Mutations affecting a surface glycoprotein, gp80, of Dictyostelium discoideum.

We isolated two independent mutations in Dictyostelium discoideum that result in the absence of the antigenic determinant recognized by monoclonal antibody E28D8. This antibody reacts with a post-translational modification on the surface glycoprotein gp80 and several other proteins. Both of the mutations occur in the same locus, modB, which was mapped to linkage group VI. The modB mutations result in sufficient alteration of gp80 that it is absent or unrecognizable by two-dimensional gel electrophoresis. Strains carrying modB mutations exhibit "contact sites A"-mediated cell-cell adhesion although more weakly than do wild-type strains and develop to fruiting bodies carrying viable spores. Although gp80 has been implicated in the mechanism of cell-cell adhesion in D. discoideum, it is clear from the behavior of these mutant strains that the determinant on gp80 recognized by E28D8 is not necessary for either morphogenesis or reduced EDTA-resistant adhesion.     

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.     

Cell-cell adhesion molecules in Dictyostelium

Multicellularity in the cellular slime mold Dictyostelium discoideum is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive adhesion sites are expressed very early in the development cycle and a surface glycoprotein of 24,000 Da is known to be responsible for these sites. The EDTA-resistant contact sites begin to accumulate on the cell surface at the aggregation stage of development. Several glycoproteins have been implicated in the EDTA-resistant type of cell-cell binding and the best characterized one has an Mr of 80,000 (gp80). gp80 mediates cell-cell binding via homophilic interaction and its cell binding site has been mapped to an octapeptide sequence. The mechanism by which gp80 mediates cell-cell adhesion will be discussed.     

Inhibition of cell-cell adhesion and morphogenesis of Dictyostelium by carnitine.

Carnitine (gamma-trimethylammonium beta-hydroxy-butyric acid) possesses the novel property of preventing cell aggregation elicited by clusterin or by fibrinogen (I.B. Fritz and K. Burdzy, J. Cell. Physiol., 140:18-28 [1989]). In investigations reported here, we show that carnitine also affects cell-cell adhesion in Dictyostelium discoideum, a cellular slime mold whose cells interact in specific and complex manners during discrete stages of development. Two types of cell adhesion systems sequentially appear on the surface of developing Dictyostelium cells, involving the surface glycoprotein gp24 which mediates EDTA-sensitive binding sites, and the surface glycoprotein gp80 which mediates the EDTA-resistant binding sites. Addition of increasing concentrations of D(+)-carnitine and L(-)-carnitine resulted in a progressive inhibition of both the EDTA-sensitive binding sites and the EDTA-resistant binding sites of Dictyostelium cells at different stages of development. In contrast, comparable or higher concentrations of choline, acetyl-beta-methylcholine, or deoxycarnitine had no detectable effects on cell aggregation. Concentrations of carnitine required for 50% inhibition of EDTA-resistant adhesion sites were found to be dependent upon levels of gp80 expressed by Dictyostelium, with greatest inhibition by carnitine of reassociation of cells containing the lowest levels of gp80. Removal of carnitine from cells by washing resulted in the rapid restoration of the ability of Dictyostelium to form aggregates and to resume normal development. We discuss possible mechanisms by which carnitine inhibits the aggregation of cells.     

Mapping of the monoclonal antibody 80L5C4 epitope on the cell adhesion molecule gp80 of Dictyostelium discoideum

EDTA-resistant cell-cell binding sites are expressed on Dictyostelium discoideum cells at the aggregation stage of development. A cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate these binding sites via homophilic interaction. We have previously raised a monospecific monoclonal antibody 80L5C4 against gp80, which blocks the cell binding site of gp80 (Siu, C.-H., Lam, T.Y. and Choi, A.H.C. (1985) J. Biol. Chem. 260, 16030-16036). To map the 80L5C4 epitope, gp80 was digested with protease V8, and the smallest proteolytic fragment that retained immunoreactivity with 80L5C4 was about 27,000 Da, corresponding to the amino-terminal fragment predicted from the cleavage sites. In addition, cDNA fragments containing different gp80 coding regions were used to construct trpE/gp80 gene fusions in the expression vector pATH10. An analysis of these fusion proteins led to the mapping of the 80L5C4 epitope to a 51 amino-acid segment between residues 123 and 173.     

