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.     

Cell-free sulfation of the contact site A glycoprotein of Dictyostelium discoideum and of a partially glycosylated precursor

An 80-kDa glycoprotein of Dictyostelium discoideum, designated contact site A, has been implicated in EDTA-stable cell adhesion. This protein is known to be the major sulfated protein of aggregation-competent cells and has been shown to contain two types of carbohydrate, sulfated type 1 and unsulfated type 2 carbohydrate moieties. Here we investigate the cell-free sulfation of this protein. In the homogenate of developing cells, [35S]sulfate was transferred by endogenous sulfotransferase from [35S]3'-phosphoadenosine-5'-phosphosulfate to the contact site A glycoprotein and to various other endogenous proteins. The sulfate was transferred to carbohydrate rather than to tyrosine residues. After differential centrifugation of the homogenate, the capacity for sulfation of the contact site A glycoprotein was barely detected in the plasma membrane-enriched 10,000 X g pellet fraction which contained the bulk of this glycoprotein, but was largely recovered in the 100,000 X g pellet fraction which contained only a small portion of this glycoprotein. After sucrose gradient centrifugation, the membranes containing the sulfation capacity were found to have a density characteristic for Golgi membranes. In immunoblots, monoclonal antibodies raised against the contact site A glycoprotein recognized not only this 80-kDa protein, but also a sulfatable 68-kDa protein found in the 100,000 X g pellet fraction. The 68-kDa protein did not react with monoclonal antibodies against type 2 carbohydrate but was converted by endoglycosidases F and H into a 53-kDa protein, indicating that it was a partially glycosylated form of the 80-kDa glycoprotein containing only type 1 carbohydrate. Isoelectric focusing showed that a substantial portion of the 68-kDa glycoprotein was unsulfated, even after cell-free sulfation. The 68-kDa glycoprotein was not found in the plasma membrane-enriched 10,000 X g pellet fraction and did not accumulate in parallel with the 80-kDa contact site A glycoprotein during cell development. We conclude that the 68-kDa glycoprotein is a precursor that is converted by attachment of type 2 carbohydrate and sulfation of type 1 carbohydrate into the mature 80-kDa glycoprotein. The precursor nature of the 68-kDa glycoprotein was supported by results obtained with mutant HL220 which is defective in glycosylation (Murray, B. A., Wheeler, S., Jongens, T., and Loomis, W. F. (1984) Mol. Cell. Biol. 4, 514-519). This mutant specifically lacks type 2 carbohydrate and produces a 68-Kda glycoprotein instead of the 80-kDa contact site A glycoprotein (Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2663-2670).(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.     

Wheat germ agglutinin binds to the contact site A glycoprotein of Dictyostelium discoideum and inhibits EDTA-stable cell adhesion

Wheat germ agglutinin (WGA), a lectin that primarily reacts with N-acetylglucosamine residues, specifically inhibits the EDTA-stable type of intercellular adhesion of aggregation competent Dictyostelium discoideum cells. The major WGA-binding protein of these cells is a developmentally-regulated glycolipoprotein of 80 kd apparent mol. wt., designated as contact site A. This glycoprotein is a target site of antibody fragments that block the EDTA-stable cell adhesion, and is characterized by sulfated carbohydrate residues. WGA does not significantly bind to glycoproteins of a mutant, HL220, which produces a 68-kd component in place of the 80-kd glycoprotein. Inhibition of N-glycosylation by tunicamycin causes wild-type cells to produce a WGA-binding but unsulfated 66-kd component and a non-binding 53-kd component. These results indicate that the 80-kd glycoprotein contains two classes of carbohydrate residues, a WGA-binding one that is defective in HL220, and another, sulfated, one that is absent from the 66-kd wild-type product; both are missing in the 53-kd protein. WGA and a monoclonal antibody that is blocked by N-acetylglucosamine were further used to probe for glycoproteins in the multicellular slug stage that share carbohydrate structures - and possibly functions - with the contact site A glycoprotein. Glycoproteins in the 95-kd range have previously been implicated in cell-to-cell adhesion during the slug stage. We distinguished a 95-kd glycoprotein that binds WGA from another one that binds antibody.     

