Possible existence of a light-inducible protein that inhibits sexual cell fusion in Dictyostelium discoideum.

Sexual cell fusion is an initial step of macrocyst formation in Dictyostelium discoideum and requires environmental conditions such as darkness, plenty of water and the presence of calcium ions. We have been analyzing the mechanism of sexual cell fusion between HM1 and NC4, heterothallic strains in D. discoideum. Cells of these strains have been shown to be fusion competent when cultured in a liquid medium in darkness, but not so when cultured on agar plates or in a liquid medium in the light. Two cell-surface proteins, gp70 and gp138, have been identified as target molecules for fusion-blocking antibodies and therefore as relevant to sexual cell fusion. In the present study, gp70 was shown to be present in HM1 cells cultured in the light, and fusion incompetent. Intact HM1 cells cultured in the light were unable to absorb the fusion-blocking activity of antibodies against membrane components of fusion-competent HM1 cells, whose activity had been shown to be absorbed by gp70, but they did so after separation of proteins in the SDS-PAGE. In addition, fusion-competent HM1 cells were found to lose their fusion competence by subsequent cultivation in the light. This loss of competence was cycloheximide sensitive, indicating that de novo synthesis of proteins was necessary for this inhibition. From these results, we presume that light induces a protein that hinders the interaction of gp70 in HM1 cells with its receptor on the NC4 cell surface and thereby inhibits the sexual process between these strains.     

Identification of the cell surface molecule involved in sexual cell fusion of Dictyostelium discoideum

Dictyostelium discoideum was used as a model system for elucidating the molecular mechanism of sexual cell fusion. In heterothallic strains NC4 and HM1 of D. discoideum, complements in mating type, amoeboid cells acquire fusion competence only under certain environmental conditions, such as the presence of excess water and a certain period of darkness, to fuse sexually. The surface of cells which acquired fusion competence was found to possess specific antigens. Monovalent antibodies prepared from rabbit antiserum against fusion-competent NC4 cells inhibit the sexual cell fusion of these cells completely. Two specific antigenic proteins, 39 and 138 k Da in relative molecular mass and specific for fusion-competent cells, were detected. Only one, the 138-k Da protein, was capable of neutralizing the fusion-inhibitory activity of the monovalent antibody. These results show that the 139-k Da protein is the one involved in the sexual cell fusion of NC4 and HM1 strains in D. discoideum.     

Fusion-inhibiting monoclonal antibodies and their relevant antigens in relation to sexual process of Dictyostelium discoideum

Sexual cell fusion occurs between NC4 and HM1, the heterothallic strains in Dictyostelium discoideum. Cells of these strains are fusion incompetent when cultured on agar plates in the light and become fusion competent upon cultivation in a liquid medium in darkness. Two cell-surface components, gp70 and gp138, have been identified and characterized as being relevant to sexual cell fusion. Both are glycoproteins, and the former is detected only in fusion-competent HM1 cells, while the latter is detected both in fusion-competent HM1 and fusion-competent NC4 cells. We therefore suspect gp 70 to be responsible for cell recognition and gp138, for membrane fusion. Therefore, NC4 cells are expected to possess specific surface molecule(s) that can be recognized by HM1 cells. In the present study, we raised monoclonal antibodies (mAbs) against membrane fractions of NC4 cells and selected fusion-inhibiting mAbs to identify novel molecules related to sexual cell fusion in D. discoideum. Out of the five mAbs we obtained three, DE1, GG6, and HH9, were characterized. DE1 recognized antigens that specifically existed in fusion-competent NC4 cells but not in fusion-incompetent NC4 or HM1 cells. GG6 recognized cell-surface proteins with approximate molecular weights of 125 and 32 kDa in both fusion-competent NC4 and fusion-competent HM1 cells. In addition GG6 also recognised other proteins commonly present in fusion-incompetent cells. The 125 kDa protein appeared to be the same as gp138. The epitope recognized by HH9 was sodium dodecyl sulfate (SDS)-sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and Characterization of Dictyostelium Mutants Defective in Sexual Cell Fusion

