Characterization and Distribution of O-Glycosylated Carbohydrates in the Cell Adhesion Molecule, Contact Site A, fromDictyostelium discoideum

This paper presents further investigation of the properties of carbohydrate II in the cell adhesion molecule, contact site A, fromDictyostelium discoideum.A purified contact site A was digested withAchromobacterprotease I to produce a 31-kDa fragment to which carbohydrate II was mainly bound and a 21-kDa fragment containing the NH₂terminus of contact site A, which was identified as Ala-Pro-Thr-Ile-Thr-Ala. The NH₂terminus of the 31-kDa fragment was Thr-Glu-Ala-Thr-Thr-Ser. It was estimated from the cDNA sequence data of contact site A that more than 20 Ser/Thr residues exist as target sites for the O-linked oligosaccharides in the 31-kDa fragment, but not for the N-linked oligosaccharides. These results suggest that carbohydrate II exists as clustered O-linked oligosaccharides in the COOH terminus of contact site A. The results of two-dimensional electrophoresis confirm that oligosaccharides of contact site A contain sialic acids. Immunoelectron microscopy was carried out to define the organelle in which O-glycosylation by carbohydrate II occurs and how carbohydrate II antigens are distributed on the cell surface. The results show that O-glycosylation can occur in the Golgi apparatus inD. discoideumas observed in other cells, although this O-glycosylation was inhibited by tunicamycin. Furthermore, gold particles were densely concentrated in cell–cell contact regions but sparsely distributed in noncontact regions.     

Replacement of the phospholipid-anchor in the contact site A glycoprotein of D. discoideum by a transmembrane region does not impede cell adhesion but reduces residence time on the cell surface

The contact site A (csA) glycoprotein of Dictyostelium discoideum, a cell adhesion molecule expressed in aggregating cells, is inserted into the plasma membrane by a ceramide-based phospholipid (PL) anchor. A carboxyterminal sequence of 25 amino acids of the primary csA translation product proved to contain the signal required for PL modification. CsA is known to be responsible for rapid, EDTA-resistant cohesion of cells in agitated suspensions. To investigate the role of the PL modification of this protein, the anchor was replaced by the transmembrane region and short cytoplasmic tail of another plasma membrane protein of D. discoideum. In cells transformed with appropriate vectors, PL-anchored or transmembrane csA was expressed under the control of an actin promoter during growth and development. The transmembrane form enabled the cells to agglutinate in the presence of shear forces, similar to the PL-anchored wild-type form. However, the transmembrane form was much more rapidly internalized and degraded. In comparison to other cell-surface glycoproteins of D. discoideum the internalization rate of the PL-anchored csA was extremely slow, most likely because of its exclusion from the clathrin-mediated pathway of pinocytosis. Thus, our results indicate that the phospholipid modification is not essential for the csA-mediated fast type of cell adhesion but guarantees long persistence of the protein on the cell surface.     

SIALIC ACID AND THE SOCIAL BEEIAVIOUR OF CELLS

1. It is suggested that specific carbohydrate side-chains of membrane glycoproteins are the sites for cell recognition or adhesion when the terminal sugar, sialic acid, is absent. 2. It is suggested that sialic acid plays a ‘protective’ or ‘blocking’ role in cell interactions so that addition of sialic acid to asialo side-chains converts them to forms inactive for recognition. This principle of ‘blocking’ by sialic acid has been observed in other situations as in covering tumour antigens and in protecting glycoproteins from uptake by the liver. It is here extended to cell-cell adhesions. 3. It is to be expected that specific ‘protective’ actions of sialic acid in membrane-bound glycoproteins will be difficult to detect. As a charged residue, sialic acid is likely to have a strong influence both on the glycoproteins on which it is borne and on their interactions with each other at the cell surface. Removal of sialic acid by enzymes could therefore perturb the structure of the cell surface in several ways and so obscure the ‘protective’ effects of sialic acid. Sialic acid is therefore suggested to have a structural role also. 4. Evidence is assembled in favour of a model in which sialysation of specific adhesive receptors affects the social behaviour of cells. This may be an effect associated with growing cells since the contact properties of mitotic cells (and populations rich in dividing cells) are decreased by the increased sialysation of receptors. One of the factors associated with malignant behaviour could be that adhesive receptors are permanently blocked by sialic acid. 5. A schematic representation of some of the points is given in Fig. 4.     