Inhibition of cell adhesion in Dictyostelium discoideum by tunicamycin is prevented by leupeptin

The carbohydrate requirement for cell adhesion of aggregation-competent cells of Dictyostelium discoideum has been examined by use of a selective glycosylation inhibitor of N-glycosyl protein, tunicamycin (TM). TM completely inhibited EDTA-stable cell adhesion and glycosylation of some membrane glycoproteins in aggregation-competent cells of D. discoideum (Yamada, H., et al. (1982) J. Biochem. 92, 399-406). The present study showed that the inhibition of EDTA-stable cell adhesion by TM was prevented significantly when the cells were treated with TM in the presence of a protease inhibitor, leupeptin (LP), whereas the inhibition of glycosylation by TM was not prevented. The cell extract of aggregation-competent cells contained acid proteases, and LP strongly inhibited acid protease from D. discoideum in vitro. On analysis by SDS-polyacrylamide gel electrophoresis (PAGE), many protein bands present in the membrane fraction of control cells disappeared or decreased on TM treatment of the cells in the absence of LP, however, some of these proteins were restored when the cells were treated with TM in the presence of LP. These results strongly support an idea that EDTA-stable cell adhesion characteristic to aggregation-competent cells is mediated by glycoproteins with asparagine-linked carbohydrate. However, the requirement for the carbohydrate moiety of the glycoprotein in cell adhesion appears to be indirect in that it acts to protect the protein moiety from proteolytic degradation.     

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)     

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.     

Biosynthesis and post-translational processing of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). N-linked glycosylation affects cell-surface expression and ligand binding

LOX-1 (lectin-like oxidized low density lipoprotein receptor-1) is a type II membrane protein belonging to the C-type lectin family that can act as a cell-surface receptor for atherogenic oxidized low density lipoprotein (Ox-LDL) and may play crucial roles in atherogenesis. In this study, we show, by pulse-chase labeling and glycosidase digestion, that LOX-1 is synthesized as a 40-kDa precursor protein with N-linked high mannose carbohydrate chains (pre-LOX-1), which is subsequently further glycosylated and processed into the 48-kDa mature form within 40 min. Furthermore, when treated with an N-glycosylation inhibitor, tunicamycin, both tumor necrosis factor-alpha-activated bovine aortic endothelial cells and CHO-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO) exclusively produced a 32-kDa deglycosylated form of LOX-1. Cell enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence confocal microscopy demonstrated that the deglycosylated form of LOX-1 is not efficiently transported to the cell surface, but is retained in the endoplasmic reticulum or Golgi apparatus in tumor necrosis factor-alpha-activated bovine aortic endothelial cells, but not in BLOX-1-CHO cells. Radiolabeled Ox-LDL binding studies revealed that the deglycosylated form of LOX-1 expressed on the cell surface of BLOX-1-CHO cells has a reduced affinity for Ox-LDL binding. Taken together, N-linked glycosylation appears to play key roles in the cell-surface expression and ligand binding of LOX-1.     

Direct implication of surface mannosyl residues in cell adhesion of Dictyostelium discoideum

In the cell adhesion of aggregation-competent Dictyostelium cells, the requirement for the carbohydrate moiety of the glycoprotein appeared to be indirect in that it acts to protect the protein moiety from proteolytic degradation; however, the effect was limited to the tunicamycin (TM)-sensitive carbohydrate moiety (Hirano, T., et al. (1983) J. Biochem. 93, 1249-1257). In the present study, we showed that the EDTA-stable adhesion of aggregation-competent Dictyostelium cells was abolished by the treatment of intact cells with jack bean alpha-mannosidase, whereas neuraminidase, beta-galactosidase, beta-N-acetylhexosaminidase, or alpha-L-fucosidase had no effect. The EDTA-stable cohesiveness of TM-treated cells in the presence of leupeptin (TM/LP cells) was also abolished by the treatment of the cells with alpha-mannosidase. The effect of alpha-mannosidase was not prevented in the presence of LP. The N-glycoside-deficient contact site A (an adhesion-mediating glycoprotein) was obtained from TM/LP cells and was shown to have a molecular weight of 70,000. This protein (p 70) was shown to still have carbohydrates as detected by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) and subsequent staining of the gel with periodic acid-silver stain. Moreover, p 70 reacted with anti-gp 68, which has a specificity against alpha-mannosyl residues of carbohydrate chains. However, p 70 treated with alpha-mannosidase showed decreased reactivity with anti-gp 68. The monovalent antibody fragment of anti-contact site A or anti-p 70 inhibited EDTA-stable cell adhesion of both control and TM/LP cells. These results indicated that TM-resistant mannosyl residues of contact site A are directly involved in EDTA-stable adhesion of aggregation-competent cells. This is the first report of the direct involvement of the carbohydrate moiety in cell adhesion of aggregation-competent Dictyostelium cells. A schematic model is presented of the role of the carbohydrate moiety in EDTA-stable cell adhesion, including the direct effect of carbohydrates.     