Sexual cell fusion in the cellular slime mold Dictyostelium discoideum occurs between cells of opposite (heterothallic system) or same (homothallic system) mating types. It also requires certain environmental conditions such as darkness and abundance of water, and thus offers an interesting model system for analyzing mechanisms of cell recognition and of cellular response to environmental factors. We have been studying the mechanism of sexual cell fusion, using two heterothallic strains, NC4 and HM1 of D. discoideum. Two cell-surface glycoproteins, gp70 and gp138, have been identified as relevant molecules in the cell fusion of these strains. The former is specific to mat a cells (HM1) and the latter, common to both mat a and mat A (NC4). Involvement of cell-surface carbohydrates has also been suggested. However, the fuctions of the above fusion-related molecules are still elusive. In the present study, we isolated fusion-deficient mutants from a mutagenized mat A strain of D. discoideum to set up combined genetic and biochemical analyses. Among the three nonconditional mutants obtained, two were normal in the fruiting-body formation, asexual development, but one was aggregateless ( agg ⁻). Further analysis of these mutants would provide detailed information on the mechanism of sexual cell fusion.     

Concanavalin A and wheat germ agglutinin binding glycoproteins associated with cell fusion and zygote differentiation in Dictyostelium discoideum: effects of calcium ions and tunicamycin on glycoprotein profiles

To determine which glycoproteins may be critical to sexual development in Dictyostelium discoideum, cell samples from different developmental stages were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and blotted to nitrocellulose. Concanavalin A (ConA) and wheat germ agglutinin (WGA) binding proteins were visualized on the blots using an immunochemical procedure employing peroxidase-antiperoxidase. ConA labelled at least 28 proteins, but only one band showed calcium-dependent changes in its expression. WGA bound at least 30 proteins and changes in several bands were observed that did not occur in calcium-deficient controls. Two WGA-binding glycoproteins which migrated at 200 and 166 kilodaltons (kDa), respectively, showed developmental changes associated with the time of cell fusion. One WGA-binding and one ConA-binding glycoprotein migrating at 130 and 126 kDa, respectively, appeared later during sexual development, in association with the phase of zygote differentiation. Several WGA- and ConA-binding glycoproteins decreased during sexual development, but were not affected by the absence of calcium ions. Tunicamycin (1 microgram/mL) inhibited cell fusion when added to sexual cultures prior to the appearance of the 166-kDa glycoprotein gp166. The effects of this inhibitor on development support the importance of glycoproteins to cell fusion during sexual development in D. discoideum.     

Protein Synthesis Initiated by Cell Fusion in Dictyostelium discoideum

D. discoideum has two alternative developmental pathways. If cells of two complement mating-type strains, NC4 and HM1, fuse sexually, a giant cell is produced which subsequently develops into a macrocyst, the sexual structure of this organism. However, if fusion fails to occur and cells are starved, a fruiting-body is produced instead of a macrocyst. In this paper, a two-dimensional polypeptide gel electrophoresis study showed that giant cells produce specific polypeptides which may possibly be involved in macrocyst development. Out of total 497 polypeptides which appeared in a giant cell during an incubation period of 13 hr, 92 were the specific for giant cells. Four of these polypeptides were appeared within only 1 hr after the cell fusion. The other 405 were non-specific polypeptides which appeared in both giant cells and NC4 or/and HM1 cells. However, the patterns and the rates of production of each polypeptide during the incubation period were different between these cells.     