Two distinct adhesion systems are responsible for EDTA-sensitive adhesion in Dictyostelium discoideum

Abstract. Early in their developmental program, Dictyostelium discoideum exhibit EDTA-sensitive and EDTA-resistant adhesion. The molecules which mediate the adhesions have been called contact sites, with contact sites A mediating EDTA-resistant adhesion and contact sites B mediating EDTA-sensitive adhesion. The studies described here have revealed that prior to aggregation, a second EDTA-sensitive adhesion system emerges. In keeping with previously established nomenclature, the molecules mediating the newly discovered adhesion system have been called contact sites C. Unlike contact sites B, contact sites C are unaffected by a contact sites B-blocking peptide. Contact sites C-mediated adhesion is also distinct from contact sites B-mediated adhesion in that contact sites C-mediated adhesion is EGTA-resistant and in the presence of EDTA it can be rescued by the addition of Mg²⁺. Thus Mg²⁺ may be the cation present under physiological conditions that is essential for contact sites C activity. Unlike contact sites B-mediated adhesion, contact sites C-mediated adhesion is not observed in growing amoebae. Contact sites C-mediated adhesion first becomes apparent within hours after the initiation of development and its strength appears to increase throughout the first 10 h of the developmental program. A mutant lacking the EDTA-resistant contact sites A exhibits normal contact sites B- and C-mediated adhesion, demonstrating that both EDTA-sensitive adhesion systems are independent of contact sites A. Thus aggregating D. discoideum amoebae possess three distinct adhesion systems, one of them is EDTA-resistant and the other two are EDTA-sensitive.     

Structural characterization of Dictyostelium discoideum prespore-specific gene D19 and of its product, cell surface glycoprotein PsA.

The Dictyostelium discoideum cell surface antigen PsA is a glycoprotein which first appears in the multicellular stage soon after tip formation and is selectively expressed on prespore cells. The D19 gene encodes an mRNA sequence which is highly enriched in prespore over prestalk cells in the slug stage. We have determined 81 amino acid residues of N-terminal sequence from immunoaffinity-purified PsA protein and shown this sequence to be identical to the predicted sequence of the D19 gene. There are several short repeat elements close to the C terminus, and unequal crossing-over within these is proposed to account for the size polymorphism observed in PsA protein isolated from different D. discoideum strains. The repeats are proline rich and show similarity to the C-terminal region of the D. discoideum cell adhesion molecule, contact sites A. The extreme C terminus, which is also homologous to contact sites A, is characteristic of proteins attached to the plasma membrane via a glycosyl-phosphatidylinositol link. We have marked the PsA gene by insertion of an oligonucleotide encoding an epitope of the human c-myc protein. A construct containing this gene and 990 base pairs of 5'-flanking region directed correct temporal and spatial mRNA accumulation. We found the marked PsA protein, detected with the human c-myc antibody, to be correctly localized on the surface of cells.     

The intracellular concentration of sialic acid regulates the polysialylation of the neural cell adhesion molecule

Sialic acids are expressed as terminal sugars in many glycoconjugates and play an important role during development and regeneration, as they are involved as polysialic acid in a variety of cell-cell interactions mediated by the neural cell adhesion molecule NCAM. The key enzyme for the biosynthesis of sialic acid is the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE). Mutations in the binding site of the feedback inhibitor CMP-sialic acid of the GNE leads to sialuria, a disease in which patients produce sialic acid in gram scale. Here, we report on the consequences after expression of a sialuria-mutated GNE. Expression of the sialuria-mutated GNE leads to a dramatic increase of both cellular sialic acid and polysialic acid on NCAM. This could also be achieved by application of the sialic acid precursor N-acetylmannosamine. Our data suggest that biosynthesis of sialic acid regulates and limits the synthesis of polysialic acid.     

