Wayne R. Springer

Discoidin I is implicated in cell-substratum attachment and ordered cell migration of dictyostelium discoideum and resembles fibronectin

All three forms of discoidin I, an endogenous N-acetylgalactosamine-binding lectin from D. discoideum, contain the amino acid sequence gly-arg-gly-asp also found in fibronectin and implicated in its attachment to cells. Synthetic peptides containing these and adjacent amino acids of discoidin I block organized streaming during aggregation of D. discoideum and, at higher concentrations, block cell attachment and spreading on a plastic surface and formation of fruiting bodies. Pure discoidin I (with or without N-acetylgalactosamine) and univalent anti-discoidin I also block formation of streams during aggregation. Two mutants of D. discoideum with low levels of discoidin I apparently reflect the deficiency of this endogenous lectin by failing to form streams or to spread on plastic and by a partial failure to enter aggregates. Together, the results indicate that discoidin I functions like fibronectin to promote cell attachment and spreading as well as ordered cellular migration during morphogenesis.

Receptor for the cell binding site of discoidin I

Discoidin I, a developmentally regulated lectin in Dictyostelium discoideum, has been implicated in cell-substratum adhesion and ordered cell migration during aggregation. This depends on the cell binding site of discoidin I, which is distinct from its carbohydrate binding site. We have isolated a receptor for the cell binding site by affinity chromatography. The receptor binds immobilized discoidin I in the presence of 0.3 M galactose and can be eluted with gly-arg-gly-asp-his-asp, a synthetic peptide the sequence of which is found in discoidin I, and which blocks cell migration into aggregates. The receptor is a developmentally regulated cell-surface glycoprotein of apparent Mr approximately 67,000. Univalent antibodies specific for this glycoprotein block aggregation.

Adhesion mutants of Dictyostelium discoideum lacking the saccharide determinant recognized by two adhesion-blocking monoclonal antibodies☆

A mutant of Dictyostelium discoideum, strain HL260, was isolated based on its failure to bind d-41, a monoclonal antibody that blocks developmentally regulated cell-cell adhesion. The mutant fails to normally acquire cell-cell adhesion as assayed with cells shaken in 10 mM EDTA, but aggregates and and constructs fruiting bodies. Other mutant strains, HL216 and HL220, previously shown to have impaired cell-cell adhesion, also lack the determinant that binds d-41. The three strains all carry mutations in a gene designated mod B, which directs a post-translational modification of several developmentally regulated D. discoideum glycoproteins. Diploids formed between independent mod B mutant haploid strains also lack this determinant and show marked impairment of cell-cell adhesion in EDTA, indicating that mutations in mod B, rather than other mutations not shared by the haploid strains, are related to the adhesion defect. The results are consistent with other evidence that an oligosaccharide carried on several developmentally regulated glycoproteins plays an essential role in EDTA-resistant cell-cell adhesion in D. discoideum. However, this type of adhesion is not essential for morphogenesis in that the only defect detected thus far in mod B mutant strains is that they construct relatively smaller fruiting bodies that contain fewer spores.

Quantification of integrin subunits on human prostatic cell lines--comparison of nontumorigenic and tumorigenic lines.

We set out to quantify integrin subunits on the surface of several prostate cell lines, including two nontumorigenic lines, in order to assess their role in tumorigenicity and metastasis.

Species-specific cell cohesion in cellular slime molds: Demonstration by several quantitative assays and with multiple species☆

Species-specific cohesion of partially differentiated cells of Dictyostelium discoideum and Dictyostelium purpureum was studied with several assays. In all cases, cells of one or both species were labelled by conjugation with either fluorescein-isothiocyanate or tetramethylrhodamine-isothiocyanate. In one assay we measured relative binding of labeled probe cells of one species to monolayers of the same or the other species. In the other assays mixtures of cells of the two species were gyrated in suspension, either for 10 min in a buffer containing ethylenediamine tetraacetic acid (EDTA) or for 40 min in a buffer without EDTA. In all cases we found species-specific cell cohesion. In other studies in which combinations of the above species, as well as Dictyostelium rosarium, Dictyostelium mucoroides and Polysphondylium pallidum were used, species-specific cell cohesion was also observed. These results cannot be due to species-specific chemotactic signals. Instead, they indicate that species specificity is a property of the cell surface.

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

Abstract 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.