Biochemical characterization and biosynthesis of the Ki-1 antigen in Hodgkin-derived and virus-transformed human B and T lymphoid cell lines

The Hodgkin-associated Ki-1 antigen was analyzed in different cell lines. In Hodgkin analogous L428 cells, biosynthetically labeled with radioactive amino acids, the Ki-1 antibody precipitated three glycoproteins with 90, 105, and 120 kDa, respectively. Surface-labeling revealed that the two larger components were membrane-associated forms of the Ki-1 antigen, although the 90-kDa molecule was shown in pulse-chase experiments to be the precursor of the 105- and 120-kDa forms. All three forms of the Ki-1 antigen possess a tunicamycin-sensitive 6-kDa N-linked carbohydrate moiety. O-Linked oligosaccharides could not be detected. Thus, the differences in m.w. are probably not due to glycosylation. The ionophore monensin prevented the appearance of the membrane-associated molecules, which demonstrated that they are assembled between the transcompartment of the Golgi complex and their insertion into the cell membrane. The 90-kDa precursor molecule cannot be generated by disulfide reduction from the two larger forms. After internal labeling with P-32, only the 105- and 120-kDa bands became visible, indicating that the Ki-1 molecule is phosphorylated after its processing into the two larger membrane-associated forms. Analysis of the Ki-1 antigens from other cell lines demonstrated that after external labeling of two other Hodgkin-derived cell lines, six Epstein-Barr virus lymphoblastoid cell lines and one human T leukemia virus I-positive T cell line, both the 105- and the 120-kDa membrane molecules could be detected, regardless of the presence or type of virus integrated.     

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.     

Purification, biochemical composition, and biosynthesis of the Mo3 activation antigen expressed on the plasma membrane of human mononuclear phagocytes.

Mo3 is an activation Ag expressed on the surface of human mononuclear phagocytes stimulated in vitro or in vivo by various activating factors. Mo3 is obtained by immunoprecipitation with anti-Mo3 mAb from lysates of PMA-stimulated U-937 cells. The Ag is a heterogeneous glycoprotein with a molecular mass range of 42 to 66 kDa (nonreducing conditions) containing N-linked carbohydrate chains. When the cells are treated with phosphatidylinositol-specific phospholipase C, greater than 60% of total precipitable gp42-66 Ag is released in the supernatant. This phosphatidylinositol-specific phospholipase C-sensitive linkage to the plasma membrane has provided a means for the one-step purification of Mo3 by immunoaffinity chromatography. The eluted soluble Mo3 (sMo3) was greater than 90% pure as documented by the appearance of a single major protein peak on reverse phase HPLC and SDS-PAGE. The average yield was 12.1 micrograms/10(8) cells. Sufficient quantities of sMo3 have been purified to permit the determination of amino acid and carbohydrate composition. Complex N-linked carbohydrates make up nearly 50% of the glycoprotein content and contribute to its heterogeneity. An anti-Mo3 polyclonal antiserum generated from sMo3 was used to immunoprecipitate Mo3 and its precursor from biosynthetically labeled, PMA-stimulated U-937 cells or LPS-stimulated monocytes. These 35S-methionine "pulse-chase" experiments demonstrated the existence of a 40- to 42-kDa endo-beta-N-acetylglucosaminidase-sensitive precursor, which over a period of 4 to 5 h gave rise to an endo-beta-N-acetylglucosaminidase-resistant, but N-glycanase-sensitive 42- to 66-kDa mature form.     

Constitutive overexpression of the contact site A glycoprotein enables growth-phase cells of Dictyostelium discoideum to aggregate.

The contact site A (csA) glycoprotein is a developmentally regulated cell adhesion molecule which mediates EDTA-stable cell contacts during the aggregation stage of Dictyostelium discoideum. A transformation vector was constructed which allows overexpression of the csA protein during the growth phase. In that stage the csA protein is normally not expressed; in the transformants it was transported to the cell surface and carried all modifications investigated, including a phospholipid anchor and two types of oligosaccharide chain. csA expression enabled the normal non-aggregative growth-phase cells to form EDTA-stable contacts in suspension and to assemble into three-dimensional aggregates when moving on a substratum. After prolonged cultivation of csA overexpressing transformants in nutrient medium the developmental program was found to be turned on, as it normally occurs only in starving cells. During later development of transformed cells, the csA glycoprotein remained present on the cell surface, while it is down-regulated in the wild type. It was detected in both the prestalk and prespore regions of the multicellular slugs made from transformed cells.     

