fragment, but not for the N-linked oligosaccharides. These results suggest that carbohydrate II exists as clustered O-linked oligosaccharides in the COOH terminus of contact site A. The results of two-dimensional electrophoresis confirm that oligosaccharides of contact site A contain sialic acids. Immunoelectron microscopy was carried out to define the organelle in which O-glycosylation by carbohydrate II occurs and how carbohydrate II antigens are distributed on the cell surface. The results show that O-glycosylation can occur in the Golgi apparatus inD. discoideumas observed in other cells, although this O-glycosylation was inhibited by tunicamycin. Furthermore, gold particles were densely concentrated in cell–cell contact regions but sparsely distributed in noncontact regions.     

Monoclonal antibodies against contact sites A of Dictyostelium discoideum: detection of modifications of the glycoprotein in tunicamycin-treated cells

Tunicamycin acts on cell aggregation in Dictyostelium discoideum by changing cell movement and by inhibiting the EDTA-stable type of intercellular adhesion. Tunicamycin-treated cells show unco-ordinated pseudopodial activity such that pseudopods are simultaneously extended from all parts of the cell surface, and the cells are unable to move in straight paths. Concurrent with the inhibition of formation of EDTA-stable contacts, N-glycosylation of a glycoprotein specific for aggregation-competent cells is inhibited. This glycoprotein, previously called contact site A, has an apparent mol. wt. of 80 kilodaltons (kd). In membranes of tunicamycin-treated cells, two components are detected that react with certain monoclonal antibodies against contact sites A: one component of 66 kd, the other of 53 kd apparent mol. wt. Another group of monoclonal antibodies reacts only with the 80-kd glycoprotein and the 66-kd component. These results are in accord with the assumption that the glycoprotein carries two carbohydrate chains, and that the antibodies differ in their requirement for glycosylation of the antigen. Despite the coincidence between blockage of EDTA-stable cell adhesion and inhibited glycosylation of contact sites A, direct involvement of the carbohydrate moieties of this glycoprotein in intercellular adhesion seems questionable. EDTA-stable cell adhesion has not been blocked by Fab fragments from antibodies that specifically react with the glycosylated protein.     

The beta 2-adrenergic receptors of human epidermoid carcinoma cells bear two different types of oligosaccharides which influence expression on the cell surface.

The beta 2-adrenergic receptors of the human epidermoid carcinoma A431 cells reside on two polypeptide chains revealed by photoaffinity labelling with [125I]iodocyanopindolol-diazirine. These proteins correspond to two distinct populations of N-asparagine-linked glycoproteins: the 55-52 kDa molecules are associated with complex carbohydrate chain(s), the 65-63 kDa component with polymannosidic carbohydrate chain(s). Both types of receptors are present in preconfluent cells, but only the polymannosidic type is found in the postconfluent cells. Moreover, complex chains appear to be associated with the receptors with the highest affinity for (-)-isoproterenol and polymannosidic chains with the receptors with the lowest affinity for this agonist. the carbohydrate moiety of the beta-adrenergic receptor is involved in the expression and function of the beta 2-adrenergic receptors at the surface of the A431 cells, since tunicamycin and monensin, complete and partial inhibitors of glycosylation respectively, diminish the number of binding sites at the cell surface and increase the total number of sites in the cell. In these conditions a diminution of cyclic AMP accumulation is also observed.     

Glycosylation of CD4. Tunicamycin inhibits surface expression

The T-cell surface glycoprotein CD4 plays an important role in mediating cellular immunity and serves as the receptor for human immunodeficiency virus. We have examined the glycosylation of CD4 and asked whether carbohydrate addition is essential for proper expression of the glycoprotein on the cell membrane. Under conditions where treatment of CD4+ human acute lymphoblastic leukemia cells (CEM-CM3 cells) with the glycosylation inhibitor tunicamycin decreased surface expression of CD4 in a time- and concentration-dependent manner, the surface expression of several other glycoproteins was unaffected. Incubation with tunicamycin for 48 h inhibited mannose incorporation by 98%, caused a 76% decrease in CD4 surface expression as judged by flow cytometry, and had little effect on methionine incorporation. Scatchard analysis showed a decrease in the total number of CD4 molecules on the cell surface from 17,000 to 8,900 after 24 h of tunicamycin treatment. Immunoprecipitation of metabolically labeled CD4 revealed the presence of an unglycosylated precursor in tunicamycin-treated cells. The observed difference between the Mr of the glycoprotein and its precursor is consistent with glycosylation at two potential N-linked sites. However, this precursor could not be detected by measuring steady state levels by immunoblotting. Also, no intracellular accumulation of CD4 in tunicamycin-treated cells was detectable using immunofluorescence microscopy. We conclude that surface expression of CD4 depends on glycosylation of the protein and that the unglycosylated precursor is preferentially degraded.     

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.     