Molecular mechanisms of cell-cell interaction in Dictyostelium discoideum

During development of the cellular slime mold Dictyostelium discoideum, cells migrate in response to cAMP to form aggregates, which give rise to fruiting bodies consisting of two major cell types: spores and stalk cells. Multicellularity is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive binding sites are expressed at the initial stage of development. At the aggregation stage, cells acquire EDTA-resistant binding sites, which are mediated by a cell-surface glycoprotein of Mr80,000 (gp80). gp80 is preferentially associated with cell surface filopodia, which are probably involved in the initiation of contact formation between cells. Covaspheres conjugated with gp80 bind specifically to aggregation-stage cells. The binding can be inhibited by precoating cells with an anti-gp80 monoclonal antibody, thus suggesting that gp80 mediates cell-cell binding via homophilic interaction. The structure of gp80 predicted from its cDNA sequence can be divided into three major domains: a membrane anchor, a hinge, and a globular region. An analysis of fusion proteins containing different gp80 segments shows that the cell-binding activity resides in the globular region. In the postaggregation stages, gp80 is replaced by other surface glycoproteins in maintaining cell-cell adhesion. One of them has a Mr of 150,000 (gp150). Anti-gp150 antibodies have no effect on aggregation-stage cells, but they disrupt cell-cell adhesion at subsequent stages. It becomes evident that the complex phenomena of cell adhesion and tissue organization involve the participation of a number of surface glycoproteins.     

The membrane glycoprotein gp150 is encoded by the lagC gene and mediates cell-cell adhesion by heterophilic binding during Dictyostelium development

gp150 is a membrane glycoprotein which has been implicated in cell-cell adhesion in the postaggregation stages of Dictyostelium development. An analysis of its tryptic peptides by mass spectrometry has identified gp150 as the product of the lagC gene, which was previously shown to play a role in morphogenesis and cell-type specification. Antibodies raised against the GST-LagC fusion protein specifically recognized gp150 in wild-type cells and showed that it is missing in lagC-null cells. Immunolocalization studies have confirmed its enrichment in cell-cell contact regions. In mutant cells that lack the aggregation stage-specific cell adhesion molecule gp80, gp150 is expressed precociously. Moreover, these cells acquire EDTA-resistant cell-cell binding during aggregation, suggesting a role for gp150 in this process. Cells in which the genes encoding gp80 and gp150 are both inactivated do not acquire EDTA-resistant cell adhesion during aggregation. Strains transformed with an actin 15::lagC construct express gp150 precociously, but do not show EDTA-resistant adhesion during early development. However, vegetative cells expressing gp150 can be recruited into aggregates of 16-h lagC-null cells. These results, together with those obtained with the cell-to-substratum binding assay, indicate that gp150 mediates cell-cell adhesion via heterophilic interactions with another component that accumulates during the aggregation stage.     

Purification and partial characterization of a cell adhesion molecule (gp150) involved in postaggregation stage cell-cell binding in Dictyostelium discoideum.

A cell surface glycoprotein of apparent Mr 150,000 (gp150) has been implicated in mediating EDTA-resistant cell-cell adhesion in Dictyostelium discoideum. A simple purification scheme making use of high-performance liquid chromatography has been devised to purify gp150 to near homogeneity. Purified gp150 was capable of neutralizing the effect of a rabbit antiserum raised against gel-purified gp150, which was previously reported to be a potent inhibitor of cell-cell adhesion (Geltosky, J. E., Weseman, J., Bakke, A., and Lerner, R. A. (1979) Cell 18, 391-398). The binding of 125I-labeled gp150 to intact cells was both dose-dependent and saturable, demonstrating the presence of specific cell surface binding sites for gp150. When reassociation of postaggregation stage cells was carried out in the presence of soluble gp150, aggregate formation was strongly inhibited. In contrast, gp150 failed to exert any effect on cells at the aggregation stage. The inhibitory effect of gp150 was sensitive to protease treatment, suggesting that the protein moiety is crucial to gp150 function. These results, taken together, provide direct evidence that gp150 is a cell-cell adhesion molecule involved in cell-cell binding in the postaggregation stage of Dictyostelium development.     