Role of carbohydrates within variant surface glycoprotein of Trypanosoma congolense. Protection against proteolytic attack

The effects of tunicamycin on different aspects of structure and biosynthesis of variant surface glycoprotein from Trypanosoma congolense have been studied. Deglycosylated variant antigen becomes synthesized in vitro, is transported through the cell, and is deposited on the cell surface in equivalent amounts compared to the glycosylated species. In contrast to the glycosylated molecule only marginal amounts of high-molecular-mass fragments can be removed from the parasitic cell by externally added proteases in the case of tunicamycin-treated cells. Most of the material removed by proteases from the cell surface of tunicamycin-treated cells has a molecular mass lower than 2 kDa. Many additional proteolytic cleavage sites become accessible after removal of the glycan chains. There is no indication that in the deglycosylated molecule the same preferential protease-sensitive site exists as is found in the glycosylated species. These results suggest that glycosylation of variant surface glycoprotein could be important for the survival of the parasite within the host organism.     

Biosynthesis of the cell adhesion molecule uvomorulin (E-cadherin) in Madin-Darby canine kidney epithelial cells

The Ca(2+)-dependent cell adhesion molecule uvomorulin is a transmembrane glycoprotein that functions at the cell surface to regulate epithelial cell recognition and adhesion. We have investigated the temporal and spatial regulation of uvomorulin biosynthesis and cell surface expression in Madin-Darby canine kidney epithelial cells. We show that uvomorulin is synthesized as a precursor polypeptide (Mr 135,000) that is core glycosylated in the endoplasmic reticulum. The precursor is processed to the mature polypeptide (Mr 120,000) shortly after addition of complex carbohydrate groups in the late Golgi complex, but prior to delivery of the polypeptide to the cell surface. However, glycosylation is not required for either efficient processing of the precursor or transport of uvomorulin to the cell surface. At the cell surface, uvomorulin is turned over rapidly (t1/2 approximately 5 h). Induction of Ca(2+)-dependent cell-cell contact results in rapid localization of cell surface uvomorulin to regions of contact and an increase in the proportion of uvomorulin that is insoluble in buffers containing Triton X-100. These results indicate several regulatory steps in the biosynthesis and cell surface expression of uvomorulin in epithelial cells.     

Protein glycosylation regulates the externalization of two distinct classes of glucocorticoid-induced glycoproteins in rat hepatoma cells

The relationship of protein glycosylation to the externalization of glucocorticoid inducible alpha1-acid glycoprotein and mouse mammary tumor virus glycoproteins was examined in M1.54, a clonal population of mouse mammary tumor virus-infected rat hepatoma cells. Multiple freeze-thaw of isolated microsomes revealed that while alpha 1-acid glycoprotein is carried through the cell as a soluble component of vesicles, extracellular viral glycoproteins are initially membrane-associated. At concentrations of tunicamycin that specifically inhibited N-linked protein glycosylation, alpha 1-acid glycoprotein fractionated between the cellular and extracellular compartments. Thus, approximately one half of the newly synthesized, nonglycosylated (22,000 Mr) alpha 1-acid glycoprotein was rapidly secreted with kinetics similar to its glycosylated counterpart (release half-time of 60 min), while the remaining species first localized in an undefined intracellular compartment prior to its slow secretion (release half-time of 24 h). The same distribution of nonglycosylated alpha 1-acid glycoprotein was observed at various absolute levels of polypeptide, suggesting that this was not due simply to the saturation of an efficient secretory pathway at high polypeptide levels. In contrast to alpha 1-acid glycoprotein, no labeled viral antigens were released by tunicamycin-treated M1.54, while a nonglycosylated viral precursor glycopolyprotein was expressed intracellularly. Taken together, these results suggest that carbohydrate attachment strongly regulates the externalization of both alpha 1-acid glycoprotein and mouse mammary tumor virus species, which represent two distinct classes of extracellular glycoproteins.     