Wheat germ agglutinin binds to the contact site A glycoprotein of Dictyostelium discoideum and inhibits EDTA-stable cell adhesion

Wheat germ agglutinin (WGA), a lectin that primarily reacts with N-acetylglucosamine residues, specifically inhibits the EDTA-stable type of intercellular adhesion of aggregation competent Dictyostelium discoideum cells. The major WGA-binding protein of these cells is a developmentally-regulated glycolipoprotein of 80 kd apparent mol. wt., designated as contact site A. This glycoprotein is a target site of antibody fragments that block the EDTA-stable cell adhesion, and is characterized by sulfated carbohydrate residues. WGA does not significantly bind to glycoproteins of a mutant, HL220, which produces a 68-kd component in place of the 80-kd glycoprotein. Inhibition of N-glycosylation by tunicamycin causes wild-type cells to produce a WGA-binding but unsulfated 66-kd component and a non-binding 53-kd component. These results indicate that the 80-kd glycoprotein contains two classes of carbohydrate residues, a WGA-binding one that is defective in HL220, and another, sulfated, one that is absent from the 66-kd wild-type product; both are missing in the 53-kd protein. WGA and a monoclonal antibody that is blocked by N-acetylglucosamine were further used to probe for glycoproteins in the multicellular slug stage that share carbohydrate structures - and possibly functions - with the contact site A glycoprotein. Glycoproteins in the 95-kd range have previously been implicated in cell-to-cell adhesion during the slug stage. We distinguished a 95-kd glycoprotein that binds WGA from another one that binds antibody.     

Cell-cell adhesion molecules in Dictyostelium

Multicellularity in the cellular slime mold Dictyostelium discoideum is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive adhesion sites are expressed very early in the development cycle and a surface glycoprotein of 24,000 Da is known to be responsible for these sites. The EDTA-resistant contact sites begin to accumulate on the cell surface at the aggregation stage of development. Several glycoproteins have been implicated in the EDTA-resistant type of cell-cell binding and the best characterized one has an Mr of 80,000 (gp80). gp80 mediates cell-cell binding via homophilic interaction and its cell binding site has been mapped to an octapeptide sequence. The mechanism by which gp80 mediates cell-cell adhesion will be discussed.     

Adhesion-induced receptor segregation and adhesion plaque formation: A model membrane study.

A model system to study the control of cell adhesion by receptor-mediated specific forces, universal interactions, and membrane elasticity is established. The plasma membrane is mimicked by reconstitution of homophilic receptor proteins into solid supported membranes and, together with lipopolymers, into giant vesicles with the polymers forming an artificial glycocalix. The homophilic cell adhesion molecule contact site A, a lipid-anchored glycoprotein from cells of the slime mold Dictyostelium discoideum, is used as receptor. The success of the reconstitution, the structure and the dynamics of the model membranes are studied by various techniques including film balance techniques, micro fluorescence, fluorescence recovery after photobleaching, electron microscopy, and phase contrast microscopy. The interaction of the functionalized giant vesicles with the supported bilayer is studied by reflection interference contrast microscopy, and the adhesion strength is evaluated quantitatively by a recently developed technique. At low receptor concentrations adhesion-induced receptor segregation in the membranes leads to decomposition of the contact zone between membranes into domains of strong (receptor-mediated) adhesion and regions of weak adhesion while continuous zones of strong adhesion form at high receptor densities. The adhesion strengths (measured in terms of the spreading pressure S) of the various states of adhesion are obtained locally by analysis of the vesicle contour near the contact line in terms of elastic boundary conditions of adhesion: the balance of tensions and moments. The spreading pressure of the weak adhesion zones is S approximately 10(-9) J/m(2) and is determined by the interplay of gravitation and undulation forces whereas the spreading pressure of the tight adhesion domains is of the order S approximately 10(-6) J/m(2).     