Protein-linked oligosaccharide implicated in cell-cell adhesion in two Dictyostelium species☆

Monoclonal antibody d-41, previously shown to block in vitro cell-cell adhesion in aggregating Dictyostelium discoideum, also blocks adhesion in aggregating D. purpureum. In both species the antibody reacts with proteins with Mr approximately 80,000, 37,000, and 27,000, presumed to be glycoproteins since the d-41 epitope is destroyed by periodate oxidation but unaffected by extensive Pronase digestion. Polyclonal antibodies raised against the mixture of d-41 reactive glycoproteins that had been purified by immunoaffinity chromatography are potent inhibitors of D. discoideum adhesion, and adhesion-blocking activity is neutralized extensively and equivalently by each of the purified glycoproteins from D. discoideum with which d-41 reacts. In contrast, polyclonal antibodies raised against the same purified glycoproteins after they had been oxidized with periodate do not block cell-cell adhesion although they react with the glycoproteins with Mr approximately 80,000, 37,000, and 27,000 and bind as extensively to the surface of aggregating D. discoideum cells as do the adhesion-blocking polyclonal antibodies. When taken together, these results raise the possibility that some component of the d-41 binding oligosaccharide participates in cell-cell adhesion.

Chapter 21 Discoidins I and II: Endogenous Lectins Involved in Cell—Substratum Adhesion and Spore Coat Formation

Publisher Summary This chapter discusses the basic biochemical and biological techniques in identifying the functions of discoidins I and II. Like soluble lectins from vertebrates, they are apparently designed to play extracellular roles—discoidin I in cell–substratum adhesion and discoidin II in spore coat formation. A number of lines of evidence point to a role for discoidin I in a specific form of cell–substratum adhesion. Antibodies against the lectin and against a 67-kDa glycoprotein with properties of a discoidin I receptor both block ordered cell migration into aggregates. A mutant deficient in discoidin I does not aggregate normally. Nor does an antisense transformant in which there is a >90% reduction in discoidin I. One interesting conclusion from studies of the cellular role of discoidin 1 is that this lectin shares several properties with fibronectin, a vertebrate molecule implicated in the adhesion of fibroblasts to substrata. Like Fibronectin, discoidin I contains the sequence Arg-Gly-Asp-x, which has been implicated in the cell-binding properties of both molecules. Both molecules also have carbohydrate-binding sites. Firbronectins binding sites for glycosaminoglycans may participate in anchoring it to the substratum. A role for discoidin II in spore coat formation is indicated by (1) its localization in vesicles which also contain a polysaccharide to bind well to this lectin, and (2) the secretion of lectin and the polysaccharide into the spore coat.

Developmentally regulated cell-cell adhesion in Dictyostelium purpureum is mediated by a glycoprotein synthesized in nonadhesive cells

Upon starvation the cellular slime mold, Dictyostelium purpureum, develops a form of cell-cell adhesion aiding in the formation of large multicellular aggregates, which are capable of further differentiation. The molecule that mediates this adhesion is a glycoprotein of Mr approximately 40,000. The protein shares a common carbohydrate epitope with another well-characterized cell adhesion molecule from Dictyostelium discoideum, contact sites A, but the polypeptides to which it is attached differ for each species. Although mediating a developmental form of adhesiveness, the protein is synthesized in vegetative cells at a time when they do not adhere. Most of the vegetative protein is associated with cell membranes and appears to be on the surface of these cells. The protein is compared to other cell adhesion molecules from other species of cellular slime molds, and possible explanations for its inability to function in vegetative cells are discussed.

Externalization of the endogenous intracellular lectin of a cellular slime mold.

Abstract Intracellular purpurin, the endogenous lectin of Dictyostelium purpureum , has previously been shown to be externalized upon exposure of the cells to anti-purpurin IgG. Externalization of additional purpurin was presumably the consequence of cross-linking of the purpurin molecules already on the cell surface by the IgG, since binding univalent anti-purpurin Fab to the cell surface did not have this effect. In the present report we show that multivalent glycoconjugates that interact with purpurin—including asialo-bovine submaxillary mucin, a bacterial galactan, and bovine albumin derivatized with multiple chains of either lactose or lacto- N -neotetraose—all elicit the externalization of intracellular purpurin. Some of the externalized lectin is bound to the cell surface and some is found in the medium, presumably bound to the glycoconjugates. A similar effect is produced by exposure of the cells to high concentrations of purified purpurin. Several plant lectins, including conA, also have some effect, whereas others are inactive. Since cells in late stages of aggregation have about three times as much cell surface purpurin as those in early stages, this externalization reaction may have significance late in development. The results suggest that intracellular purpurin may be released in response to cross-linking of the endogenous cell surface glycoconjugates that are: (1) already bound to endogenous purpurin; (2) capable of binding purpurin; (3) capable of binding certain other lectins. The intracellular lectin may be a reserve pool, that functions only upon externalization.