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.     

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.     

Cell adhesion during the migratory slug stage of Dictyostelium discoideum

Prespore-specific Antigen (PsA) is selectively expressed on the surface of prespore cells at the multicellular migratory slug stage of Dictyostelium discoideum development. It is a developmentally regulated glycoprotein that is anchored to the cell membrane through a glycosyl phosphatidylinositol (GPI) anchor. We present the results of an in vitro immunological investigation of the hypothesis that PsA functions as a cell adhesion molecule (CAM), and of a ligand-binding assay indicating that PsA has cell membrane binding partner(s). This is the first evidence to implicate a direct role for a putative CAM in cell-cell adhesion during the multicellular migratory slug stage of D. discoideum development. Cell-cell adhesion assays were carried out in the presence or absence of the monoclonal antibody (mAb) MUD1 that has a single antigenic determinant: a peptide epitope on PsA. These assays showed specific inhibition of cell-cell adhesion by MUD1. Further, it was found that a purified recombinant form of PsA (rPsA), can neutralize the inhibitory effect of MUD1; the inhibitory effect on cell-cell adhesion is primarily due to the blocking of PsA by the mAb. The resistance of aggregates to dissociation in the presence of 10 mM EDTA (ethylenediamintetraacetic acid) indicates that PsA mediates EDTA-stable cell-cell contacts, and that PsA-mediated cell adhesion is likely to be independent of divalent cations such as Ca(2+) or Mg(2+).     

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.     

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.     

The 80L5C4 epitope overlaps with the homophilic binding site of the cell adhesion molecule gp80 of Dictyostelium.

The monoclonal antibody (mAb) 80L5C4 is a potent inhibitor of the cell adhesion molecule gp80 of Dictyostelium discoideum. To map the exact location of the epitope recognized by mAb 80L5C4, overlapping hexapeptides were synthesized on plastic pins and the binding p6 mAb 80L5C4 to these peptides was monitored by enzyme-linked immunosorbent assay. The 80L5C4 epitope is mapped to a single hexapeptide sequence GYKLNV, which shares five amino acid residues with the octapeptide sequence YKLNVNDS involved in gp80 homophilic binding. Analogue studies indicate that the hydrophobic residues within this sequence are crucial for antigen recognition.     

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.     

Identification of sialic acids in cell adhesion molecule, contact site A from Dictyostelium discoideum.

It has been believed that Dictyostelium discoideum cell membranes contain no sialic acid. In this study, however, we found that contact site A, the cell adhesion molecule of D. discoideum, is a major glycoprotein containing sialic acids. This suggests that sialic acid in non-reducing terminal plays an important role in the cell adhesion in which contact site A is involved.     

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.     

Regulation of the anterior-like cell state by ammonia in Dictyostelium discoideum

Ammonia appears to be an important regulatory signal for several aspects of the Dictyostelium life cycle. The postulated role of ammonia in the determination of the prespore pathway in cells of the slug stage has led us to examine the effect of ammonia on the prestalk/prespore ratio of migrating slugs. In the presence of 10(-3) M ammonium chloride, the volume of the prestalk region decreases by 40.8%. The kinetics of the process make it unlikely that this is due to a shift in the differentiation pathway. A test of the hypothesis that the decrease in volume of the prestalk region is due to the conversion of prestalk cells to anterior-like cells shows that the percent of anterior-like cells in the posterior region increases by the amount predicted by the hypothesis. This suggests that ammonia may be the molecular signal, produced by the tip, that prevents anterior-like cells from chemotactically migrating to the tip and thereby becoming anterior cells. The effect of enzymatic removal of ammonia from vitally stained migrating slugs is the appearance of a series of dark stripes beginning at the posterior end and progressing forward. We interpret this as a result of progressive removal of anterior-like cells from tip dominance and essentially as the formation of new potential tips. Indeed, in a few cases one or even two of the stripes separate from the posterior of the cell mass and form small fruiting bodies. We consider the phenomenon of stripe formation further evidence that the tip acts on anterior-like cells through ammonia.     

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.     

The cell adhesion molecule DdCAD-1 regulates morphogenesis through differential spatiotemporal expression in Dictyostelium discoideum

During development of Dictyostelium, multiple cell types are formed and undergo a coordinated series of morphogenetic movements guided by their adhesive properties and other cellular factors. DdCAD-1 is a unique homophilic cell adhesion molecule encoded by the cadA gene. It is synthesized in the cytoplasm and transported to the plasma membrane by contractile vacuoles. In chimeras developed on soil plates, DdCAD-1-expressing cells showed greater propensity to develop into spores than did cadA-null cells. When development was performed on non-nutrient agar, wild-type cells sorted from the cadA-null cells and moved to the anterior zone. They differentiated mostly into stalk cells and eventually died, whereas the cadA-null cells survived as spores. To assess the role of DdCAD-1 in this novel behavior of wild-type and mutant cells, cadA-null cells were rescued by the ectopic expression of DdCAD-1-GFP. Morphological studies have revealed major spatiotemporal changes in the subcellular distribution of DdCAD-1 during development. Whereas DdCAD-1 became internalized in most cells in the post-aggregation stages, it was prominent in the contact regions of anterior cells. Cell sorting was also restored in cadA(-) slugs by exogenous recombinant DdCAD-1. Remarkably, DdCAD-1 remained on the surface of anterior cells, whereas it was internalized in the posterior cells. Additionally, DdCAD-1-expressing cells migrated slower than cadA(-) cells and sorted to the anterior region of chimeric slugs. These results show that DdCAD-1 influences the sorting behavior of cells in slugs by its differential distribution on the prestalk and prespore cells.     