Discrete interactions in cell adhesion measured by single-molecule force spectroscopy

Cell-cell adhesion mediated by specific cell-surface molecules is essential for multicellular development. Here we quantify de-adhesion forces at the resolution of individual cell-adhesion molecules, by controlling the interactions between single cells and combining single-molecule force spectroscopy with genetic manipulation. Our measurements are focused on a glycoprotein, contact site A (csA), as a prototype of cell-adhesion proteins. csA is expressed in aggregating cells of Dictyostelium discoideum, which are engaged in development of a multicellular organism. Adhesion between two adjacent cell surfaces involves discrete interactions characterized by an unbinding force of 23 +/- 8 pN, measured at a rupture rate of 2.5 +/- 0.5 microm s-1.     

Cross-reactivity of contact site A to antibody produced against a stage-specific antigen from a phenol/water extract of Dictyostelium discoideum

The stage-specific antigen, gp68 (Hirano, T., Yamada, H., & Miyazaki, T. (1983) Biochim. Biophys. Acta 742, 224-234), was purified from a phenol/water extract of aggregation-competent cells of Dictyostelium discoideum by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Anti-gp68 was produced against purified gp68 which was determined to be homogeneous by silver staining on analysis by SDS-polyacrylamide gel electrophoresis. The cross-reactivity of anti-gp68 against cellular antigens was estimated by immuno-affinity chromatography on anti-gp68 immunoglobulin G (IgG)/Sepharose. When the whole cell lysate was applied to this affinity column, three major proteins, with molecular weights of 80,000, 68,000, and 56,000, were obtained in the absorbed fraction. When the butanol extract, which was enriched in contact site A, an adhesion mediating glycoprotein, was applied to the same column, two major proteins with molecular weights of 80,000 (corresponding to contact site A) and 56,000 were obtained in the absorbed fraction but, however, gp68 was negligible. Reversely, when the phenol/water extract was applied to anti-contact site A-IgG/Sepharose, only gp68 was obtained in the absobed fraction. Moreover, contact site A was seen to compete with [3H]mannose-labeled gp68 in a competition radioimmunoassay using anti-gp68 serum. The effect of Pronase or exo-alpha-mannosidase digestion on the antigenic activity of gp68 was examined by radioimmunoassaying. The results indicated that the alpha-mannosyl residue of the non-reducing terminal in the carbohydrate moiety of gp68 was a major immunodeterminant. However, the polypeptide chain did not participate in the antigenic reactivity against anti-gp68. Both anti-gp68 and anti-contact site A agglutinated heat killed-yeast cells. Also, both anti-sera inhibited EDTA-stable cell adhesion of aggregation-competent cells in the presence of Fab from goat anti-rabbit IgG. These results indicate that gp68 and contact site A have a common antigenic determinant against anti-gp68, and that the target antigen of anti-gp68 was somehow involved in cell adhesion.     

Carbohydrate moieties of N-cadherin from human melanoma cell lines

Expression of N-cadherin an adhesion molecule of the cadherin family, in tumor cells is associated with their increased invasive potential. Many studies suggested the role of N-linked oligosaccharides as important factors that contribute to metastasis by influencing tumor cell invasion and adhesion. N-cadherin is a heavily glycosylated protein. We have analysed the carbohydrate profile of this protein synthesized in human melanoma cell lines: WM35 from the primary tumor site and WM239, WM9, and A375 from different metastatic sites. N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterisation of its carbohydrate moieties was carried out by SDS/PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and analysis of their glycans using highly specific digoxigenin or biotin labelled lectins. The positive reaction of N-cadherin from the WM35 cell line with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and Sambucus nigra agglutinin (SNA) indicated the presence of high-mannose type glycans and biantennary complex type oligosaccharides with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines gave a positive reaction with Phaseolus vulgaris leukoagglutinin (L-PHA) and lotus Tetragonolobus purpureas agglutinin (LTA). This indicated the presence of tri- or tetra-antennary complex type glycans with alpha-fucose. In addition, N-cadherin from WM9 (lymphomodus metastatic site) and A375 (solid tumor metastatic site) contained complex type chains with alpha2-3 sialic acid (positive reaction with Maackia amurensis agglutinin--MAA). The results demonstrated that N-glycans of N-cadherin are altered in metastatic melanomas in a way characteristic for invasive tumor cells.     