Mapping of a cell-binding domain in the cell adhesion molecule gp80 of Dictyostelium discoideum

At the aggregation stage of Dictyostelium discoideum development, a cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate the EDTA-resistant type of cell-cell adhesion via homophilic interaction (Siu, C.-H., A. Cho, and A. H. C. Choi. 1987. J. Cell Biol. 105:2523-2533). To investigate the structure-function relationships of gp80, we have isolated full length cDNA clones for gp80 and determined the DNA sequence. The deduced structure of gp80 showed three major domains. An amino-terminal globular domain composed of the bulk of the protein is supported by a short stalk region, which is followed by a membrane anchor at the carboxy terminus. Structural analysis suggested that the cell-binding domain of gp80 resides within the globular domain near the amino terminus. To investigate the relationship of the cell-binding activity to this region of the polypeptide, three protein A/gp80 (PA80) gene fusions were constructed using the expression vector pRIT2T. These PA80 fusion proteins were assayed for their ability to bind to aggregation stage cells. Binding of 125I-labeled fusion proteins PA80I (containing the Val123 to Ile514 fragment of gp80) and PA80II (Val123 to Ala258) was dosage dependent and could be inhibited by precoating cells with the cell cohesion-blocking mAb 80L5C4. On the other hand, there was no appreciable binding of PA80III (Ile174 to Ile514) to cells. Reassociation of cells was significantly inhibited in the presence of PA80I or PA80II. In addition, 125I-labeled PA80II exhibited homophilic interaction with immobilized PA80I, PA80II, or gp80. The results of these studies lead to the mapping of a cell-binding domain in the region between Val123 and Leu173 of gp80 and provide direct evidence that the cell-binding activity of gp80 resides in the protein moiety.     

Cell-cell adhesion and morphogenesis in Dictyostelium discoideum

During development of Dictyostelium discoideum, cells acquire EDTA-resistant cell-cell adhesion at the aggregation stage. The EDTA-resistant cell binding activity is associated with a cell surface glycoprotein of Mr 80,000 (gp80), which mediates cell-cell binding via homophilic interaction. Analysis of the structure of gp80 deduced from cDNA sequence reveals the presence of three internally homologous segments in the NH2-terminal domain, which also contains regions with homology to the neural cell adhesion molecule. Secondary structure predictions show an abundance of beta-structures and very few alpha-helices. This is confirmed by circular dichroism measurements. It is likely that the homologous segments are organized into globular structures, extended from the cell surface by a Pro-rich stalk domain. The cell binding activity of gp80 resides within the first globular repeat of the NH2-terminal domain and has been mapped to a 51 amino acid region between Val123 and Leu173. Synthetic oligopeptides corresponding to sequences within this region have been prepared and assayed for their ability to bind to cell surface gp80. Results lead to identification of the homophilic binding site to an octapeptide sequence within this region. Synthetic peptides containing this octapeptide sequence and univalent antibodies directed against this site block the formation of organized cell streams during aggregation. Although cell aggregates are eventually formed, most fail to undergo further development to give rise to slugs and fruiting bodies, indicating that cell-cell adhesion involving gp80 is an important step in normal morphogenesis.     

Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum.

WE have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp80) of Dictyostelium discoideum. gp80 has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp80. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp80 affinity purified on immobilized monoclonal antibody was able to neutralize the adhesion-blocking effect of rabbit antiserum to gp80. Although gp80 itself may not be essential for cell-cell adhesion, it appears to carry the determinants associated with adhesion.     

