The endocytosis of concanavalin A by Dictyostelium discoideum cells

Differentiating cells of D. discoideum in suspension bind ConA. The proportion of the bound lectin which is competitively removed by methyl-α- mannopyranoside decreases with the time of exposure. Ferritin conjugated ConA is seen to bind both to the cell surface and to be taken into the cells, the proportion of the ConA inside the cells increasing with time. The surface bound ConA is removed by washing with methyl-α- mannopyranoside while the endocytosed ConA appears unaffected. It is suggested that much of the [¹²⁵I]ConA, uncompetable by methyl-α- mannopyranoside in our and other binding studies, may be this intracellular ConA.     

Cell surface mobility of a bacterial R-form glycolipid, mR595, after binding to rat cells, and its effect on the mobility of concanavalin A receptors

Binding of small amounts of glycolipid mR595 to rat cells, followed by sequential incubation of cells at 37 °C with rabbit anti-glycolipid mR595 and fluorescein-conjugated sheep anti-rabbit γ-globulin antisera results in the localization of fluorescence at one pole of the cell surface (capping). Binding of higher amounts of glycolipid mR595 to cells not only inhibits formation of glycolipid caps but those of the ConA receptor-fluorescent ConA complex as well. Glycolipid mR595 binding does not alter [³H]ConA binding to cells but cell agglutination by ConA is inhibited in a competitive fashion. Binding of small amounts of ConA to cells does not affect glycolipid capping. Colchicine and cytochalasin B (CB) treatment of cells inhibits glycolipid cap formation.     

Rearrangement of concanavalin A receptor sites on cells tagged with dinitrofluorobenzene: Evidence for the chemical induction of a change usually associated with malignant transformation

Normal human peripheral blood granulocytes which are tagged with 1-fluoro-2,4-dinitrobenzene (DNFB) are agglutinated by concanavalin A (ConA) in a way which resembles the pattern of reactivity displayed by leukemic cells. The present study further defines this reaction. The binding of ConA to untagged and DNP-tagged granulocytes, treated with DNFB at a ratio of 10¹¹ molecules/cell, was quantified by isotopic dilution experiments employing [³H]ConA. Similar amounts of the lectin were bound to untagged and DNP-tagged cells following incubation for 5 min at 4 °C or 30 min at 24 °C: 1.1 × 10⁵ molecules/cell, 4.6 × 10²/μ² of surface area, and 1.6 × 10³/μg of protein. The binding of [³H]ConA to both untagged and DNP-tagged cells was inhibited to the same degree by α-methylglucopyranoside (α-MG). Fixation with either glutaraldehyde or formaldehyde, which immobilizes ConA receptor sites, completely inhibited the agglutination of both untagged and DNP-tagged cells although lectin binding was unchanged. This suggests that the inhibition of agglutination was not due to the blocking of ConA-binding sites by aldehyde groups but rather to the immobilization of lectin receptors. We conclude that dinitrophenylation of normal granulocytes facilitates the rearrangement of lectin receptors in a way which resembles the ConA-induced clustering of sites which have been observed with malignant and transformed cells.     

The Cell Surface of Trypanosoma musculi Bloodstream Forms. II. Lectin and Immunologic Studies*

