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.     

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.     

Sequence and expression of the discoidin I gene family in Dictyostelium discoideum

We have isolated recombinant phage and plasmids containing the four developmentally regulated discoidin I genes of Dictyostelium discoideum. Two of the genes are linked within 0.5 × 10³ bases with the same polarity. S¹ nuclease mapping shows that at least three members of the gene family are expressed and that the 5′ ends of the mRNAs start at equivalent sites. The genes have homologous 5′ untranslated regions and extremely divergent 3′ untranslated regions. In addition, some of the genes are flanked by homologous repeat sequences. The genes encode three different isoelectric forms of the protein. Examination of nucleotide sequences in the protein coding region shows that most nucleotide changes in the 5′ half of the gene result in amino acid substitutions while most base substitutions in the 3′ half are neutral.     

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.     

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.     

Spatiotemporal Patterning of discoidin I and II during Development of Dictyostelium discoideum

We have examined the distribution of Dictyostelium lectins (discoidin I and II) during development by means of a sample preparation method of a whole mount. Monoclonal antibodies which were bound to discoidins revealed unique patterns of discoidin distribution. Discoidin I was localized mainly at the periphery of the aggregates, while the base of the aggregates was devoid of discoidin I staining. Discoidin I was not prominent in the body of the aggregates but when a migrating slug culminated, discoidin I staining appeared in the prestalk region, this suggested that prestalk cells begin to express discoidin I at the onset of culmination. During fruit formation we observed discoidin I staining at the foremost anterior prestalk region of the culminant, which implies a heterogeneity of discoidin I expression among prestalk cells; such a heterogenous pattern has also been found in other prestalk-specific proteins. In addition, anterior-like cells (ALC), which were sorted at the apex and basal parts of a spore mass during culmination, were also strongly stained with anti-discoidin I mAb; interestingly, we observed the staining of ALC from the slug stage through fruit formation. No discoidin II was observed in a migrating slug that had already accumulated prespore antigen ligands for discoidin II; it appeared in prespore cells after the onset of culmination. The present results indicate that, in addition to the early expression of discoidin I, both discoidin I and II are expressed during culmination, and these lectins also seem to be involved in the late development of Dictyostelium .     

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.     

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.     

An analysis of developmental timing in Dictyostelium discoideum

A new method has been developed to assess the minimum complexity and relationships of those pathways (developmental timers) which time the consecutive stages of a developing system (Soll, 1983). This method has been applied to the morphogenetic program of Dictyostelium discoideum and has resulted in (1) a minimum estimate of the number of components comprising the timers for the first seven stages of morphogenesis, (2) a characterization of the temperature sensitivities of these components including demonstration of a reversible timer component, (3) detailed temporal definition of a number of transition points between rate-limiting components including a major branch point for the onset of several independent timer components coincident with the onset of aggregation, and (4) a temporal model for the relationships between the timers of the seven consecutive morphogenetic stages, including several examples of parallel timers.     

Genomic instability and mobile genetic elements in regions surrounding two discoidin I genes of Dictyostelium discoideum.

We have found that the genomic regions surrounding the linked discoidin I genes of various Dictyostelium discoideum strains have undergone rapid changes. Wild-type strain NC-4 has three complete discoidin I genes; its axenic derivative strain Ax-3L has duplicated a region starting approximately 1 kilobase upstream from the two linked genes and extending for at least 8 kilobases past the genes. A separately maintained stock, strain Ax-3K, does not have this duplication but has undergone a different rearrangement approximately 3 kilobases farther upstream. We show that there are repeat elements in these rapidly changing regions. At least two of these elements, Tdd-2 and Tdd-3, have characteristics associated with mobile genetic elements. The Tdd-3 element is found in different locations in related strains and causes a 9- to 10-base-pair duplication of the target site DNA. The Tdd-2 and Tdd-3 elements do not cross-hybridize, but they share a 22-base-pair homology near one end. At two separate sites, the Tdd-3 element has transposed into the Tdd-2 element, directly adjacent to the 22-base-pair homology. The Tdd-3 element may use this 22-base-pair region as a preferential site of insertion.     