Fusion of cell ghosts with intact cells in Dictyostelium discoideum: Differential response of opposite mating-type cells

The sexual cycle of Dictyostelium discoideum is initiated by the fusion of cells that are of opposite mating types (e.g. NC4- and HM1-type cells). Cells grown in light on agar plates are not capable of sexual cell fusion, but become capable when cultured in the dark in a liquid medium. Cells in the incapable state are called fusion-incompetent cells, and cells in the latter state, fusion-competent cells. To gain some understanding of the mechanism of cell fusion, cell ghosts prepared by freeze-thawing intact cells were incubated with intact cells. The cell ghosts killed the intact cells by directly fusing with them, the extent of fusion depending on the particular strains employed and the fusion-competency of the intact cells and of the cells from which the cell ghosts had been prepared. A detailed examination revealed that fusion-competent NC4 cells were always more easily killed by cell ghosts than fusion-incompetent NC4 cells. It also became apparent that cell ghosts prepared from fusion-competent NC4 cells killed all cell types far more efficiently than did those prepared from fusion-incompetent NC4 cells. However, fusion-competent and fusion-incompetent HM1 cells were equally sensitive to cell ghosts, and cell ghosts prepared from fusion-competent HM1 cells had the same ability to kill as those prepared from fusion-incompetent HM1 cells. From these findings, it thus appears that opposite mating-type cells have distinct membrane properties related to sexual cell fusion.     

Lectin binding and inhibition studies reveal the importance of D-glucose, D-mannose and N-acetylglucosamine during early sexual development of Dictyostelium discoideum

Fluorescein-conjugated and non-conjugated lectins were used to determine which surface sugars are involved in the early events of sexual (macrocyst) development in Dictyostelium discoideum. Only zygote giant cells showed unique binding of FITC-WGA and FITC-PNA while all cell types (amoebae, gametes, binucleates, giant cells) showed identical patterns of FITC-Con A, -Gorse and -RCA II binding. In spite of its non-selective labelling of all cell types, Con A inhibited macrocyst formation. The temporal addition of Con A with and without specific hapten sugars indicates the importance of both D-mannose and D-glucose in phagocytosis and, possibly, cell fusion. WGA also inhibited macrocyst formation. Varying the time of addition of the lectin plus/minus its primary hapten sugar implicates N-acetylglucosamine as being important in cell fusion. Neither Gorse, RCA II nor PNA had any detectable inhibitory effects on macrocyst development leaving the appearance of increased PNA receptors at the giant cell surface as an enigma.     

High mannose type N-linked oligosaccharides on endothelial cells may influence β2 integrin mediated neutrophil adherence in vitro

We report herein on the role of N-linked oligosaccharide processing of endothelial cell surface proteins on the adhesion of neutrophils. Monolayers of human umbilical vein endothelial cells were treated for 24 h with deoxymannojirimycin (DMJ), an inhibitor of golgi mannosidase I, which results in changes in glycoprotein processing, and then incubated with neutrophils to examine their ability to adhere to the treated endothelial cells. Treatment with DMJ, which leads to accumulation of high mannose type oligosaccharides, resulted in a twofold increase in adherence of phorbol ester (PMA) activated neutrophils compared to attachment to untreated endothelial cells. This adherence was likely mediated by the β2 integrin, Mac-1, and could be specifically inhibited with monoclonal antibodies to ICAM-1 and to the integrin β2 subunit. Similarly, IL-1 treatment resulted in a β2 integrin mediated increase in neutrophil adherence to the DMJ treated endothelial cells in a dose dependent manner. However, the IL-1 induced adherence was not significantly inhibited by the anti-ICAM-1 antibody, thus, suggesting the presence of other inducible components on the endothelial cell surface. Our results demonstrate that alterations in glycosylation of N-linked oligosaccharides, resulting in the synthesis of high mannose type sugars on molecules that may interact with the β2 integrins, leads to an increased adherence of PMA activated neutrophils to endothelial cells. © 1993 Wiley-Liss, Inc.     

