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.     

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.     

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.     

Characterization of antigen 117. A developmentally regulated cell surface glycoprotein of Dictyostelium discoideum

Antigen 117 is involved in the process of intercellular cohesion in Dictyostelium discoideum (Brodie, C., Klein, C., and Swierkosz, J. Cell 32, 1115-1123 (1983]. The antigen was shown to arise from a 62,000-64,000-dalton precursor. The mature antigen consists of two forms of molecular weights, 69,000 and 72,000. These forms are glycosylated, phosphorylated, acylated, and sulfated. Developmental changes in the cellular and cell surface levels of the antigen reflect changes in its rate of synthesis. All aggregating cells express antigen 117 on their surfaces. Antigen 117 then disappears from the surface of all cells when tip formation occurs. The antigen is re-expressed briefly again on cells undergoing culmination.     

Mutations causing rapid development of Dictyostelium discoideum

A mutation affecting the speed of slime mold development has been genetically analyzed. Strain FR17 carries a recessive mutation on linkage group IV. A selection procedure for isolating more mutants of this type has been developed and new mutations have been tested for complementation. The aberrant morphology of these strains can be partially corrected by development in the presence of glucose.     

A polymorphic, prespore-specific cell surface glycoprotein is present in the extracellular matrix of Dictyostelium discoideum.

Polymorphisms of a major developmentally regulated prespore-specific protein (PsA) in Dictyostelium discoideum slugs are described. These polymorphisms allowed discrimination between PsA (found on the cell surface and in the extracellular matrix) and a similar extracellular but nonpolymorphic protein, ShA. The two proteins were also distinguished by their differing reactivities with a range of monoclonal antibodies and by their sensitivity to release from the sheath with cellulase. The results are discussed in terms of the molecular and genetic relationships between the cell surface and the extracellular matrix during development.     

A surface glycoprotein indispensable for gamete fusion in the social amoeba Dictyostelium discoideum

Sexual reproduction is essential for the maintenance of species in a wide variety of multicellular organisms, and even unicellular organisms that normally proliferate asexually possess a sexual cycle because of its contribution to increased genetic diversity. Information concerning the molecules involved in fertilization is accumulating for many species of the metazoan, plant, and fungal lineages, and the evolutionary consideration of sexual reproduction systems is now an interesting issue. Macrocyst formation in the social amoeba Dictyostelium discoideum is a sexual process in which cells become sexually mature under dark and submerged conditions and fuse with complementary mating-type cells. In the present study, we isolated D. discoideum insertional mutants defective in sexual cell fusion and identified the relevant gene, macA, which encodes a highly glycosylated, 2,041-amino-acid membrane protein (MacA). Although its overall similarity is restricted to proteins of unknown function within dictyostelids, it contains LamGL and discoidin domains, which are implicated in cell adhesion. The growth and development of macA-null mutants were indistinguishable from those of the parental strain. The overexpression of macA using the V18 promoter in a macA-null mutant completely restored its sexual defects. Although the macA gene encoded exactly the same protein in a complementary mating-type strain, it was expressed at a much lower level. These results suggest that MacA is indispensable for gamete interactions in D. discoideum, probably via cell adhesion. There is a possibility that it is controlled in a mating-type-dependent manner.     

Structural characterization of Dictyostelium discoideum prespore-specific gene D19 and of its product, cell surface glycoprotein PsA.

The Dictyostelium discoideum cell surface antigen PsA is a glycoprotein which first appears in the multicellular stage soon after tip formation and is selectively expressed on prespore cells. The D19 gene encodes an mRNA sequence which is highly enriched in prespore over prestalk cells in the slug stage. We have determined 81 amino acid residues of N-terminal sequence from immunoaffinity-purified PsA protein and shown this sequence to be identical to the predicted sequence of the D19 gene. There are several short repeat elements close to the C terminus, and unequal crossing-over within these is proposed to account for the size polymorphism observed in PsA protein isolated from different D. discoideum strains. The repeats are proline rich and show similarity to the C-terminal region of the D. discoideum cell adhesion molecule, contact sites A. The extreme C terminus, which is also homologous to contact sites A, is characteristic of proteins attached to the plasma membrane via a glycosyl-phosphatidylinositol link. We have marked the PsA gene by insertion of an oligonucleotide encoding an epitope of the human c-myc protein. A construct containing this gene and 990 base pairs of 5'-flanking region directed correct temporal and spatial mRNA accumulation. We found the marked PsA protein, detected with the human c-myc antibody, to be correctly localized on the surface of cells.     

