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.     

Two-step glycosylation of the contact site A protein of Dictyostelium discoideum and transport of an incompletely glycosylated form to the cell surface

Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cells of the 53-kDa precursor with no detectable exposure of this protein on the cell surface. It is concluded that type 1 carbohydrate, which is cotranslationally added in N-glycosidic linkages, is neither required for transport of the protein to the Golgi apparatus nor for type 2 glycosylation or protection of the protein against proteolytic degradation. Incapability of tunicamycin-treated cells of forming EDTA-stable cell contacts suggests a role for type 1 carbohydrate in cell adhesion. Type 2 carbohydrate is added posttranslationally. It is required in the absence of type 1 glycosylation for transport of the protein to the cell surface.     

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.     

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

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

Mediation of cell-cell adhesion by the altered contact site A glycoprotein expressed in modB mutants of Dictyostelium discoideum

In Dictyostelium discoideum, a surface glycoprotein with Mr 80,000 (gp80) has been found to mediate the EDTA-resistant contact sites A at the aggregation stage of development. To evaluate the role of the carbohydrate moiety in cell-cell adhesion, we have examined the accumulation and activity of an altered gp80 molecule in two glycosylation (modB) mutants. Both mutants synthesize an altered gp80 of lower molecular size. This modB-gp80 can be detected by the monoclonal antibody 80L5C4, which is capable of blocking cell-cell adhesion (C. -H. Siu, T. Y. Lam, and A. Choi, (1985) J. Biol. Chem. 260, 16,030-16,036). The mutant cells exhibit both EDTA-sensitive and EDTA-resistant types of cell-cell binding, though to a lesser extent than that of the parental strain, and the EDTA-resistant binding sites are blocked in the presence of 80L5C4 Fab. Mutant cells can also bind Covaspheres conjugated with gp80. These results suggest that the modB-gp80 protein still retains the domain essential for its cell binding activity and the carbohydrate moiety affected by the modB mutation is not directly involved in cell-cell adhesion.     

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

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

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.     

EDTA treatment alters protein glycosylation in the cellular slime mold Dictyostelium discoideum

We have found that treatment of cells with EDTA resulted in the accumulation of lower molecular weight forms of two cell-type-specific glycoproteins. These new glycoproteins lacked a developmentally regulated glycoantigen defined by monoclonal antibody 54.2. Since EDTA dissociated the cells, the possible involvement of cell separation was tested by immobilizing cells in soft agarose. Glycoantigen expression on these proteins was found to be dependent on cAMP and high oxygen tension but not on cell contact, and was reversibly sensitive to EDTA regardless of the state of cell association. The EDTA effect was mimicked by other soluble, but not particulate, membrane impermeable chelators, could be competed by Zn2+ better than Mg2+, and appeared to involve an intracellular mechanism. Studies with [14C]EDTA showed that EDTA equilibrated with a cellular compartment in a temperature-dependent, Zn2+-insensitive fashion with half-time kinetics of loading and unloading of 30-40 min. If the compartment was assumed to be labeled with the same concentration of EDTA as was present extracellularly, calculations showed that its volume was circa 2% of the total cell volume. This compartment probably consists of intracellular vesicles based on the similar labeling of this compartment with a bulk fluid phase marker, inulin. The data suggest that this step in glycosylation, which was found to be delayed 1 or more hours subsequent to protein synthesis, involves an intracellular, transition metal ion-dependent process which can be modulated by chelators entering the cell through the endocytic pathway.     

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.     

Function of the carbohydrates in contact site A glycoprotein of Dictyostelium discoideum affected by tunicamycin

1. The relationship between glycosylation of contact site A (csA) of 80 kDa with two types of N-linked carbohydrates, I and II, and EDTA-resistant cell contact of Dictyostelium was investigated by tunicamycin treatment. 2. Carbohydrate I glycosylation, involved in a shift of csA from 66 to 80 kDa, was more sensitive to tunicamycin than carbohydrate II glycosylation in its shift from 53 to 66 kDa. 3. The appearance of csA of 80 kDa corresponded to that of EDTA-resistant cell contact. Carbohydrate I may be essential for EDTA-resistant cell contact. 4. In starved cells treated with tunicamycin, only 4-8% of moieties labeled with wheat germ agglutinin in carbohydrate II were modified.     