SYNOPSIS. Living Trypanosoma musculi bloodstream trypomastigotes were agglutinated specifically with concanavalin A (ConA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose-binding protein (FBP). The agglutination with these lectins of living cells from which the coat was removed by trypsinization was the same as with intact trypanosomes. Glutaraldehyde or formalin fixation did not affect the results with regard to agglutination with WGA, SBA, and FBP, but lower agglutination with ConA was observed upon fixation. By using a dense iron-dextran marker many fewer ConA marker particles were localized at the fine structural level in the intact than in trypsin-treated trypanosomes. On the basis of the results obtained by agglutination and electron microscopy, it is likely that fixation cross-links intact surface-coat components associated with the ConA binding sites. It is evident from the studies in which lectins were employed that ligands containing α-D-mannose, N-acetylglucosamine, N-acetylgalactosamine, and α-L-fucose are randomly distributed in the outer surface of the pellicular and flagellar membranes of T. musculi trypomastigotes. Results obtained with α-amylase- and dextranase-treated trypanosomes suggested that lectin-binding sugar ligands in the cell surface were not directly associated with α-1,4 or repetitive α-1,6 glucan-bonded polysaccharide moieties. Similar conclusions can be drawn on the basis of neuraminidase treatment with regard to N-acetylated neuraminic acids. After thorough washing, intact, but not trypsin-treated trypomastigotes were agglutinated specifically with antisera against whole mouse serum and against mouse IgG. Evidently, adsorbed constituents of mouse serum are regular components of the T. musculi surface coat. After incubation in dilute whole mouse serum or in mouse IgG solutions, also the trypsinized cells were agglutinated by the 2 antisera. No such results were obtained with trypsinized cells incubated in serum-free buffers. It was concluded that mouse serum proteins were readily readsorbed on, and firmly bound to the trypsinized cells' surfaces. Specific agglutinations were obtained with trypsinized cells after incubation in dilute rat, rabbit, bovine, and human sera and in solutions of rat and rabbit IgG in reactions with the corresponding antisera. It seems, therefore, that the host serum proteins are adsorbed nonspecifically to the cell surface of trypsinized T. musculi bloodstream forms. When examined by electron microscopy, the intact trypomastigotes were covered by an ununiform, slightly granular, fibrillar extracellular coat, applied to the entire outer lamina of the pellicular and flagellar membranes. No indication of such a coat was noted in the trypsinized organisms. Flocculent surface coat-like matrix could, however, be discerned in cells which, after trypsinization, were incubated in various sera.     

Exocytosis in mammalian cells : I. Interaction of Concanavalin A and Wheat Germ Agglutinin with isolated acinar cells

The agglutination of isolated pancreas acinar cells has been induced by Wheat Germ Agglutinin (WGA) and Concanavalin A (ConA). This agglutination is significantly reduced by glutaraldehyde fixation of the membrane. The binding of ConA has been further studied in detail. The rate of ConA-cell complex formation is very rapid and the saturation curve shows a plateau at 100 μg/ml. Scatchard plot of these data revealed the presence of a class of high affinity sites which corresponds to an average of 1.5 × 10⁷ sites/cell. There is good correspondence between the agglutination curve and the ConA saturation curve. Membrane labelling with Ferritin-ConA (Fer-ConA) conjugate shows a uniform distribution over the entire cell surface. Clusters of particles were also found but they were sparsely distributed on the plasma membrane. This tracer can be used as a probe to study polysaccharides topography and especially its modification associated with the secretory process.     

Binding of concanavalin A to intact cells and spheroplasts ofAnacystis nidulans

Native cells of the cyanobacterium (blue-green alga)Anacystis nidulans did not bind fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) as measured by fluorescent spectrophotometry. By contrast, spheroplasts ofA. nidulans underwent rapid and specific agglutination in the presence of ConA thus showing appreciable affinity towards the lectin. After treatment with 0.01–0.05% (wt/vol) cetyltrimethylammonium bromide (CTAB) intact cells also became liable to ConA binding, which was not accompanied by significant agglutination. Detergents, other than CTAB, were far less effective. Specific and nonspecific binding was discriminated, as usual, with the aid of methyl -d-mannoside. Conditions are described that allow specific binding of up to 7×10⁴ molecules of FITC-ConA per cell. The binding of ConA to pretreated cells ofA. nidulans was verified by freeze-etching electron microscopy using ferritin-ConA conjugate. Our results appear to be first to demonstrate lectin binding to a cyanobacterium.     

Identification and characterization of the adenosine 3′,5′-cyclic monophosphate binding proteins appearing during the development of Dictyostelium discoideum

A photosensitive, radioactive analogue of cyclic adenosine monophosphate, 8-azido-adenosine 3′,5′-[³²P]monophosphate (8-N₃-cyclic AMP), was used to label the cyclic AMP binding proteins of Dictyostelium discoideum. During development cytosolic proteins appear which are specifically labeled by the photoaffinity agent. The proteins are developmentally regulated since they are only found in starved, developing cells. Unlabeled cyclic AMP competes specifically with the labeled analogue for protein binding sites in contrast to unlabeled 5′-AMP which does not compete. A mutant which develops spores but is deficient in stalk cell production produces a different set of cyclic AMP binding proteins from the parent strain.     

