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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.

Antibodies↗

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.

Carrier Proteins↗

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.

Antibodies, Monoclonal↗

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-state 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 preadsorption 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.

Carrier Proteins↗

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)

Asialoglycoproteins↗

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.

Binding Sites↗

RasG regulates discoidin gene expression during Dictyostelium growth.

Activated rasG, rasG(G12T), was expressed in Dictyostelium cells under the control of the folate-repressible discoidin promoter (pVEII-rasG(G12T)) and found to have a unique pattern of expression when cells were transferred to folate-deficient media: an initial increase of RasG(G12T) resulting from the removal of folate, followed by a rapid decline while cells were still in the early exponential phase of growth. Discoidin levels were considerably lower and declined more rapidly in the pVEII-rasG(G12T) transformant than they did in the wild type, suggesting that RasG(G12T) represses discoidin expression. This was independently confirmed by placing the rasG(G12T) gene under the control of the ribonucleotide reductase (rnrB) promoter. Exposure of cells to 10 mM methyl methanesulfonate (MMS) rapidly generated RasG(G12T) and this was accompanied by an equally rapid decrease in discoidin mRNA levels. rasG null cells also contained decreased levels of discoidin under all conditions tested, indicating that RasG is essential for optimum discoidin expression. However, rasG null cells showed normal regulation of discoidin expression in response to PSF, CMF, folate, bacteria, and axenic media, indicating that RasG is not necessary for any of these responses. These results reveal a role for RasG in regulating discoidin gene expression and add a further level of complexity to the regulation of the discoidin promoter.

Animals↗

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)

Affinity Labels↗

Molecular analysis of collagen binding by the human discoidin domain receptors, DDR1 and DDR2. Identification of collagen binding sites in DDR2.

The widely expressed mammalian discoidin domain receptors (DDRs), DDR1 and DDR2, are unique among receptor tyrosine kinases in that they are activated by the extracellular matrix protein collagen. Various collagen types bind to and activate the DDRs, but the molecular details of collagen recognition have not been well defined. In this study, recombinant extracellular domains of DDR1 and DDR2 were produced to explore DDR-collagen binding in detail. In solid phase assays, both DDRs bound collagen I with high affinity. DDR1 recognized collagen I only as a dimeric and not as a monomeric construct, indicating a requirement for receptor dimerization in the DDR1-collagen interaction. The DDRs contain a discoidin homology domain in their extracellular domains, and the isolated discoidin domain of DDR2 bound collagen I with high affinity. Furthermore, the discoidin domain of DDR2, but not of DDR1, was sufficient for transmembrane receptor signaling. To map the collagen binding site within the discoidin domain of DDR2, mutant constructs were created, in which potential surface-exposed loops in DDR2 were exchanged for the corresponding loops of functionally unrelated discoidin domains. Three spatially adjacent surface loops within the DDR2 discoidin domain were found to be critically involved in collagen binding of the isolated DDR2 extracellular domain. In addition, the same loops were required for collagen-dependent receptor activation. It is concluded that the loop region opposite to the polypeptide chain termini of the DDR2 discoidin domain constitutes the collagen recognition site.

Amino Acid Sequence↗

Expression of discoidin domain receptor 1 during mouse brain development follows the progress of myelination.

Discoidin domain receptor 1 is a tyrosine kinase receptor expressed in a variety of tissues including the brain. This study describes mRNA and protein expression of discoidin domain receptor 1 in mouse brain during development and provides new insights into its role during gliogenesis and neurogenesis. We performed in situ hybridization for discoidin domain receptor 1 in mouse brains at embryonic day 18, postnatal days 5, 9, 15, 21 and adulthood and observed a diffuse pattern in the proliferative areas during embryogenesis. From postnatal day 5 onwards, a defined cellular expression pattern of discoidin domain receptor 1 was observed, mainly located in white matter tracts and following a spatio-temporal pattern that overlapped the progress of myelination. Next, we performed double-labeling reactions (in situ hybridization followed by immunohistochemistry) that confirmed that discoidin domain receptor 1 was expressed by mature oligodendrocytes. We observed that cells positive for discoidin domain receptor 1 also expressed carnosine and anti-adenomatous polyposis coli, two mature oligodendrocyte markers. Based on the localization of discoidin domain receptor 1 specifically in the white matter fiber tracts during postnatal development, we suggest that discoidin domain receptor 1 participates in the development and maintenance of the myelin sheath.