Identification of a major heparin and cell binding site in the LG4 module of the laminin alpha 5 chain

The G domain of the laminin alpha chains consists of five homologous G modules (LG1-5) and has been implicated in various biological functions. In this study, we identified an active site for cell and heparin binding within the laminin alpha5 G domain using recombinant proteins and synthetic peptides. Recombinant LG4, LG5, and LG4-5 modules were generated using a mammalian expression system. The LG4 and LG4-5 modules were highly active for cell binding, whereas the LG5 module alone showed only weak binding. Heparin inhibited cell binding to the LG4-5 module, whereas no inhibition was observed with EDTA or antibodies against the integrin beta(1) subunit. These results suggest that the LG4-5 module interacts with a cell surface receptor containing heparan sulfate but not with integrins. Solid-phase assays and surface plasmon resonance measurements demonstrated strong binding of the LG4 and LG4-5 modules to heparin with K(D) values in the nanomolar range, whereas a 16-fold lower value was determined for the LG5 module. Treatment with glycosidases demonstrated that N-linked carbohydrates on the LG5 module are complex-type oligosaccharides. The LG4-5 module, devoid of N-linked carbohydrates, exhibited similar binding kinetics toward heparin. Furthermore, cell binding was unaffected by removal of N-linked glycosylation. To localize active sites on the LG4 module, various synthetic peptides were used to compete with binding of the tandem module to heparin and cells. Peptide F4 (AGQWHRVSVRWG) inhibited binding, whereas a scrambled peptide of F4 failed to compete binding. Alanine replacements demonstrated that one arginine residue within F4 was important for cell and heparin binding. Our results suggest a critical role of the LG4 module for heparan sulfate-containing receptor binding within the laminin alpha5 chain.     

Characterization and distribution of multiple antigens on N-linked oligosaccharides of Dictyostelium discoideum proteins

Monoclonal antibodies were prepared against a mixture of purified lysosomal enzymes from Dictyostelium discoideum. Three classes of antibodies were found which recognized distinct antigenic determinants on N-linked oligosaccharides of multiple proteins. The structure of the determinants was studied by competition assays using monosaccharides and oligosaccharide/glycopeptide fractions prepared from one Dictyostelium lysosomal enzyme or other sources. The results of these studies suggest that one class of antibody recognizes an epitope containing residues of Man-6-SO4, another recognizes a domain containing a modified GlcNAc, and the third class recognizes an undefined determinant that involves the oligosaccharide. The three determinants are found on multiple overlapping, but nonidentical sets of glycoproteins. The ability to produce monoclonal antibodies against unusual N-linked oligosaccharides offers a powerful tool which can be used to investigate the occurrence, structure, biosynthesis, and the biological roles of these highly immunogenic saccharides.     

Cell fusion activities of Hantaan virus envelope glycoproteins

Hantaan virus (HTNV)-infected Vero E6 cells undergo cell fusion with both infected and uninfected cells under low-pH conditions. Flow cytometry and fluorescence microscopy of HTNV-infected Vero E6 cells showed that envelope glycoproteins (GPs) were located both on the cell surface and in the cytoplasm. Neutralizing monoclonal antibodies (MAbs) against the G1 and G2 envelope GPs inhibited cell fusion, whereas nonneutralizing MAbs against G1 or G2 and MAbs against the nucleocapsid protein (NP) did not. Transfected Vero E6 cells that expressed GPs but not those that expressed NP fused and formed syncytia. These results indicate that HTNV GPs act as fusogens at the cell surface. No fusion activity was observed either in infected Vero cells that were passaged more than 150 times or in BHK-21 cells, although GPs appeared to localize to the cell surface. This variability in fusion induction suggests the involvement of host cell factors in the process of cell membrane fusion.     

Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins.

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin-Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N-linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N-linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N-linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal-mediated in polarized and non-polarized cells.     

Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells

Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands at 55 and 51 kDa for the 55-kDa TNF receptor and a major 75-kDa and a minor 65-kDa band for the 75-kDa TNF receptor. All these bands specifically bound TNF alpha and TNF beta in ligand blot experiments. The exclusive specificity of monoclonal antibodies of the utr series for the 75.65-kDa bands and of the htr series for the 55.51-kDa bands was demonstrated with the purified antigens on Western blots. Both TNF receptor types were found to contain N-linked carbohydrates. N-terminal amino acid sequence analysis of the 55- and 51-kDa bands of the 55-kDa TNF receptor revealed identical sequences suggesting a possible truncation at the C-terminal end. Two different N-terminal sequences were determined for the 65-kDa band. One corresponded to the published sequence of ubiquitin; the other was therefore assumed to be a unique sequence of the 75-kDa TNF receptor. Additional internal sequences of this receptor were determined after proteolytic cleavage.     