Mutants of Dictyostelium discoideum with altered carbohydrate moieties of contact site A.

Mutants of Dictyostelium discoideum were isolated and found to be defective in the epitope recognized by the monoclonal antibody 120 against the carbohydrate moieties of an integral membrane glycoprotein, contact site A, with the apparent molecular mass of 80 x 10(3). One mutant, HG764, did not express any contact site A and had lost cell contact resistant to EDTA. The others, including HG794, expressed a 68-kDa form of contact site A. In comparison with the parental strain HG592, HG794 showed weaker EDTA-resistant cell contact and the same degree of EDTA-sensitive cell contact. This suggested that the moieties which HG794 lacked were involved in EDTA-resistant cell contact. The 68-kDa contact site A in HG794 could be labeled with wheat germ agglutinin and incorporated [35S] sulfate. The modB mutant HL220 also expresses 68-kDa contact site A, although it cannot be labeled with wheat germ agglutinin. Therefore, the mutants HG794 and HL220 were compared by a complementation test. The diploid strain DG701 expressed 80-kDa contact site A and showed the same degree of EDTA-resistant cell contact as strain HG592. In its EDTA-resistant cell contact, HG794 was stronger than HL220. These results suggest that HG794 is a new mutant, and that there might be at least two processes in the glycosylation of 68-kDa contact site A to the 80-kDa form. The carbohydrate moieties recognized by monoclonal antibody 120 and by wheat germ agglutinin might be involved in EDTA-resistant cell contact.     

Replacement of the phospholipid-anchor in the contact site A glycoprotein of D. discoideum by a transmembrane region does not impede cell adhesion but reduces residence time on the cell surface

The contact site A (csA) glycoprotein of Dictyostelium discoideum, a cell adhesion molecule expressed in aggregating cells, is inserted into the plasma membrane by a ceramide-based phospholipid (PL) anchor. A carboxyterminal sequence of 25 amino acids of the primary csA translation product proved to contain the signal required for PL modification. CsA is known to be responsible for rapid, EDTA-resistant cohesion of cells in agitated suspensions. To investigate the role of the PL modification of this protein, the anchor was replaced by the transmembrane region and short cytoplasmic tail of another plasma membrane protein of D. discoideum. In cells transformed with appropriate vectors, PL-anchored or transmembrane csA was expressed under the control of an actin promoter during growth and development. The transmembrane form enabled the cells to agglutinate in the presence of shear forces, similar to the PL-anchored wild-type form. However, the transmembrane form was much more rapidly internalized and degraded. In comparison to other cell-surface glycoproteins of D. discoideum the internalization rate of the PL-anchored csA was extremely slow, most likely because of its exclusion from the clathrin-mediated pathway of pinocytosis. Thus, our results indicate that the phospholipid modification is not essential for the csA-mediated fast type of cell adhesion but guarantees long persistence of the protein on the cell surface.     

A RAS-encoded protein in Dictyostelium discoideum is acylated and membrane-associated

Dictyostelium dfscoideum synthesizes a 23000 M_r protein, p23^dd-ras, closely related to the mammalian oncogene-encoded protein p21^ras. To investigate the subcellular localization of P23^dd-ras, conditions were optimized to reduce protein degradation following cell breakage. Subcellular fractionation of D. discoideum showed that p23^dd-ras was associated predominantly with the membrane fraction during both vegetative growth and differentiation, in the absence of suitable protease inhibitors considerable amounts of a truncated form of p23^dd-ras were recovered in the cytosol fraction, suggesting that intact p23^dd-ras is attached to the membrane by a short terminal peptide sequence. Radio-isotope labelilng of D. discoideum with myristic acid or palmitic acid in the presence of excess un-labelled acetate resulted in radio-isotope incorporation into a select group of proteins including p23^dd-ras. No acyl label appeared in the truncated cytoplasmic form of p23^dd-ras when ceil breakage was performed In the absence of suitable protease inhibitors, indicating that the acyl group is associated with the short terminal peptide that is cleaved. These data suggest that p23^dd-ras like its mammailan counterpart, is acylated and associated with the plasma membrane. There was no evidence during a 30-minute pulse of methionine for a cytoplasmic precursor to the membrane-bound p23^dd-ras suggesting that the turnover of the presumptive precursor must be much more rapid in D. discoideum than for pro-p21^ras in mammalian cells.     