Guanine nucleotides modulate cell surface cAMP-binding sites in membranes from Dictyostelium discoideum

D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (K_d = 15 nM, $\text{t}\text{1}\text{2}\text{= 15 s}$). A second class is fast dissociating ($\text{t}\text{1}\text{2}$ about 1 s) and is composed of high affinity binding sites H (K_d ≈ 60 nM), and low affinity binding sites L (K_d = ≈ 450 nM) which interconvert during the binding reaction. Guanine nucleotides affect these three binding types in membranes prepared by shearing D.discoideum cells through Nucleopore filters. The affinity of S for cAMP is reduced by guanine nucleotides from 13 nM to 25 nM, and the number of S-sites is reduced about 50%. The number of fast dissociating sites is not altered by guanine nucleotides, but these sites are mainly in the low affinity state. Half-maximal effects are obtained at about 1 μM GTP, 2 μM GDP and 10 μM Gpp(NH)p(guanyl-5′-yl-imidodiphosphate); ATP and ADP are without effect up to 1 mM. These results indicate that D.discoideum cells have a functionally active guanine nucleotide binding protein involved in the transduction of extracellular cAMP signals via cell surface cAMP receptors.     

Serological investigations of cell adhesion in the slime molds,Dictyostelium discoideum,Dictyostelium purpureum,and Polysphondylium violaceum

1. Antibodies to slime molds were produced by injecting D. discoideum and D. purpureum amebas from 48 hour cultures into rabbits. 2. Anti-D. discoideum and anti-D. purpureum sera caused agglutination of homologous amebas from 24 to 26 hour cultures, agglutination of certain heterologous amebas from 30 to 36 hour cultures, and agglutination of all heterologous amebas from 43 to 48 hour cultures. 3. The data show that new surface antigens are formed in cultures after 26 hours and it is suggested that the new antigens are concerned with cell adhesion. 4. The probable role of surface antigens in the interaction of cells of different species of slime molds was discussed.     

Distinct lectin activities from six species of cellular slime molds.

Extracts of cohesive cells of four species of cellular slime mold, D. mucoroides, D. purpureum, D. rosarium and P. violaceum agglutinate erythrocytes in a manner that is similar to that previously observed with extracts of D. discoideum and P. pallidum. We determined inhibitory activity of a series of sugars on the agglutination activity of each of these extracts, using both semiquantitative and quantitative agglutination assays. The inhibitory potency of this series of sugars was distinct for each extract, although only slight differences were found between several species, especially D. discoideum and P. violaceum. A possible role of these agglutinins in species-specific cell cohesion is considered.     

Agglutination of growing and differentiating cells of Dictyostelium discoideum by concanavalin A

Cells of Dictyostelium discoideum are agglutinated by by concanavalin A (Con A). Agglutination is dependent upon Con A concentration and is inhibited by preincubation with α-methyl-glucoside. Agglutination by Con A has no adverse effect on cell viability. Cells harvested from exponential growth phase are agglutinated by lower concentrations of Con A, than are cells harvested during the stationary growth phase or during differentiation. The possible significance of these findings to the process of differentiation in D. discoideum is discussed.     

Crucial role of poly (ADP-ribose) polymerase (PARP-1) in cellular proliferation of Dictyostelium discoideum

The unicellular, as well as multicellular stages of Dictyostelium discoideum’s life cycle, make it an excellent model system for cell type determination, differentiation, development, and cell death studies. Our preliminary results show the involvement of poly (ADP-ribose) polymerase-1 (PARP-1) during D. discoideum growth by its constitutive downregulation as well as by its ortholog overexpression. The current study now analyzes and strengthens the role of the PARP-1 ortholog in cellular proliferation of D. discoideum. ADPRT1A was knocked out (KO) from D. discoideum and studied for its effect on cell growth, cell cycle, morphology, and oxidative stress. The present findings show that ADPRT1A KO ( A KO) cells exhibited reduced cellular proliferation, stressed phenotype, and cell cycle arrest in G2-M phase. Under oxidative stress, A KO cells exhibited slower growth and DNA damage. This is the first report where the involvement of ADPRT1A in growth in D. discoideum is established.     