Transport and metabolism of 5'-nucleotidase in a rat hepatoma cell line

The biosynthesis of the ectoenzyme 5'-nucleotidase in the rat hepatoma cell line H4S has been studied by pulse-labeling with [35S]methionine and subsequent immunoprecipitation of the cell lysate. 5'-Nucleotidase is a membrane glycoprotein with an apparent molecular mass on SDS-gels of 72 kDa. The enzyme is initially synthesized as a 68-kDa precursor which is converted to the mature 72-kDa form in 15-60 min (t1/2 = 25 min). The molecular mass of the unglycosylated enzyme is approximately 58 kDa. Culturing the cells in the presence of varying concentrations of tunicamycin, an inhibitor of N-glycosylation, revealed six species of 5'-nucleotidase after sodium dodecyl sulfate/polyacrylamide electrophoresis. This indicates the presence of five N-linked oligosaccharide chains accounting for the difference between the 58-kDa polypeptide backbone and the 68-kDa species. The 68-kDa precursor is susceptible to cleavage by endo-beta-N-acetylglycosaminidase H; the 72-kDa mature protein is converted to several bands upon this treatment. This result indicates that part of 5'-nucleotidase keeps one or two high-mannose or hybrid chains in the mature form, even after prolonged pulse-chase labeling. The newly synthesized mature enzyme reaches the cell surface after 20-30 min. The half-life of 5'-nucleotidase is about 30 h in H4S cells. No immunoprecipitable 5'-nucleosidase is released into the culture medium.     

The structural proteins of infectious pancreatic virus are not glycosylated.

The major capsid protein, VP2, of infectious pancreatic necrosis virus, a nonenveloped icosahedral virus, contains six N-glycosylation consensus sequences (Asn-X-[Thr/Ser]). Since VP2 contains the major virus-neutralizing epitopes, the possible role for glycosylation in capsid formation and antigenicity was examined. The carbohydrate content of the virion proteins was determined by chemical detection, pulse-chase experiments,[3H]mannose labeling, and alteration of protein migration on sodium dodecyl sulfate-polyacrylamide gels after tunicamycin treatment. No glycosylation of any virion protein was observed when the carbohydrate nature of the glycoprotein of infectious hematopoietic necrosis virus was detected.     

In vivo sulfation of the contact site A glycoprotein of Dictyostelium discoideum

During the development of Dictyostelium discoideum from the growth phase to the aggregation stage, a glycoprotein with an apparent mol. wt. of 80 kd is known to be expressed on the cell surface. This glycoprotein, referred to as contact site A, has been implicated in the formation of species-specific, EDTA-stable contacts of aggregating cells. When developing cells were labeled in vivo with [³⁵S]sulfate, the 80-kd glycoprotein was found to be the most prominently sulfated protein. Another strongly sulfated protein had an apparent mol. wt. of 130 kd and was, like the 80-kd glycoprotein, developmentally regulated and associated with the particulate fraction of the cells. The [³⁵S]sulfate incorporated into the 80-kd and 130-kd proteins was not present as tyrosine-O-sulfate, a modified amino acid found in many proteins of mammalian cells. D. discoideum cells incubated with [³⁵S]sulfate in the presence of tunicamycin, an inhibitor of N-glycosylation, produced a 66-kd protein that reacted with monoclonal antibodies raised against the 80-kd glycoprotein, but no longer contained [³⁵S]sulfate. These results suggest that sulfation of the 80-kd glycoprotein occurred on carbohydrate residues. The possible importance of sulfation for a role of the 80-kd glycoprotein in cell adhesion is discussed.     

Effect of tunicamycin on the synthesis, processing, and secretion of pro-opiomelanocortin peptides in mouse pituitary cells

Pro-opiomelanocortin (POMC) is glycosylated and proteolytically cleaved to produce a number of smaller peptide hormones including adrenocorticotropic hormone (ACTH) and endorphin in mammalian pituitary and the mouse pituitary cell line AtT-20/D16v. When glycosylation of POMC is inhibited in AtT-20 cells with the drug tunicamycin, a 26,000-dalton protein appears in place of the glycosylated 29,000- and 32,000-dalton forms of POMC. The 26,000-dalton form found in tunicamycin-treated cells has the same [35S]methionine tryptic peptides as 29,000- and 32,000-dalton POMC, indicating that the decrease in apparent mass is most likely due to loss of carbohydrate and not to changes in the peptide backbone. The 4,500-dalton form of alpha(1-39)ACTH and the 3,000- and 11,000-dalton forms of endorphin are all present in tunicamycin-treated cells. The glycosylated form of alpha(1-39)ACTH, however, is missing and the glycosylated ACTH intermediates are replaced by unglycosylated ACTH intermediates. Pulse-chase studies demonstrate that the 26,000-dalton unglycosylated POMC is the precursor of the smaller ACTH and endorphin molecules in tunicamycin-treated cells. Furthermore, all of the forms of ACTH and endorphin found in tunicamycin-treated cells are secreted. Thus, it appears that glycosylation is not an essential step for correct cleavage or secretion of POMC or its products.