Common Cell-Surface Receptors for Discoidin I and Discoidin II in Dictyostelium discoideum

Following nutrient depletion, cells of the cellular slime mould Dictyostelium discoideum become cohesive and aggregate to form multicellular complexes. Several proteins that accumulate on the cell surface during this period have been implicated in mediating aggregative-phase cell cohesion, namely contact sites A (CsA), gp 150, and two endogenous lectins (discoidin I and discoidin II). The aggregating cells also possess receptors for both discoidin I and discoidin II but these have not yet been isolated and characterised for both lectins.
In the present study we investigated the relationship between the receptors for these lectins, in particular to what extent discoidin I and discoidin II receptors are common. Radio-iodinated discoidin I and discoidin II were purified and used in binding assays for lectin receptors on the surface of aggregated (10 h stage of development) D. discoideum NC4 cells. Sugar competition of ¹²⁵I-labelled discoidin I and ¹²⁵I-labelled discoidin II binding indicated distinct but overlapping sugar specificities for these lectins when binding to their in vivo receptors. Competition of the binding of radio-iodinated lectin with either unlabelled discoidin I or unlabelled discoidin II showed that at least 50% of the cell-surface binding sites for these lectins are in common and for these receptors the binding affinity of discoidin I is 9–20 times higher than for discoidin II. Approximately 35% of discoidin II binding sites appear to be unavailable for discoidin I binding.     

Localization of soluble endogenous lectins and their ligands at specific extracellular sites

Soluble lectins of chicken, rat, frog, and the cellular slime mold, Dictyostelium discoideum, were purified and specific antibodies raised against these proteins were used to immunohistochemically localize the lectins in and around the tissues in which they were synthesized. Within cells, some of these soluble lectins (chicken-lactose-lectin-II in intestinal goblet cells, discoidin II in prespore cells) appear to be concentrated within vesicles whereas others (e.g., rat beta-galactoside lectin in pulmonary alveolar and smooth muscle cells) appear to be free in the cytoplasm. All of these lectins are eventually secreted to extracellular sites in developing or adult tissues. The sites include mucin (chicken-lactose-lectin-II in intestine); developing extracellular matrix (chicken-lactose-lectin-I in muscle; Xenopus laevis lectin in blastula stage embryos); slime (discoidin I); developing spore coat (discoidin II); and a specialized extracellular matrix, elastic fibers (rat beta-galactoside lectin in lung). In cases where this has been studied in detail (discoidin I, discoidin II, and chicken-lactose-lectin-II), the lectin is associated with a complementary extracellular ligand, at least transiently. Lectin-ligand interactions presumably confer specialized properties in these particular extracellular domains.     

Purification and comparison of two developmentally regulated lectins from Dictyostelium discoideum. Discoidin I and II.

When cells of the cellular slime mold Dictyostelium discoideum differentiate from a nonsocial amoeboid form to a cohesive, aggregating form, they synthesize a lectin-like protein called discoidin, which is present on the cell surface. It is now reported that discoidin consists of two distinct lectins, designated discoidin I and discoidin II, which, although similar in some respects, differ in their electrophoretic mobilities, isoelectric points, subunit molecular weights, amino acid compositions, tryptic peptide maps, the erythrocyte species which they agglutinate, and the sensitivity of their agglutination activity to inhibition by monosaccharides. Furthermore, discoidins I and II differ in their developmental regulation as evidenced by the distinct time courses of their appearance during differentiation.     

Immunochemical Analysis of Discoidins I and II at the Cell Surface in Wild Type and Aggregation-Defective Mutants of Dictyostelium discoideum

The endogenous lectins discoidins I and II are believed to be primary components of the morphogenetic cell cohesion system of D discoideum. We have developed two immunochemical methods to analyze the association of the discoidins with the cell surface. One method is a two-stage specific antibody binding assay in which intact cells are incubated on ice with rabbit serum (either control serum or antidiscoidin I and II), washed, then incubated with ¹²⁵I-Protein A. Specific antibody binding is defined as the difference between percent radioactivity bound with antidiscoidin versus control serum during the first stage. Substantial specific binding was observed with developed A3 cells but not with vegetative cells, and nearly all of the activity could be removed by pread-sorption of the antiserum with discoidin-Sepharose. As a complementary method, quantitative immunoadsorption analysis was performed in which we tested the ability of intact cells to remove antibodies reactive with purified ¹²⁵I-discoidin I or II. Developed cells, but not vegetative cells, were capable of adsorbing antibodies reactive with discoidin I as well as those reactive with discoidin II. This represents the first demonstration that both lectins are present on the surface of cohesive cells. These procedures, coupled with other methods to analyze soluble discoidin in cell extracts, were used to study discoidin expression in wild type cells and in two newly isolated aggregation-defective mutants. Strain EB-32 fails to aggregate and displays little or no discoidin in cell extracts or at the cell surface. On the other hand, strain EB-18 forms loose amorphous mounds, and expresses substantial quantities of the discoidins, both in cell extracts and at the cell surface. These mutants should prove valuable in studying the organization and regulation of discoidins I and II at the surface of aggregating cells.     