Cleavage of the polysialosyl units of brain glycoproteins by a bacteriophage endosialidase. Involvement of a long oligosaccharide segment in molecular interactions of polysialic acid

Polysialosyl chains containing alpha 2-8-linked N-acetylneuraminic acid have been suggested to modulate the biological activity of a neural cell adhesion molecule. Polysialosyl glycopeptides isolated from developing brain were incubated with a bacteriophage containing endosialidase. Sialic acid oligomers up to 7 residues long were liberated both from the glycopeptides and colominic acid. The substrate specificity of the endosialidase was studied with sialic acid oligomers of different sizes prepared from colominic acid. It was found that the endosialidase required the simultaneous presence adjacent to the site of cleavage a minimum of 3 sialic acid residues on the distal side and a minimum of 5 sialic acid residues on the proximal (reducing end) side. From the fragments liberated by the enzyme the existence of polysialic acid chains up to at least 12 residues long in the glycopeptides were concluded. This was also supported by the interaction of the glycopeptides with a meningococcal group B polysaccharide antiserum, which was found to require 10 residues or more for binding. The results indicate that the brain polysialosyl glycopeptides contain a long polysialic acid segment, which is also specifically needed for certain molecular interactions. The implications of the findings for the biological properties of the neural cell adhesion molecule are discussed.     

The Sialoadhesins — A family of sialic acid-dependent cellular recognition molecules within the immunoglobulin superfamily

For many years evidence has accumulated that sialic acids function in cellular interactions either by masking or as a recognition site. However, receptors or adhesion molecules mediating such functions between eukaryotic cells were unknown until about 5 years ago, when it was found that the members of the Selectin family mediate adhesion of leukocytes to specific endothelia through binding to sialylated glycans like sialyl Lewis^x. More recently, the Sialoadhesin family of sialic acid-dependent adhesion molecules was defined within the superfamily of immunoglobulin-like molecules. So far, it has been shown that sialoadhesin (Sn), CD22, CD33, the myelin-associated glycoprotein (MAG) and the Schwann cell myelin protein (SMP) belong to this family. In contrast to the Selectins, these proteins are associated with diverse biological processes, i.e. hemopoiesis, neuronal development and immunity. In this review their properties, carbohydrate specificities and potential biological functions are discussed. Finally, we provide perspectives with respect to the nature of ligands, implications of sialic acid modifications and future research.Abbreviations IgSF immunoglobulin superfamily - MAG myelin-associated glycoprotein - Sia sialic acid - SMP Schwann cell myelin protein - Sn sialoadhesin     

The contact site A glycoprotein of Dictyostelium discoideum carries a phospholipid anchor of a novel type.

The contact site A glycoprotein, a cell adhesion protein of aggregating Dictyostelium cells, was labeled with fatty acid, myo-inositol, phosphate and ethanolamine in vivo, indicating that the protein is anchored in the membrane by a lipid. This lipid was not susceptible to phosphatidyl inositol specific phospholipase C. When cleaved with nitrous acid or when subjected to acetolysis, the anchor released lipids which were different from those released from Trypanosoma variant cell surface glycoprotein, a protein with a known phosphatidyl inositol-glycan anchor. Resistance to weak and sensitivity to strong alkali indicated that the fatty acid in the contact site A glycolipid anchor was in an amide bond. On incubation with sphingomyelinase, a lipid with the chromatographic behavior of ceramide was released. These results suggest that the contact site A glycoprotein is anchored by a ceramide based lipid glycan.     