Sulfate suicide selection of Dictyostelium discoideum mutants defective in protein glycosylation.

The assembly and processing of glycoprotein-linked oligosaccharides in Dictyostelium discoideum has been shown to generate a wide array of glycan structures which undergo dramatic developmental regulation. As late steps in processing of these oligosaccharides involve sulfation, a sulfate suicide selection procedure was developed to select for temperature-sensitive glycoprotein-processing mutants. Of 673 clones derived from the survivors of suicide selection, 99 were classified by replica-plating fluorography as temperature sensitive for sulfate transport or incorporation. Of these, 74 were unable to complete the developmental program to the fruiting body stage at the restrictive temperature, 29 being blocked in some aspect of aggregation and 45 being blocked at some postaggregation stage. Quantitative metabolic labeling experiments with representative clones showed that they incorporated wild-type levels of [35S]methionine but reduced levels of sulfate at the restrictive temperature. The specific incorporation patterns in the mutants suggest that distinct oligosaccharide-processing steps are involved in different developmental events.     

Spore coat is altered in modB glycosylation mutants of Dictyostelium discoideum.

The modB mutation eliminates specific carbohydrate epitopes from glycoproteins which are expressed primarily in prespore and spore cells of differentiating Dictyostelium discoideum. Spores formed by the mutant show several phenotypes. Whereas mutant spores germinate efficiently after heat activation, they germinate poorly after urea activation. Following germination, at least one glycosylation-defective glycoprotein is cleaved, and the larger fragment is released in soluble form from the spore coat. However, an earlier difference in the spore coat can be traced back to the nongerminated spore coat, as detected by the elutability of protein from intact spores by chemical extraction. An altered character of the pregermination spore coat is also suggested by increased labeling by a fluorescent lectin which binds to its interior. The findings are consistent with a change in the character of certain molecular contacts leading to altered characteristics of the mutant spore coat, which are specific because they are distinctive from changes observed in another glycosylation mutant which affects a different epitope.     

A cAMP-dependent protein kinase is present in differentiating Dictyostelium discoideum cells

We demonstrate the occurrence of a cAMP-dependent protein kinase in Dictyostelium discoideum cells at the terminal stage of differentiation. A cAMP-binding component was purified to homogeneity by affinity chromatography. This subunit inhibits the activity of purified catalytic subunit from beef heart protein kinase; the inhibition is reversed upon addition of cAMP. The protein is highly specific for cAMP and has a dissociation constant of 4 nM. The isolated regulatory subunit is a monomer of 39 K, with a sedimentation coefficient of 3.5S and a frictional coefficient of 1.24. The differences between this regulatory subunit and regulatory subunits of protein kinases from other sources are discussed.     

Nhe1 is essential for potassium but not calcium facilitation of cell motility and the monovalent cation requirement for chemotactic orientation in Dictyostelium discoideum

In Dictyostelium discoideum, extracellular K+ or Ca2+ at a concentration of 40 or 20 mM, respectively, facilitates motility in the absence or presence of a spatial gradient of chemoattractant. Facilitation results in maximum velocity, cellular elongation, persistent translocation, suppression of lateral pseudopod formation, and myosin II localization in the posterior cortex. A lower threshold concentration of 15 mM K+ or Na or 5 mM Ca2+ is required for chemotactic orientation. Although the common buffer solutions used by D. discoideum researchers to study chemotaxis contain sufficient concentrations of cations for chemotactic orientation, the majority contain insufficient levels to facilitate motility. Here it has been demonstrated that Nhe1, a plasma membrane protein, is required for K+ but not Ca2+ facilitation of cell motility and for the lower K+ but not Ca2+ requirement for chemotactic orientation.