Assay and characterization of carbohydrate binding by the lectin discoidin I immobilized on nitrocellulose

Discoidin I is the most abundant galactose binding lectin produced by the cellular slime mold Dictyostelium discoideum and has been implicated in cell-substratum adhesion. We have developed an assay of carbohydrate binding activity utilizing binding of 125I-asialofetuin to discoidin I, or to other lectins, immobilized on nitrocellulose. Among the proteins examined, only lectins exhibited the ability to bind asialofetuin. Specificity of asialofetuin binding was demonstrated by competition with monosaccharides, which inhibited binding consistent with the known sugar specificity of the lectins examined. Experiments with fetuin and derivatives differing in their oligosaccharide structure indicated a requirement for terminal galactosyl residues for probe binding to discoidin I. We have used this assay to characterize the carbohydrate binding behavior of discoidin I. The extent of asialofetuin binding to discoidin I was dependent on the concentrations of both lectin and ligand. Interpretation of equilibrium binding data suggested that, under saturating conditions, 1 mol of oligosaccharide was bound per mole discoidin I monomer. Furthermore, discoidin I in solution and discoidin I on nitrocellulose were equally effective at competing for soluble asialofetuin, suggesting that immobilization had no effect on the carbohydrate binding behavior of discoidin I. Binding was strongly inhibited by ethylenediaminetetraacetic acid; both Ca2+ and Mn2+ could overcome that inhibition, but Mg2+ could not. Preincubation of discoidin I at 60 degrees C stimulated asialofetuin binding 2-fold by increasing the affinity, while preincubation at higher temperatures resulted in a complete loss of activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colocalization of discoidin-binding ligands with discoidin in developing Dictyostelium discoideum

The Dictyostelium discoideum lectins, discoidin I and discoidin II, and the endogenous ligands to which they bind were immunohistochemically localized in sections of this organism at successive stages of development. For these studies, an axenic strain, AX3, was grown in a macromolecule-depleted medium rather than on bacteria, which themselves contain discoidin-binding ligands. Discoidin I-binding sites (endogenous ligands) in sections of D. discoideum were concentrated in the slime coat around aggregates, whereas discoidin II-binding sites were observed in a vesicle-like distribution in prespore cells and also in spore coats. In contrast, discoidin II did not bind to the slime coat and discoidin I bound relatively poorly to prespore cells and spore coats. The distributions of the endogenous lectins themselves were the same in axenically grown cells as previously reported for cells raised on bacteria. Discoidin I was concentrated in the slime coat and around stalk cells, and discoidin II was prominent in and around prespore cells. The congruent localization of each lectin with its endogenous ligand suggests that discoidin I normally functions in association with glycoconjugates in the slime around aggregates, and discoidin II with the galactose-rich spore coat polysaccharide.     

Monoclonal antibodies against discoidin I and discoidin II of the cellular slime mold, Dictyostelium discoideum

The preparation and properties of monoclonal antibodies against carbohydrate-binding proteins (discoidin I and discoidin II) in the cellular slime mold, Dictyostelium discoideum are described. Monoclonal antibody (mAb) ndI,II-1 bound both discoidins I and II specifically. mAb nI-1 and mAb dI-1 bound only discoidin I but their binding specificities were different: nI-1 recognized the native form and dI-1 the denatured form. mAb dII-1 bound only denatured discoidin II. In preliminary work mAbs dII-1 and nI-1 were found to be useful for localizing discoidins I and II immunohistochemically.     

