Salvatore Bozzaro

Carbohydrate and other epitopes of contact site A glycoprotein of Dictyostelium discoideum as characterized by monoclonal antibodies

A series of monoclonal antibodies against a developmentally regulated protein of Dictyostelium discoideum, the contact site A glycoprotein, were used in immunoblots to label proteins of cells harvested at three stages of development: during the growth phase, at the aggregation competent stage, and at the slug stage. The antibodies fell into two groups according to their reactivity with partially or fully deglycosylated forms of the 80 kDa glycoprotein. Group A antibodies reacted not only with a 66 kDa, but also with a 53 kDa product of tunicamycin-treated wild-type cells, and they reacted with a 68 kDa component produced by HL220, a mutant that carries a specific defect in glycosylation. The 68 kDa product of the mutant was not completely unglycosylated. Like the 80 kDa glycoprotein of the wild type, which carried sulfate at carbohydrate residues, the mutant product was sulfated. In the presence of tunicamycin, the mutant produced a 53 kDa component indistinguishable from that of the wild type, which represents, most likely, the non-N-glycosylated protein portion of the contact site A glycoprotein. The group A antibodies showed almost no cross-reactivity with other proteins of the developmental stages tested, in accord with their postulated specificity for the protein moiety of the contact site A molecule. Group B antibodies did not react with the 53 kDa product of tunicamycin-treated cells, nor with the 68 kDa component of mutant HL220. These antibodies were of varying specificity. Some of them were almost as specific as group A antibodies, others cross-reacted with many proteins, particularly of the slug stage. Competition or non-competition between various group B antibodies for binding to the contact site A glycoprotein allowed sub-classification of these antibodies. According to two criteria, group B antibodies were characterized as anti-carbohydrate antibodies: (1) some of these antibodies were blocked by N-acetylglucosamine; (2) none of them reacted with the 68 kDa product or any other protein of mutant HL220. These results indicate that the 80 kDa glycoprotein carries two types of carbohydrate: type 1 carbohydrate that is sulfated and present on the 68 kDa product of mutant HL220, and type 2 carbohydrate that reacts with group B antibodies and is present on the 66 kDa product of tunicamycin-treated wild-type cells. Type 2 carbohydrate moieties are also present on many glycoproteins that are enriched in the prespore area of the slugs.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell adhesion: its quantification, assay of the molecules involved, and selection of defective mutants in Dictyostelium and Polysphondylium.

Publisher Summary Cells of Dictyostelium discoideum adhere to substrata such as glass or plastic surfaces, to bacteria, to each other, and to cells of related species. These various contact interactions have different biological functions and different specificities. Adhesion to substratum is a prerequisite of movement of single cells and of their chemotactic response during aggregation. Attachment of bacteria precedes their phagocytosis and is important for nutrition of D.discoideum cells if the bacteria are suspended rather than fixed to a substratum. Homotypic cell–cell adhesion is involved in the aggregation process and in the maintenance of multicellularity in the slug and culmination stages. The chapter describes methods applicable to either one of these contact interactions and presents the cell adhesion in a related species, Polysphondylium pallidum . In the currently used methods, coulter counting and agglutinometer assays, the agglutination of suspended cells under conditions of constant shear are determined. One strategy to identify molecules involved in intercellular adhesion is based on the use of univalent antibody fragments (Fab) against total membrane antigens for the blockage of intercellular adhesion.

Monoclonal antibodies against Dictyostelium plasma membranes: their binding to simple sugars

Monoclonal antibodies raised against purified membranes from Dictyostelium discoideum were classified according to three criteria: type of antigen as revealed in immunoblots, developmental regulation of the target antigens, and location of the antigens on the cell surface. Some antibodies reacted with myosin, two with glycolipids. One group of antibodies bound to the protein moiety of the contact site A glycoprotein, whereas another group reacted with carbohydrate epitopes that the contact site A glycoprotein shared with a few other membrane glycoproteins. Binding of the latter antibodies to their antigens was either specifically blocked by N-acetylglucosamine or by maltose as well as methyl-alpha-mannoside and N-acetylglucosamine. These anti-carbohydrate antibodies bound specifically to agarose beads derivatized with some sugars. These results and competition studies with several carbohydrates suggest that the epitope recognized by the antibodies contains as major components N-acetylglucosamine, maltose and alpha-mannose residues. One monoclonal antibody, which reacts with N-acetylglucosamine, was used for affinity purification of the contact site A glycoprotein from a crude membrane extract. N-acetylglucosamine was used as a mild eluent of the antigen from the antibody column. No detergents were added during the entire purification procedure.

Cell surface carbohydrates and cell recognition in Dictyostelium

Carbohydrate ligands and complementary receptors have been detected on the surface of Dictyostelium cells, using lectins, monoclonal antibodies, and immobilized sugar probes. They have been implicated in cell recognition processes, such as phagocytosis and intercellular adhesion, and could act as membrane signals for differentiation. Specific glycoproteins have been proposed to mediate intercellular adhesion in Dictyostelium discoideum and Polysphondylium pallidum and to account for the species-specificity of adhesion displayed by these species. Recent studies with the inhibitor of N-glycosylation, tunicamycin, and with glycosylation defective mutants suggest that some carbohydrate groups in these glycoproteins play a role in cell adhesion.

Cell differentiation in the absence of intracellular and extracellular cyclic AMP pulses in Dictyostelium discoideum

Abstract The mutant HSB1 was obtained by colony immunoblotting of mutagenized Dictyostelium discoideum cells with a monoclonal antibody against contact site A, a membrane glycoprotein. HSB1 cells undergo early development in the absence of intracellular and extracellular cAMP pulses. The receptor-mediated activation of adenylate cyclase is defective, but the basal activity increases normally during the first hours of development. Other preaggregative gene products, such as the inhibitor of phosphodiesterase, the contact site A glycoprotein, EDTA-stable contacts, and four developmentally regulated gene transcripts are also expressed in the mutant. Postaggregative gene transcripts fail to accumulate, even after pulsatile addition of cAMP. However, complete development of mutant cells is obtained by complementation with cells of the parent strain AX2.

Cell--cell contact, cyclic AMP, and gene expression during development of Dictyostelium discoideum.

Publisher Summary Dictyostelium discoideum exhibits many features of development seen in more complex eukaryotic organisms: specific cell–cell contacts are found; a homogeneous cell population differentiates into discrete cell types; and there is specific cell migration and pattern formation. These morphogenetic changes are accompanied by major changes in the pattern of gene expression. This chapter discusses the transcription of these genes, which is controlled by many of the same factors that affect gene expression in other differentiating systems, in particular, by cell- cell contact and by an extracellular hormone, cyclic AMP. Cell–cell contact in Dictyostelium is essential for stabilizing a large class of regulated mRNAs. cAMP also induces or accelerates at least part of the developmental program and may also induce the cytodifferentiation and segregation of prestalk and prespore cells. cAMP increases the synthesis and the stability of the 2500 mRNA species that are induced at aggregation; addition of this compound to disaggregated cells restores synthesis of these mRNAs.

A plasma membrane factor inhibiting intercellular adhesion in Polysphondylium pallidum

Abstract A cell adhesion-inhibiting activity was released by sonication of purified plasma membranes from Polysphondylium pallidum aggregation-competent cells. The activity was found to be periodate-sensitive but insensitive to trypsin treatment and relatively heat-stable. Adhesion of Dictyostelium discoideum cells was not affected by the released factor suggesting its species specificity.