Adenomatous Polyposis Coli Protein↗

Mapping of epitopes in discoidin domain receptor 1 critical for collagen binding.

The binding and activation of the discoidin domain receptor 1 by collagen has led to the conclusion that proteins from the extracellular matrix can directly induce receptor tyrosine kinase-mediated signaling cascades. A region in the extracellular domain of DDR1 homologous to the Dictyostelium discoideum protein discoidin-I is also present in the secreted human protein RS1. Mutations in RS1 cause retinoschisis, a genetic disorder characterized by ablation of the retina. By introducing point mutations into the discoidin domain of DDR1 at positions homologous to the retinoschisis mutations, ligand binding epitopes in the discoidin domain of DDR1 were mapped. Surprisingly, some residues only affected receptor phosphorylation, whereas others influenced both collagen-binding and receptor activation. Furthermore, two truncated DDR1 variants, lacking either the discoidin domain or the stalk region between the discoidin and transmembrane domain, were generated. We showed that (i) the discoidin domain was necessary and sufficient for collagen binding, (ii) only the region between discoidin and transmembrane domain was glycosylated, and (iii) the entire extracellular domain was essential for transmembrane signaling. Using these results, we were able to predict key sites in the collagen-binding epitope of DDR1 and to suggest a potential mechanism of signaling.

Amino Acid Sequence↗

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.

Dictyostelium↗

Translational control of discoidin lectin expression in drsA suppressor mutants of Dictyostelium discoideum.

Genetic analysis in Dictyostelium discoideum has identified regulatory genes which control the developmental expression of the discoidin lectin multigene family. Among these, the drsA mutation is a dominant second-site suppressor of another mutation, disB, which has the discoidinless phenotype. We now demonstrate a novel mechanism by which the drsA allele exerts its suppressive effect on the disB mutation. Interestingly, drsA does not merely bypass the disB mutation and restore the wild-type pattern of lectin expression. Rather, drsA mutant cells have high levels of discoidin lectin synthesis during growth but do not express lectins during aggregation. In contrast, wild-type cells only express lectin protein during the aggregation period of development. Phenocopies of the drsA mutation show a pattern of discoidin expression similar to that seen in the bona fide mutant. These data suggest that there may be a mechanism of negative feedback, resulting from the high levels of discoidin lectin made during growth, which inhibits further discoidin lectin expression during development. Northern (RNA) analysis of developing drsA mutant cells shows that these cells contain high levels of discoidin mRNA, although no discoidin lectin protein is being translated from these messages. Therefore, expression of the discoidin gene family can be controlled at the level of translation as well as transcription.

Blotting, Northern↗

Cell surface species-specific high affinity receptors for discoidin: developmental regulation in Dictyostelium discoideum.

Vegetative (noncohesive) D. discoideum cells and cells at several stages during the development of cohesiveness were fixed with glutaraldehyde, and their agglutinability by purified carbohydrate-binding proteins (lectins) from slime molds and plants was determined. The two purified lectins from D. discoideum, called discoidin I and II, were poor agglutinins of fixed vegetative D. discoideum cells and potent agglutinins of fixed cohesive D. discoideum cells. The increased agglutinability of D. discoideum cells by discoidin I or discoidin II with differentiation paralleled discoidin appearance in cell extracts and development of cohesiveness. The lectin of another slime mold, Polysphondylium pallidum (pallidin) and the lectin of Ricinus communis (RCA-I) were also more potent agglutinins of fixed differentiated D. discoideum cells than of fixed vegetative D. discoideum cells. However, wheat germ agglutinin was much more potent with fixed vegetative cells. The association constant (Ka) of discoidin I and discoidin II for fixed differentiated D. discoideum cells was in the range of 10(9) M-1, more than twenty-fold higher than the Ka of these lectins for fixed vegetative cells. The Ka of discoidin I and discoidin II for fixed differentiated P. pallidum cells was an order of magnitude lower than for fixed differentiated D. discoideum cells. The Ka of pallidin for fixed differentiated P. pallidum cells was about 4 X 10(9) M-1 and was an order of magnitude lower for fixed differentiated D. discoideum cells. Since previous studies showed that the lectin of each of these slime mold species is detectable on the surface of cohesive cells, the observation of species-specific receptors on the surface of cohesive cells suggests a basis for species-specific cohesion.