Concanavalin a and wheat germ agglutinin receptors on dictyostelium: Their visualization by scanning electron microscopy with microspheres

The cellular slime mold, Dictyostelium discoideum, is a convenient model for studying cellular interactions during development. Evidence that specific cell surface components are involved in cellular interactions during its development has been obtained by Gerisch and co-workers (1, 2) using immunological techniques. Smart and Hynes (3) have shown that a cell surface protein can be iodinated on cells in aggregation phase, but not in vegetative phase, by the lactoperoxidase procedure. Recently, McMahon et al. (4), and Hoffman and McMahon have demonstrated, by SDS gel electrophoresis, considerable differences in cell surface proteins and glycoproteins of plasma membranes isolated from cells at different stages of development. Plant lectins have also been used to monitor changes in cell surface properties of D. discoideum cells during development. Weeks and co-workers (5, 6) have detected differences in the binding and agglutination of cells by concanavalin A (Con A). Gillette and Filosa (7) have shown that Con A inhibits cell aggregation and prematurely induces cyclic AMP phosphodiesterase. Capping of Con A receptors has also been reported (8). Reitherman et al. (9) have recently reported that agglutination of cells by several plant lectins and the slime mold agglutination, discoidin, changes during development. Such studies indicate that differences in surface properties exist for cells at various stages of development. However, owing to the uncertainties in the factors which contribute to lectin-induced cell agglutination (10), the molecular basis for these observations remain to be determined. In this study, we have used microspheres (11-14) coupled to either Con A or wheat germ agglutinin (WGA) as visual markers to study by scanning electron microscopy the topographical distribution of lectin receptors on D. discoideum cells fixed at different stages of development. We also describe the effect of labeling on the distribution of lectin receptors and on the morphology of the cell surface.     

Bacterial glycoconjugates are natural ligands for the carbohydrate binding site of discoidin I and influence its cellular compartmentalization

Klebsiella pneumoniae, Escherichia coli, and Bacillus subtilis, bacteria commonly eaten by Dictyostelium discoideum, contain glycoconjugates that bind discoidin I, a lectin synthesized by the slime mold as it differentiates. In cells fed bacteria that contain abundant discoidin I-binding glycoconjugates, these ligands and endogenous discoidin I accumulated in specialized structures called multilamellar bodies. In contrast, in cells fed bacteria that had been treated to thoroughly deplete them of discoidin I-binding glycoconjugates, neither endogenous discoidin I nor complementary glycoconjugates were found in the multilamellar bodies. In such cells discoidin I was located in the cytoplasm, as indicated by both immunohistochemistry with the electron microscope and immunoassay of subcellular fractions. The results indicate that a function of the carbohydrate-binding site of discoidin I is to interact with bacterial glycoconjugates, which the slime mold does not degrade. This interaction directs compartmentalization of the lectin in multilamellar bodies and its externalization from the cell in these structures.     

An analysis of discoidin I binding sites in Dictyostelium discoideum (NC4).

Vegetative wild-type (strain NC4) D. discoideum cells and cells at the 10h stage of development (aggregation) were harvested in the presence of 0.5 M-galactose to remove any endogenous discoidin I already bound to the cell surface, and fixed with glutaraldehyde. Affinity-purified 125I-labelled discoidin I bound to these fixed cells in a specific manner, greater than or equal to 95% of binding being inhibited by 0.5 M-galactose. Binding of 125I-labelled discoidin I was essentially complete in 90 min at 22 degrees C. Based on specific radioactivity measurements, vegetative (0h) D. discoideum (NC4) cells bind approx. 8.4 x 10(5) discoidin I tetramers/cell and aggregated (10h) cells bind 5.1 x 10(5) discoidin I tetramers/cell, each exhibiting apparent positive co-operativity of binding with highest limiting affinity constants (Ka) of approx. 1 x 10(7) and 2 x 10(7) M-1, respectively. Klebsiella aerogenes, the food source used for growth of D. discoideum NC4 amoebae, also binds 125I-labelled discoidin I and this is greater than 99% inhibited by 0.5 M-galactose. However, at the levels of bacterial contamination present, greater than 97% of 125I-labelled discoidin I binding to D. discoideum cell preparations was to the cells themselves. Confirmation of the number of discoidin I tetramers bound per D. discoideum cell was obtained by elution of bound 125I-labelled discoidin I followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and then quantification by scanning of stained discoidin I bands.     

Pallidin. Purification and characterization of a carbohydrate-binding protein from Polysphondylium pallidum implicated in intercellular adhesion.