Effect of recombinant Mce4A protein of Mycobacterium bovis on expression of TNF-α, iNOS, IL-6, and IL-12 in bovine alveolar macrophages

CD38 is a type II transmembrane protein with 25% of its molecular mass consisting of glycosyl moieties. It has long been predicted that the carbohydrate moieties of glycoproteins play important roles in the physical function and structural stability of the proteins on cell surfaces. To determine the structural/functional significance of glycosylation of the human CD38, the four potential N-linked glycosylation sites asparagine residues, N100, N164, N209 and N219 were mutated. The mutant (CD38mu) and wild-type (CD38wt) were expressed separately in Escherichia coli, HeLa, and MCF-7 cells. SDS-polyacrylamide gel electrophoresis under reducing conditions and western blotting indicated that the molecular mass of the CD38wt is 45 kDa, and that of the CD38mu is 34 kDa in HeLa cells. Importantly, the CD38mu protein expressed in HeLa cells, showed the high molecular weight oligomers in addition to the 34 kDa monomeric form. Similarly, in E. coli, the CD38wt formed dimers and other oligomers besides the monomeric form. Moreover, MCF-7 cells stably transfected with CD38wt cDNA, also revealed the presence of cross-linked oligomers when treated with a N-linked glycosylation inhibitor tunicamycin (TM). These results suggested that the N-linked glycosylation of CD38 plays a crucial role in the structure stability by preventing the formation inter-molecular cross-links. In addition, immunostaining, enzyme activity (cyclase), and western blotting data revealed that the glycosylation of human CD38 protein is not required for its localization to the cell membrane.     

Effect of tunicamycin on glycosylation of a 50 kDa protein and thermotolerance development.

We investigated whether or not a 50 kDa glycoprotein might play an important role in protein synthesis-independent thermotolerance development in CHO cells. When cells were heated for 10 min at 45.5 degrees C, they became thermotolerant to a heat treatment at 45.5 degrees C administered 12 hr later. The thermotolerance ratio at 10(-3) isosurvival was 4.4. The cellular heat shock response leads to enhanced glycosylation of a 50 kDa protein. The glycosylation of proteins including a 50 kDa glycoprotein was inhibited by treatment with various concentrations of tunicamycin (0.2-2 micrograms/ml). The development of thermotolerance was not affected by treatment with tunicamycin after the initial heat treatment, although 2 micrograms/ml tunicamycin inhibited glycosylation by 95%. However, inhibiting protein synthesis with cycloheximide (10 micrograms/ml) after the initial heat treatment partially inhibited the development of thermotolerance. Nevertheless, there was no further reduction of thermotolerance development by treatment with a combination of 2 micrograms/ml tunicamycin and 10 micrograms/ml cycloheximide. These data suggest that development of thermotolerance, especially protein synthesis-independent thermotolerance, is not correlated with increased glycosylation of the 50 kDa protein.     

Effect of glycosylation inhibitors on the structure and function of the murine transferrin receptor

The murine transferrin receptor is a disulphide-linked dimer with three N-glycosylation sites. We have investigated the structural and functional properties of the transferrin receptor from murine plasmacytoma cells (NS-1 cells) treated with the glycosylation inhibitor, tunicamycin and the glycosylation-processing inhibitors, swainsonine and castanospermine. 1. Tunicamycin (1 microgram/ml) inhibited mannose incorporation in NS-1 cells by greater than 90%, but also inhibited methionine incorporation by up to 50%. Both swainsonine (1 microgram/ml) and castanospermine (50 micrograms/ml) resulted in mannose incorporation greater than 100% of untreated cells and neither drug affected methionine incorporation. 2. Incubation of NS-1 cells with tunicamycin resulted in a shift in the apparent molecular mass of the transferrin receptor from 96 kDa and 94 kDa to approximately 82 kDa. 3. Peptide N-glycosidase F digestion of the receptor from untreated cells resulted in the fully deglycosylated 82 kDa component as well as an 87 kDa component which represents partially deglycosylated receptor resistant to peptide N-glycosidase F digestion. 4. The receptor from swainsonine-treated cells was equally sensitive to peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H (endo H; resulting in both 87-kDa and 82-kDa components), whereas the receptor from castanospermine-treated cells was only partially sensitive to endo H. 5. Analysis of mannose- and fucose-labelled cellular glycopeptides by concanavalin-A--Sepharose chromatography showed that swainsonine (1 microgram/ml) treatment resulted in approximately 90% inhibition of the synthesis of complex N-glycans and an accumulation of fucosylated hybrid structures. In contrast, castanospermine (100 micrograms/ml) treatment resulted in only partial inhibition (60%) of the synthesis of complex N-glycans. 6. Analysis of the receptor from tunicamycin, swainsonine and castanospermine treated cells under nonreducing conditions showed a single component corresponding to the dimer, indicating that dimerisation of newly synthesised murine receptor is independent of carbohydrate. 7. The non-glycosylated receptor from tunicamycin-treated cells appears to bind transferrin as demonstrated by interaction with transferrin-Sepharose. 8. Surface expression of the receptor was not significantly altered in the presence of either swainsonine or castanospermine as judged by flow cytometry.