Identification of an octapeptide involved in homophilic interaction of the cell adhesion molecule gp80 of dictyostelium discoideum

During development of Dictyostelium discoideum, a surface glycoprotein of Mr 80,000 (gp80) is known to mediate EDTA-resistant cell-cell adhesion via homophilic interaction. Antibodies directed against a 13 amino acid sequence (13-mer) near the NH2 terminus of the protein were found to inhibit cell reassociation. This 13-mer also inhibited gp80-cell interaction and gp80-gp80 interaction. The cell binding site was mapped to the octapeptide sequence YKLNVNDS by using shorter peptide sequences to inhibit gp80 interaction. High salt concentrations inhibited homophilic interactions of both the 13-mer and gp80, suggesting that ionic interactions are involved in the forward binding reaction. Since disruption of homophilic interactions between the bound molecules required the presence of Triton X-100, hydrophobic interactions may occur after the initial ionic binding.     

Cell adhesion in transformed D. discoideum cells: expression of gp80 and its biochemical characterization

Dictyostelium discoideum amoebae were transformed with an expression vector for the gp80, a protein believed to mediate EDTA-resistant cell adhesion in developmental cells. Vegetative cells, that do not normally contain gp80, expressed the protein and this expression was correlated with the formation of cell-cell adhesions. These contacts exhibited minimal EDTA-resistance. Biochemical analyses of the protein synthesized by vegetative cells suggested that it is identical to that produced by aggregation-competent cells, including the presence of a glycolipid anchor. Additional experiments indicated that the anchor was insensitive to hydrolysis by exogenous (glycosly)phosphatidylinositol-specific phospholipase C [G)PI-PLCs) but was sensitive to the endogenous anchor degrading enzyme. This enzyme, initially described in aggregating cells (da Silva and Klein, Exp. Cell Res., in press) was found to be present also in vegetative amoebae.     

The contact site A glycoprotein mediates cell-cell adhesion by homophilic binding in Dictyostelium discoideum

Dictyostelium discoideum expresses a developmentally regulated cell surface glycoprotein of Mr 80,000 (gp80), which has been implicated in the formation of the EDTA-resistant contact sites A at the cell aggregation stage. To determine whether gp80 participates directly in cell binding and, if so, its mode of action, we conjugated purified gp80 to Covaspheres (Covalent Technology Corp., Ann Arbor, MI) and investigated their ability to bind to cells. The binding of gp80- Covaspheres was dependent on the developmental stage of the cells, with maximal interaction at the late aggregation stage. Scanning electron microscopic studies revealed the clustering of gp80-Covaspheres at the polar ends of these cells, similar to the pattern of gp80 distribution on the cell surface as reported earlier (Choi, A. H. C., and Siu, C.- H., 1987, J. Cell Biol., 104:1375-1387). Precoating cells with an adhesion-blocking anti-gp80 monoclonal antibody inhibited the binding of gp80-Covaspheres, suggesting that Covasphere-associated gp80 might undergo homophilic interaction with gp80 on the cell surface. Quantitative binding of 125I-labeled gp80 to intact cells gave an estimate of 1.5 X 10(5) binding sites per cell at the aggregation stage. Binding of soluble gp80 to cells was blocked by precoating cells with the anti-gp80 monoclonal antibody. The ability of gp80 to undergo homophilic interaction was further tested in a filter-binding assay, which showed that 125I-labeled gp80 was able to interact with gp80 bound on nitrocellulose in a dosage-dependent manner. In addition, reassociation of cells was significantly inhibited in the presence of soluble gp80, suggesting that gp80 has a single cell-binding site. These results are consistent with the notion that gp80 mediates cell- cell binding at the aggregation stage of development via homophilic interaction.     