Preparation and characterization of monoclonal antibodies to an N-linked oligosaccharide

Two monoclonal antibodies to an N-linked oligosaccharide, MT-5 and MT-9, have been prepared by immunization with a pyridylaminated, asialylated, galactosylated, fucosylated, bisected biantennary sugar. The reactivity of these antibodies was monitored by their reaction with human asialoglycophorin in a solid-phase enzyme-linked immunosorbent assay. Both antibodies reacted with the sugar chains of various human glycoproteins such as immunoglobulin G, transferrin, gamma-glutamyl transpeptidase, alpha 1-acid glycoprotein, and alpha-fetoprotein. Treatment of asialoglycophorin with beta-N-acetylhexosaminidase or alpha-mannosidase resulted in reduction of the binding to these antibodies. The reactivity of MT-5 to asialoglycophorin was slightly inhibited by D-mannose and N-acetylglucosamine, whereas that of MT-9 was inhibited by D-mannose, N-acetyl-D-glucosamine, chitobiose, and L-fucose. The epitope specificity of MT-5 appears to be a sugar chain containing biantennary N-acetyl-D-glucosamine residues, the bisected N-acetyl-D-glucosamine residue, and a trimannosyl core. The epitope to which MT-9 is directed may be a complex made up of beta-mannose, chitobiose, and L-fucose. These studies indicate that immunization with pyridylaminated sugars can produce antibodies that recognize N-linked oligosaccharides. Monoclonal/polyclonal antibodies to the N-linked sugar chains of glycopeptides would be useful in such studies of proteins.     

Nuclear Behavior in Artificially Induced Multi-Nuclear Cells of Dictyostelium discoideum

The culture medium of the strain CK-8 of the cellular slime mold Polysphondylium pallidum contains a cell-fusion induction factor. Cells of the two opposite mating type strains NC-4 and HM1 of Dictyostelium discoideum were treated to induce cell fusion with the diluted fraction of CK-8 cultures, F2, which contains the factor and consequently numerous multinuclear cells were produced. NC-4 and HM1 usually fuse in the sexual cycle and form large multinuclear cells, called giant cells, which develop into macrocysts. These cells are very similar in morphology to the multinuclear cells produced following F2 treatment, however, the latter cells did not develop into macrocysts. In the sexually formed multinuclear cells, only two haploid nuclei fused to form a diploid nucleus and all others degenerate as previously reported. However, in the artificially produced multinuclear cells, no nuclear-fusion and degeneration took place. They stayed as heterokaryons and seem to lyse within 20 h incubation.     

Depletion of glycoprotein gp85 from virosomes made with Epstein-Barr virus proteins abolishes their ability to fuse with virus receptor-bearing cells.

Entry of an enveloped virus such as Epstein-Barr virus (EBV) into host cells involves fusion of the virion envelope with host cell membranes either at the surface of the cell or within endocytic vesicles. Previous work has indirectly implicated the EBV glycoprotein gp85 in this fusion process. A neutralizing monoclonal antibody to gp85, F-2-1, failed to inhibit binding of EBV to its receptor but interfered with virus fusion as measured with the self-quenching fluorophore octadecyl rhodamine B chloride (R18) (N. Miller and L. M. Hutt-Fletcher, J. Virol. 62:2366-2372, 1988). To test further the hypothesis that gp85 functions as a fusion protein, EBV virion proteins including or depleted of gp85 were incorporated into lipid vesicles to form virosomes. Virosomes were labeled with R18, and those that were made with undepleted protein were shown to behave in a manner similar to that of R18-labeled virus. They bound to receptor-positive but not to receptor-negative cells and fused with Raji cells but not with receptor-positive, fusion-incompetent Molt 4 cells; monoclonal antibodies that inhibited binding or fusion of virus inhibited binding and fusion of virosomes, and virus competed with virosomes for attachment to cells. In contrast, virosomes made from virus proteins depleted of gp85 by immunoaffinity chromatography remained capable of binding to receptor-positive cells but failed to fuse. These results are compatible with the hypothesis that gp85 is actively involved in the fusion of EBV with lymphoblatoid cell lines and suggest that the ability of antibody F-2-1 to neutralize infectivity of EBV represents a direct effect on the function of gp85 as a fusion protein.     