Phosphorylation of the contact site A glycoprotein (gp80) of Dictyostelium discoideum

Developmentally regulated cohesion of Dictyostelium discoideum can be blocked by the Fab fragment of antiserum prepared against a glycoprotein of about 80,000 daltons, gp80, purified from the membranes of developing amoebae. Immunoprecipitation of gp80 with this serum, from ³²P-labeled cell extracts of aggregating D. discoideum amoebae showed it to be a phosphoprotein. Serine phosphate was found in the molecule. All multiple isoelectric forms of gp80 were phosphorylated. Synthesis of the phosphorylated form of gp80 was found to be limited to the period of aggregation and coincided with the period of incorporation of [³⁵S]methionine into gp80. Phosphorylation could be rapidly inhibited by cycloheximide suggesting that phosphorylation occurs only on newly made gp80. No unphosphorylated gp80 could be detected in cell extracts.     

Surface glycoprotein, gp24, involved in early adhesion of Dictyostelium discoideum

A membrane glycoprotein of 24,000 Da (gp24) was purified from developed cells of Dictyostelium discoideum and shown to neutralize a crude antiserum (R695) that blocks EDTA-sensitive cell-cell adhesion during the early developmental stages of this organism. Purified gp24 was used to raise rabbit polyclonal antibodies and mouse monoclonal antibodies. Rabbit antiserum R851 was shown to be highly specific to gp24 by both Western analysis and immunoprecipitation. IgG of R851 is able to block adhesion of dissociated cells swirled in suspension. Adhesion of wild-type cells is blocked by R851 antibodies during the first 8 hr of development but not thereafter when other adhesion mechanisms come into play. The glycoprotein gp80 plays an essential role in the second adhesion system that appears during the aggregation stage of D. discoideum. By adding both anti-gp24 and anti-gp80 antibodies, adhesion of aggregation stage cells could be blocked. Late in development a third adhesion mechanism appears that is not blocked by either antibodies to gp24 or gp80 or both antibodies together. Western analysis and immunoprecipitation with monoclonal antibody mLJ11, specific for gp24, indicated that gp24 is absent in cells growing exponentially on bacteria but is rapidly synthesized and accumulated following the initiation of development. Synthesis of gp24 is maximal during the first 4 hr of development and then continues at a reduced rate throughout the remainder of development. The coordinate appearance of gp24 and EDTA-sensitive cell-cell adhesion as well as the ability of this glycoprotein to neutralize the adhesion blocking activity of R695 and R851 antibodies indicates that it plays a role in early cell-cell adhesion.     

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.     

Length regulation in the Dictyostelium discoideum slug is a late event.

Time-lapse video light microscopy was used to study the emergence and maturation of the migratory slug from a D. discoideum aggregate. The anterior part, the tip of this simple multicellular organism, establishes migration prior to the definition of the rear, and hence the length of the slug. It was found that newly formed slugs of wild-type strain WS380B can reach lengths greater than 1 cm, yet mature slugs of this strain are rarely longer than 2-3 mumm. Often the tip extended out of the aggregation mound upon an arching pillar of cells. After the tip first touched the substratum, it commenced migration with a rapid succession of movement steps. Here we show that at the initiation of migration, a differential rate of cell movement along the developing slug axis results in a series of complicated changes, before the stable and mature shape of the slug is formed. Our results lead to new conclusions about D. discoideum slug formation and shape maintenance. Evidence is presented for regulation of slug length.     

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.     

Differentiation, cell sorting and proportion regulation in the slug stage of Dictyostelium discoideum

Recent experimental work suggests that under normal conditions cell sorting plays an important part in maintaining and re-establishing the axial pattern of cell types in the slug stage of the cellular slime mold Dictyostelium discoideum. Following removal of the anterior zone of the slug, anterior-like cells that are normally distributed throughout the posterior of the slug rapidly migrate to the anterior end of the transected slug, and new anterior-like cells appear in the posterior portion. These results provide evidence that the direct linkage between spatial location and differentiation hypothesized in positional information models of spatial pattern formation is not universal. In this paper we develop and analyze a class of mathematical models of the slug in which cell determination can be less rigidly tied to spatial location, and which involve chemotactic cell sorting to re-establish and maintain the spatial pattern of cell types. We show that these models can reproduce the qualitative aspects of the experimental observations and that sorting takes place on the observed time scale when reasonable values of the parameters are used.