Binding Sites↗

Specific cell-cell contact serves as the developmental signal to deactivate discoidin I gene expression in Dictyostelium discoideum.

Specific cell-cell contact is a major regulatory signal controlling cell differentiation in Dictyostelium discoideum, causing dramatic changes in the developmental program of gene expression. In this report, we focus on the relationships between specific cell-cell contact and the activity of the genes for discoidin I, an endogenous lectin that has been implicated in the cell-cell cohesion process. By performing quantitative RNA dot-hybridization assays and RNA gel blot-hybridization analyses, using as a probe a recombinant plasmid containing a discoidin I cDNA insert, we have measured changes in discoiding I mRNA levels during normal development and in response to specific manipulations of the state of cellular aggregation. Our major findings are as follows. (i) During normal development on filters, there is a close temporal correspondence between the establishment of specific cell-cell contacts and the decline in discoidin I mRNA levels. By the tight-aggregate stage, discoidin I mRNA is barely detectable. (ii) When tight aggregates are disaggregated and the cells are maintained in the disaggregated state, there is a dramatic rise in discoidin I mRNA content. (iii) When cells are developed in suspension (conditions that interfere with the establishment of tight cell-cell contacts), discoidin I mRNA accumulates to abnormally high levels, and these persist well after the levels in filter-developed cells have declined. Taken together, these results strongly suggest that cell-cell contact is the normal developmental signal to deactivate discoidin I gene expression; thus, a contact-deactivated gene for which a recombinant DNA probe is available has now been identified. Furthermore, we demonstrate that exogenous cAMP almost completely blocks the disaggregation-induced reactivation of discoidin I gene expression. Possible mechanistic relationships between specific cell-cell contact, intracellular cAMP levels, and developmental gene expression are discussed.

Journal Article↗

Mutants of Dictyostelium discoideum with defects in the regulation of discoidin I expression.

Discoidins are proteins, coded by a multigene family, which are regulated by extracellular factors during growth and development of Dictyostelium discoideum. In this paper we describe the isolation and characterization of mutants which misregulate the expression of the discoidin I subgroup. One mutant (III29) induces discoidin I during late growth phase but does not express it during development. Another mutant (VI41) has significantly reduced discoidin levels under all conditions tested, while two mutants (VI88 and X27) express discoidin early during exponential growth and accumulate more discoidin protein than the wild type. The defects are due to abnormal regulation of transcription in all mutants except VI41. Experiments in which mutants and wild type are mixed suggest that the mutant phenotypes are not caused by changes in extracellular signals. Since multiple members of the multigene family are affected, it can be concluded that the intracellular signals regulating discoidin expression are changed rather than the genes themselves. The mutants are thus likely to have defects in the reception or intracellular processing of environmental signals.

Animals↗

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.

Carrier Proteins↗

Identification of endogenous binding proteins for the lectin discoidin-I in Dictyostelium discoideum.

Recent biochemical and genetic evidence has shown that the endogenous lectin discoidin-I is involved in intercellular adhesion during development of the cellular slime mold Dictyostelium discoideum. We have prepared discoidin-I affinity columns and used them to isolate the lectin receptors. By using the cell surface radioiodination method, 11 discoidin-I binding proteins were identified in wild-type NC4 cells by gel electrophoresis, with apparent molecular weights of 95,000, 85,000, 78,000, 72,000, 60,000, 33,000, 31,000, 28,000, 25,000, 21,000, and 15,000. Only three (gp33, gp31, and gp28) were under developmental regulation. The amount of gp31 increased 8- to 10-fold during aggregation, and it was the predominant discoidin-I binding protein synthesized at the aggregation stage. Discoidin-I binding proteins derived from aggregation stage cells were potent inhibitors of discoidin-I in a hemagglutination assay in vitro. The same preparation was found to promote aggregation of cells bearing discoidin-I on the surface, suggesting a multivalent interaction.

Carrier Proteins↗