A carbohydrate-binding protein from Polysphondylium pallidum, a species of cellular slime mold, was purified to homogeneity by adsorption to formalinized erythrocytes and elution with D-galactose. The protein, for which we propose the name PALLIDIN, is assayed by its activity as an agglutinin of erythrocytes. It was previously shown to have different carbohydrate-binding specificities than discoidin, a carbohydrate-binding protein from Dictyostelium discoideum, another species of slime mold. Evidence has been presented previously that each of these proteins is detectable on the cell surface. In the present report we show that the physico-chemical properties of pallidin are different from discoidin. Pallidin has a subunit molecular weight of 24 800 +/- 1100 determined by polyacrylamide electrophoresis in the presence of dodecyl sulfate and 2-mercaptoethanol, compared to 26 100 +/- 1000 for discoidin. The weight-average molecular weight of pallidin is 250 000 +/- 50 000 determined by equilibrium sedimentation in the presence of D-galactose compared to 100 000 +/- 2000 for discoidin. In equilibrium sedimentation studies, pallidin exhibited some heterogeneity at equilibrium while discoidin was homogeneous. The amino acid composition of pallidin is generally similar but clearly different from the composition of discoidin. The isoelectric point of pallidin is 7.0 compared to 6.1 for discoidin. Like discoidin, pallidin contains no detectable hexosamine or neutral sugar. These results establish that agglutinins from two species of cellular slime molds are distinct. The different properties of the cell-surface agglutinins, pallidin and discoidin, are consistent with their suggested role in species-specific cellular recognition and adhesion in the species of slime mold from which they are derived.     

The Discoidin I Gene Family of Dictyostelium Discoideum Is Linked to Genes Regulating Its Expression

The discoidin I protein has been studied extensively as a marker of early development in the cellular slime mold Dictyostelium discoideum. However, like most other developmentally regulated proteins in this system, no reliable information was available on the linkage of the discoidin genes to other known genes. Analysis of the linkage of the discoidin I genes by use of restriction fragment length polymorphisms revealed that all three discoidin I genes as well as a pseudogene are located on linkage group II. This evidence is consistent with the discoidin I genes forming a gene cluster that may be under the control of a single regulatory element. The discoidin I genes are linked to three genetic loci (disA, motA, daxA) that affect the expression of the discoidin I protein. Linkage of the gene family members to regulatory loci may be important in the coordinate maintenance of the gene family and regulatory loci. A duplication affecting the entire discoidin gene family is also linked to group II; this appears to be a small tandem duplication. This duplication was mapped using a DNA polymorphism generated by insertion of the Tdd-3 mobile genetic element into a Tdd-2 element flanking the gamma gene. A probe for Tdd-2 identified a restriction fragment length polymorphism in strain AX3K that was consistent with generation by a previously proposed Tdd-3 insertion event. A putative duplication or rearrangement of a second Tdd-2 element on linkage group IV of strain AX3K was also identified. This is the first linkage information available for mobile genetic elements in D. discoideum.     

Discoidin I and discoidin II are localized differently in developing Dictyostelium discoideum

The distribution of discoidin I and discoidin II, developmentally regulated lectins in Dictyostelium discoideum, was determined immunohistochemically at various stages of development. Discoidin I was first prominent as focal clumps in aggregating cells, then accumulated on the surface of aggregates and around them. Discoidin II became prominent later and ultimately localized in what appear to be prespore vesicles. The results indicate that discoidin I and discoidin II have different and possibly multiple functions.     

Mutants of Dictyostelium discoideum blocked in expression of all members of the developmentally regulated discoidin multigene family

Mutant strains of D. discoideum are described that can complete morphogenesis and cytodifferentiation but which express vastly reduced levels of the galactose-binding lectins discoidin I and II (less than 1% and 1%-2% respectively) compared to the wild-type control. Mutant cells proceeding through development lack lectin activity, lectin protein, and specific lectin mRNA. In contrast, the genes encoding these proteins are present in their wild-type configurations in the genome. Since these proteins are encoded by four to five discrete genes, the mutations in these strains are most likely in genes involved in the regulation of the expression of members of this multigene family. The results also indicate that the discoidin lectins may not be required for fruiting body construction in this organism. Finally, coupled with the recent ability to transform D. discoideum, these mutants open the way to identification and isolation of regulatory genes and their products.     

Multiple regulatory genes control expression of a gene family during development of Dictyostelium discoideum.

Mutant strains of Dictyostelium discoideum carrying dis mutations fail to transcribe specifically the family of developmentally regulated discoidin lectin genes during morphogenesis. The phenotypes of these mutants strongly suggested that the mutations reside in regulatory genes. Using these mutant strains, we showed that multiple regulatory genes are required for the expression of the lectin structural genes and that these regulatory genes (the dis+ alleles) act in trans to regulate this gene family. These regulatory genes fall into two complementation groups (disA and disB) and map to linkage groups II and III, respectively. A further regulatory locus was defined by the identification of an unlinked supressor gene, drsA (discoidin restoring), which is epistatic to disB, but not disA, and results in the restoration of lectin expression in cells carrying the disB mutation. Mutant cells carrying the drsA allele express the discoidin lectin gene family during growth and development, in contrast to wild-type cells which express it only during development. Therefore, the suppressor activity of the drsA allele appears to function by making the expression of the discoidin lectins constitutive and no longer strictly developmentally regulated. The data indicate that normal expression of the discoidin lectins is dependent on the sequential action of the disB+, drsA+, and disA+ gene products. Thus, we described an interacting network of regulatory genes which in turn controls the developmental expression of a family of genes during the morphogenesis of D. discoideum.     