F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes

F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin-membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge.     

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.     

Re-expression of 117 antigen, a cell surface glycoprotein of aggregating cells, during terminal differentiation of Dictyostelium discoideum prespore cells

117 antigen is a glycoprotein expressed on the surface of D. discoideum cells at aggregation. It then disappears and is later re-expressed on the surface of a subpopulation of cells at culmination, the terminal differentiation stage (Sadeghi et al. 1987). A cDNA clone was used to show that the appearance of cell surface 117 antigen accurately reflects the expression of the 117 gene as measured by mRNA levels. It was also shown that during multicellular development there is a reciprocal relationship between the levels of 117 mRNA and the mRNA which codes for prespore surface glycoprotein, PsA. Dual parameter flow cytometry was used to demonstrate that the 117 antigen is found on the surface of maturing prespore cells after the PsA glycoprotein disappears, but that it is not found on mature spores. Using three monoclonal antibodies which identify respectively 117 antigen, PsA, and MUD3 antigen (a spore coat glycoprotein--probably Sp96), two new stages of final spore maturation were defined. These results indicate that there is a recapitulation of at least one aggregative cell surface glycoprotein in the prespore subpopulation of cells as they rise up the stalk during final spore development. This raises the possibility that culmination, which involves complex three dimensional morphogenetic movements not unlike those observed during animal embryogenesis, involves components of the two-dimensional pattern seen during aggregation.     

Purification of a membrane glycoprotein with an inositol-containing phospholipid anchor from Dictyostelium discoideum.

Large-scale purification of a Dictyostelium discoideum cell surface glycoprotein, which is anchored in the membrane via a glycosylphosphatidylinositol (GPI) moiety, is described. The purification protocol involved four steps: separation of crude cell membranes by low-speed centrifugation, delipidization of these membranes using acetone, extraction of the membrane proteins using the detergent Octyl beta-D-thioglucopyranoside (OTP), and purification of a specific membrane protein by monoclonal antibody immunoaffinity chromatography. The protein purified, PsA (prespore-specific antigen), is a developmentally regulated membrane glycoprotein found on a subset of cells from the cellular slime mould, D. discoideum. The protocol provides an efficient, economical, and technically simple way to purify GPI proteins in sufficient quantities for structural and functional studies. PsA was recovered at a yield of about 60%; with a purity of 97%, the extraction of 1 x 10(10) cells (1.1 g dry weight) yielded about 0.5 mg PsA glycoprotein. Techniques are described for growing kilogram quantities of D. discoideum cells in stainless steel trays at little cost. D. discoideum has considerable potential as a novel expression system for the production of foreign membrane-associated proteins. The purification strategy provides a means of purifying other GPI proteins, including those produced by protein engineering techniques.     

The contact site A glycoprotein of Dictyostelium discoideum carries a phospholipid anchor of a novel type.

The contact site A glycoprotein, a cell adhesion protein of aggregating Dictyostelium cells, was labeled with fatty acid, myo-inositol, phosphate and ethanolamine in vivo, indicating that the protein is anchored in the membrane by a lipid. This lipid was not susceptible to phosphatidyl inositol specific phospholipase C. When cleaved with nitrous acid or when subjected to acetolysis, the anchor released lipids which were different from those released from Trypanosoma variant cell surface glycoprotein, a protein with a known phosphatidyl inositol-glycan anchor. Resistance to weak and sensitivity to strong alkali indicated that the fatty acid in the contact site A glycolipid anchor was in an amide bond. On incubation with sphingomyelinase, a lipid with the chromatographic behavior of ceramide was released. These results suggest that the contact site A glycoprotein is anchored by a ceramide based lipid glycan.     

Amphiphilic properties of the avian myeloblastosis virus major glycoprotein, gp 80

The major glycoprotein (gp 80) from avian myeloblastosis virus (AMV) displays significant lipophilic properties, as shown by its strong interactions with acetylated uncharged decylamino agarose in hydrophobic chromatography. In effect, release from binding was achieved only by the added presence of a polarity reducing agent (ethylene glycol) and the strong anionic detergent sodium dodecyl sulfate. The hydrophobic behavior of the glycoprotein, coupled to the high content of hydrophilic carbohydrates, indicates its amphiphilic character. Confirmation of the amphiphilic nature of the AMV gp 80 was obtained by charge shift electrophoresis and crossed hydrophobic interaction immunoelectrophoresis. In both instances, the electrophoretic behavior of the glycoprotein was dependent on the presence of detergents. The AMV gp 80 displays the properties of integral membrane proteins.