Inhibition of cell adhesion in Dictyostelium discoideum by tunicamycin is prevented by leupeptin

The carbohydrate requirement for cell adhesion of aggregation-competent cells of Dictyostelium discoideum has been examined by use of a selective glycosylation inhibitor of N-glycosyl protein, tunicamycin (TM). TM completely inhibited EDTA-stable cell adhesion and glycosylation of some membrane glycoproteins in aggregation-competent cells of D. discoideum (Yamada, H., et al. (1982) J. Biochem. 92, 399-406). The present study showed that the inhibition of EDTA-stable cell adhesion by TM was prevented significantly when the cells were treated with TM in the presence of a protease inhibitor, leupeptin (LP), whereas the inhibition of glycosylation by TM was not prevented. The cell extract of aggregation-competent cells contained acid proteases, and LP strongly inhibited acid protease from D. discoideum in vitro. On analysis by SDS-polyacrylamide gel electrophoresis (PAGE), many protein bands present in the membrane fraction of control cells disappeared or decreased on TM treatment of the cells in the absence of LP, however, some of these proteins were restored when the cells were treated with TM in the presence of LP. These results strongly support an idea that EDTA-stable cell adhesion characteristic to aggregation-competent cells is mediated by glycoproteins with asparagine-linked carbohydrate. However, the requirement for the carbohydrate moiety of the glycoprotein in cell adhesion appears to be indirect in that it acts to protect the protein moiety from proteolytic degradation.     

Mutants of Dictyostelium discoideum with altered carbohydrate moieties of contact site A.

Mutants of Dictyostelium discoideum were isolated and found to be defective in the epitope recognized by the monoclonal antibody 120 against the carbohydrate moieties of an integral membrane glycoprotein, contact site A, with the apparent molecular mass of 80 x 10(3). One mutant, HG764, did not express any contact site A and had lost cell contact resistant to EDTA. The others, including HG794, expressed a 68-kDa form of contact site A. In comparison with the parental strain HG592, HG794 showed weaker EDTA-resistant cell contact and the same degree of EDTA-sensitive cell contact. This suggested that the moieties which HG794 lacked were involved in EDTA-resistant cell contact. The 68-kDa contact site A in HG794 could be labeled with wheat germ agglutinin and incorporated [35S] sulfate. The modB mutant HL220 also expresses 68-kDa contact site A, although it cannot be labeled with wheat germ agglutinin. Therefore, the mutants HG794 and HL220 were compared by a complementation test. The diploid strain DG701 expressed 80-kDa contact site A and showed the same degree of EDTA-resistant cell contact as strain HG592. In its EDTA-resistant cell contact, HG794 was stronger than HL220. These results suggest that HG794 is a new mutant, and that there might be at least two processes in the glycosylation of 68-kDa contact site A to the 80-kDa form. The carbohydrate moieties recognized by monoclonal antibody 120 and by wheat germ agglutinin might be involved in EDTA-resistant cell contact.     

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.     

Role of UDP-N-Acetylglucosamine2-Epimerase/N-Acetylmannosamine Kinase (GNE) in β1-Integrin-Mediated Cell Adhesion

Hereditary inclusion body myopathy (GNE myopathy) is a neuromuscular disorder due to mutation in key sialic acid biosynthetic enzyme, GNE. The pathomechanism of the disease is poorly understood as GNE is involved in other cellular functions beside sialic acid synthesis. In the present study, a HEK293 cell-based model system has been established where GNE is either knocked down or over-expressed along with pathologically relevant GNE mutants (D176V and V572L). The subcellular distribution of recombinant GNE and its mutant showed differential localization in the cell. The effect of mutation on GNE function was investigated by studying hyposialylation of cell membrane receptor, β1-integrin. Hyposialylated β1-integrin localized to internal vesicles that was restored upon supplementation with sialic acid. Fibronectin stimulation caused migration of hyposialylated β1-integrin to the cell membrane and co-localization with focal adhesion kinase (FAK) leading to increased focal adhesion formation. This further activated FAK and Src, downstream signaling molecules and led to increased cell adhesion. This is the first report to show that mutation in GNE affects β1-integrin-mediated cell adhesion process in GNE mutant cells.