Developmental regulation of a prestalk- and stalk-enriched protein in Dictyostelium discoideum

Abstract. Monoclonal antibodies reactive with proteins specifically present either in the prespore cells or the prestalk cells of Dictyostelium discoideum were obtained. Four of them recognized prespore-enriched proteins, as shown by both immunoblotting assays and immunofluorescent staining. The other monoclonal antibody ( mab150 ) produced more than 10 protein bands when reacted with both prespore and prestalk cell extracts in immunoblotting assays. However, a protein band with molecular weight 35 000 (st35) was specifically detected in prestalk cells as well as mature stalk cells. St35 was solubilized from the Triton X-100 insoluble fraction of mature stalks by sodium dodecyl sulfate (SDS). The purified sample gave a single spot on two-dimensional gel electrophoresis, with pI of 5.0. During development, st35 first appeared at the tipped aggregate stage and accumulated up to stalk-cell formation without modification. The protein was not lost even when slugs were disaggregated. The importance of the tipped aggregate stage for prestalk differentiation as well as prespore differentiation is discussed.     

Developmental changes in glycosylation and targeting of lysosomal proteins in Dictyostelium discoideum

Abstract. Changes during the development of Dictyostelium discoideum , in the abundance, synthesis, and cell-type-specific distribution of modifications on N-linked oligosaccharides, were measured using specific affinity probes for N-linked moeities. Total proteins and individual lysosomal enzymes were reacted with three monoclonal antibodies raised against Dictyostelium proteins (recognizing epitopes containing mannose 6-sulfate, sulfated N-acetylglucosamine, and an undefined but unsulfated N-linked group, respectively), the mammalian 215-kDa phosphomannosyl receptor, and Con A. Independent and dramatic changes in the reaction of the antibodies and phosphomannosyl receptor with protein were observed during development, whereas modest changes were observed in Con A binding. The two sulfated antigens, but not the other moeities, were reduced preferentially in prestalk and mature stalk cells. The lysosomal enzyme β-glucosidase, which is synthesized late in development, binds poorly to the phosphomannosyl receptor and contains little of the three antigens. The subcellular transport of lysosomal enzymes also changes during development, as most are not targeted to lysosomes as is normal, but are secreted in precursor form.     

Targeted disruption of genes for gp138, a cell-fusion-related protein in Dictyostelium discoideum, revealed the existence of a third gene

The sexual cycle of the cellular slime mold, Dictyostelium discoideum , offers a suitable experimental system to analyze sexual cell interactions. We have been analyzing molecular mechanisms involved in sexual cell fusion using complementary heterothallic strains in D. discoideum and have identified several cell surface proteins involved in the process. One of them, gp138 is present in strains of both mating types and considered to be responsible for membrane fusion itself. Two genes with high mutual homology, GP 138 A and GP 138 B , have been identified so far as encoding this protein. Expression of antisense RNA for GP 138 B has been shown to suppress sexual cell fusion, confirming the critical importance of these genes in sexual cell fusion. However, neither the functional relationship of the two gp138 genes nor the possibility of the existence of more genes that encode gp138 has been determined yet. In the present study, GP 138 A and GP 138 B were disrupted by homologous recombination in an effort to clarify these points. Analysis of the double knock-out mutants suggested the presence of a third gene for gp138.