Cell-surface discoidin in aggregating cells of Dictyostelium discoideum.

Both discoidin I and discoidin II have been detected on the surface of aggregating (10 h developmental stage) cells of Dictyostelium discoideum NC4 by radioiodination of the cell-surface followed by immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis. Approx. 92% of cell-surface discoidin I and 72% of cell-surface discoidin II can be eluted with 0.5 M-galactose, showing that most of each endogenous lectin is not present as integral membrane protein but rather is bound to cell-surface discoidin receptors. Two-dimensional polyacrylamide-gel-electrophoretic analysis of discoidin I suggests that the native tetramer may be a hetero-multimer composed of both Ia and Ib subunits. Cell-surface discoidin I also contains both types of subunit, but it is not clear whether both subunits have corresponding cell-surface receptors.     

Ion dependence of the discoidin I lectin from Dictyostelium discoideum

The lectin discoidin I from Dictyostelium discoideum requires divalent cations for binding activity. The data indicate that calcium is the preferred ion in vitro. In contrast, the lectin activity of discoidin II is independent of divalent ions.     

Ion dependence of the discoidin I lectin from Dictyostelium discoideum

Abstract. The lectin discoidin I from Dictyostelium discoideum requires divalent cations for binding activity. The data indicate that calcium is the preferred ion in vitro. In contrast, the lectin activity of discoidin II is independent of divalent ions.     

Monoclonal antibody against cytoplasmic lectins of Dictyostelium discoideum: cross-reactivity with a membrane glycoprotein, contact site A, and with E. coli beta-galactosidase and lac repressor

Monoclonal antibodies were raised against two soluble, galactose-binding lectins from cells of Dictyostelium discoideum, discoidin I and II. These antibodies reacted not only with both discoidins, but also with a plasma membrane glycoprotein of aggregation competent cells, called contact site A, and with two carbohydrate-binding proteins of E. coli, beta-galactosidase and lac repressor. The possibility that the antibody recognizes a structure common to different carbohydrate-binding proteins is discussed. The two carbohydrate-binding proteins of E. coli share with discoidin I the sequence -Ser-X-X-Ile-His(Pro)-Pro(His)-Leu-Thr- which might be responsible for the cross-reactivity.     

Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide

N-(4-Azidosalicyl)galactosamine (GalNASA), a photoactivatable, radioiodinatable analogue of N-acetylgalactosamine (GalNAc), has been prepared and characterized. We have used this reagent for labeling of the carbohydrate binding site of discoidin I, an endogenous lectin produced by Dictyostelium discoideum. GalNASA behaved as a ligand for discoidin I, as judged by its ability to compete in an assay measuring the carbohydrate binding activity of discoidin I. In this assay, it exhibited a Ki,app of 800 microM, comparable to that of GalNAc. The Ki,app of GalNASA decreased to 40 microM upon prior photolysis with ultraviolet light. In contrast, N-(4-azidosalicyl)ethanolamine produced no inhibition of carbohydrate binding regardless of photolysis. Covalent labeling of discoidin I with 125I-GalNASA was entirely dependent upon ultraviolet light. A portion of the labeling, representing 40-60% of the total, was sensitive to reagents which were known to inhibit carbohydrate binding by discoidin I, including GalNAc, asialofetuin, and ethylenediaminetetraacetic acid. N-Acetylglucosamine, which is not a ligand of discoidin I, was without effect. As a control, no carbohydrate-sensitive labeling was observed upon incubation of 125I-GalNASA with bovine serum albumin. The carbohydrate-sensitive fraction of discoidin I photolabeling with 125I-GalNASA exhibited a Kd of 15-40 microM, in agreement with the Ki,app of prephotolyzed GalNASA observed in the carbohydrate binding assay. Some labeling occurred if 125I-GalNASA was photolyzed prior to incubation with discoidin I, suggesting the involvement of long-lived species in the labeling reaction. Partial proteolytic digestion of photolabeled discoidin I revealed specific fragments whose labeling was completely blocked by GalNAc.(ABSTRACT TRUNCATED AT 250 WORDS)