The role of membrane lectins in Dictyostelium discoideum aggregation as ascertained by specific univalent antibodies against discoidin I

Antibodies against pure discoidin I have been used as a tool to ascertain the role of this lectin in aggregation of Dictyostelium discoideum. Discoidin I is widely expressed over the cell surface of aggregation-competent AX-2 cells, as ascertained by indirect immunofluorescence with specific (antidiscoidin I) antibodies. Univalent antidiscoidin I antibodies (Fab fragments) inhibit the aggregation-specific intercellular adhesion of D discoideum AX-2 cells in an in vitro assay. This inhibition depends on antibody concentration and cell density; a 50% inhibition of cell aggregation was obtained at antidiscoidin I Fab concentration of 4.5 mg/ml and 1 X 10(6) cells/ml. Aggregation and morphogenesis on solid support is also effectively inhibited when AX-2 cells are starved in the presence of antidiscoidin I Fab fragments. The inhibition of morphogenesis is also dose dependent and more effective than in the in vitro assay. No inhibition of aggregation either in the in vitro assay or on morphogenesis on solid support was observed with preimmune Fab fragments at any of the concentrations tested (up to 9.6 mg/ml).     

Conserved structural features are found upstream from the three co-ordinately regulated discoidin I genes of Dictyostelium discoideum

The discoidin I genes of Dictyostelium form a small, co-ordinately regulated multigene family. We have sequenced and compared the upstream regions of the DiscI-alpha, -beta and -gamma genes. For the most part the upstream regions of the three genes are non-homologous. The upstream sequences of the beta and gamma genes are exceedingly A + T-rich, while those of the alpha gene are less so. All three genes have a relatively G + C-rich region 20 to 40 base-pairs in length, found approximately 200 base-pairs 5' to the messenger RNA start site. This G + C-rich region 5' to the beta and gamma genes is flanked by short inverted repeats. Within this region, there is an 11 base-pair exact homology between the alpha and gamma genes, and a less perfect homology between these genes and the beta gene. The homology is flanked at a short distance by interspersed G and T residues. The gamma gene is greater than 90% A + T for greater than 800 base-pairs upstream. Further upstream there is a G + C-rich region that is also found inverted approximately 3.5 X 10(3) base-pairs away. The gamma and beta genes are tandemly linked, and the entire approximately 500 base-pair intergene region between the 3' end of the gamma gene and the 5' end of the beta gene is A + T-rich (approximately 90%) with the exception of the homology region 5' to the gamma gene. We demonstrate also the presence of a discoidin I pseudogene fragment having only 139 base-pairs of discoidin homology with greater than 8% mismatch. It is flanked upstream by five 39 base-pair G + C-rich repeats, and downstream by sequences that are extremely A + T-rich. We discuss the possible significance of the conserved G + C-rich structures on discoidin I gene expression.     

Reconstitution of discoidin hemagglutination activity by lipid extracts of Dictyostelium discoideum cells.

The developmentally regulated carbohydrate binding protein discoidin (from Dictyostelium discoideum) has been purified in a nonagglutinating form. While substantial agglutination activity is present in cell lysates, this activity is consistently lost upon affinity purification of discoidin. The lack of agglutination activity is not due to a mutational event or a nutritional deficiency. The carbohydrate binding site of the protein is functional, and dissociation of the oligomeric protein into subunits has not occurred. The addition of aqueous dispersions of a CHCl3/CH3OH extract of a slime-mold particulate fraction to the purified discoidin reconstitutes agglutination activity in a concentration-dependent manner. The reconstituted agglutination activity has the specificity of discoidin's carbohydrate binding sites. The reconstitutive ability of the CHCl3/CH3OH extract is due to a lipid component. Treatments of the lipid extract and fractionation of the active species suggest that it may be unsaturated fatty acid. Of many purified lipids tested, only high concentrations of cisvaccenic acid (C18:1 delta11) or oleic acid (C18:1 delta9) significantly reconstituted agglutination activity.