Regulation of spatiotemporal expression of cell-cell adhesion molecules during development of Dictyostelium discoideum

The social amoeba Dictyostelium discoideum is a simple but powerful model organism for the study of cell-cell adhesion molecules and their role in morphogenesis during development. Three adhesive systems have been characterized and studied in detail. The spatiotemporal expression of these adhesion proteins is stringently regulated, often coinciding with major shifts in the morphological complexity of development. At the onset of development, amoeboid cells express the Ca(2+) -dependent cell-cell adhesion molecule DdCAD-1, which initiates weak homophilic interactions between cells and assists in the recruitment of individuals into cell streams. DdCAD-1 is unique because it is synthesized as a soluble protein in the cytoplasm. It is targeted for presentation on the cell surface by an unconventional protein transport mechanism via the contractile vacuole. Concomitant with the aggregation stage is the expression of the contact sites A glycoprotein csA/gp80 and TgrC1, both of which mediate Ca(2+) /Mg(2+) -independent cell-cell adhesion. Whereas csA/gp80 is a homophilic binding protein, TgrC1 binds to a heterophilic receptor on the cell. During cell aggregation, csA/gp80 associates preferentially with lipid rafts, which facilitate the rapid assembly of adhesion complexes. TgrC1 is synthesized at low levels during aggregation and rapid accumulation occurs initially in the peripheral cells of loose mounds. The extracellular portion of TgrC1 is shed and becomes part of the extracellular matrix. Additionally, analyses of knockout mutants have revealed important biological roles played by these adhesion proteins, including size regulation, cell sorting and cell-type proportioning.     

Cell-cell adhesion in Dictyostelium discoideum

Three separate mechanisms of cell-cell adhesion have been shown to appear at different stages of development in Dictyostelium discoideum. During the first few hours of development, the cells synthesize and accumulate a glycoprotein of 24,000 daltons (gp24) that is positioned in the membrane. The time of appearance of gp24 correlates exactly with the time of appearance of cell-cell adhesion in two strains in which temporal control varies by several hours. Antibodies specific to gp24 are able to block cell-cell adhesion during the first few hours of development but not during later development. By 8 hr of development, another glycoprotein, gp80, that is not recognized by antibodies to gp24 accumulates on the surface of cells. This membrane protein mediates an independent adhesion mechanism during the aggregation stage that is resistant to 10 mM EDTA. Antibodies specific to gp80 can block EDTA-resistant adhesion during this stage. During subsequent development, gp80 is removed from the cell surface and replaced by another adhesion mechanism that is insensitive to antibodies to either gp24 or gp80. A lambda gt11 expression vector carrying a Dictyostelium cDNA insert was isolated that directs the synthesis of a fusion protein recognized by antibodies specific to gp24. This cDNA was used to probe a genomic library. A clone carrying a 1.4-kb insert of genomic DNA was recognized by the cDNA and shown to hybridize to a 0.7-kb mRNA that accumulates early in development. This unusually small RNA could code for the small protein, gp24. Southern analysis of restriction fragments generated by various enzymes on Dictyostelium DNA with both the cDNA and genomic clones indicated the presence of two tandem copies of the gene. This may account for the failure to recover mutations resulting in the lack of gp24. Mutations have been recovered that result in the lack of accumulation of gp80, and cells carrying these mutations have been shown to be missing the second adhesion mechanism. These mutant strains are able to complete development because the other adhesion mechanisms are not impaired. Sequential addition of adhesion mechanisms provides a means for the formation of multicellular organisms from previously solitary cells.     

Adhesion-blocking antibodies prepared against gp150 react with gp80 of Dictyostelium

A membrane glycoprotein of 150000 D, gp150, has been implicated in the mechanism of cell-cell adhesion which arises during development of Dictyostelium discoideum. This conclusion was founded on the observation that monovalent Fab′ fragments prepared from an antiserum raised against partially purified gp150 are able to block cell-cell adhesion. We show that this serum contains antibodies to a distinct membrane glycoprotein, gp80, previously implicated in cell-cell adhesion. Reaction of Fab′ to this surface molecule can account for the adhesion-blocking activity in the antiserum to gp150. Moreover, binding of gp80 neutralized Fab′ to gp150 does not block adhesion. If gp150 carries other determinants which bind adhesion-blocking Fab′, these determinants must also be present on gp80. Thus, it is not clear that gp150 is directly involved in cell-cell adhesion of Dictyostelium.