Biochemical and morphological effects of sodium butyrate on Dictyostelium discoideum development

Pretreatment of proliferating D. discoideum amoebae with 10 mM butyrate for at least 8 h (one duplicating time) induced a reversible and dose dependent premature expression of several developmental parameters when the cells were starved in the absence of the fatty acid. The aggregative phase of the morphogenetic cycle was reduced in 2 h and the appearance of mature fruiting bodies and spores took place 4 h earlier as a result of butyrate pretreatment. Some developmentally regulated proteins, such as contact-sites A, cell surface lectins and cyclic AMP phosphodiesterase were also expressed 2 h earlier in butyrate pretreated cells than in controls. The level of extracellular cyclic AMP was reduced in butyrate pretreated cells, while other parameters of cyclic AMP metabolism were not affected. Butyrate also caused a partial inhibition of growth and the hyperacetylation of histone H4 in growing amoeba. These results suggest that butyrate acts as an inducer of differentiation in D. discoideum and can therefore be used as an experimental tool in order to explore regulatory mechanisms operating in slime mold differentiation.Abbreviations MES 2-N-morpholinoethanesulfonate - EDTA ethylendiaminotetracetate - TCA trichloroacetate - DTT dithiothreitol - SDS sodium dodecylsulfate     

Discoidin I-membrane interactions I. Discoidin I binds to two types of receptor on fixed Dictyostelium discoideum cells

Under physiological buffer conditions (17 mM P_i, pH 6.3), the endogenous lectin of Dictyostelium discoideum, discoidin I, binds to two types of receptors on the surface of glutaraldehyde-fixed, wild-type (NC-4) D. discoideum cells. We have designated these two types of receptors the carbohydrate or C sites and the ionic or I sites. Binding to the C sites is saturable with respect to discoidin I and is inhibited by hapten sugars (such as $\text{N-}\text{acetyl}\text{-}\text{d}\text{-}\text{galactosamine}$), but not by increasing buffer ionic strength with NaCl or polyelectrolytes. The number of C sites increases about 4-fold during the first 8.5 h of suspension differentiation, reaching a capacity for about 2–10⁴ discoidin I tetramers per cell. The binding activity of the C sites is reduced about 50% by sequential NaIO₄ oxidation/NaBH₄ reduction of the fixed cells, but it is not reduced by CHCl₃-CH₃OH extraction of the fixed cells. In marked contrast, binding to the I sites appears nonsaturable with respect to discoidin I, and it is inhibited by increasing buffer ionic strength with NaCl or polyelectrolytes (such as poly-l-glutamic acid or heparin), but not by hapten sugars. The I sites are present on both vegetative and differentiated fixed cells and can bind more than 10⁶ discoidin I tetramers per cell. The binding activity of the I sites on fixed cells is not reduced by sequential NaIO₄ oxidation/NaBH₄ reduction, but is reduced 70 to 90% by CHCl₃-CH₃OH extraction. The data suggest that the I sites represent ionic lipids that bind discoidin I electrostatically.     

Lectin-binding properties of hamster egg zona pellucida and plasma membrane during maturation and preimplantation development

The structure and organization of the zona pellucida and plasma membrane of the hamster egg at various stages of maturation and development were examined using lectin-mediated agglutination and the binding of fluorescent-labeled lectins. Ricinus communis I and Dolichos biflorus lectins specifically agglutinated the zona pellucida of both unfertilized and fertilized eggs, while wheat germ agglutinin (WGA) only agglutinated eggs which had been pretreated with protease. Six other lectins failed to agglutinate even eggs pretreated with protease. A comparison of the lectin-binding sites on the zona pellucida of eggs in various stages of maturation and development revealed that the intensity of binding and distribution of fluorescent-labeled lectins remain unchanged. Zona-free eggs were agglutinated by every lectin tested except those recognizing -fucose-like residues. Fertilized zona-free eggs were slightly more agglutinable by concanavalin A (ConA), Lens culinaris and WGA than unfertilized eggs. When the surfaces of zona-free eggs were examined with fluorescent ConA, Ricinus communis I and WGA, maximal binding was seen when eggs reached full maturity and binding decreased during the later stages of preimplantation development.     

Inhibition of ConA erythroagglutination by alkali-treated lipopolysaccharide

Bacterial lipopolysaccharide treated by hydrolysis in basic solution (hLPS) and then added to suspensions of human erythrocytes markedly inhibited erythroagglutination by concanavalin A (ConA), soybean agglutinin (SBA), and Phaseolus vulgaris phytohemagglutinin (PHA). Likewise, red cells presensitized by exposure to hLPS and then suspended in hLPS-free buffer were not agglutinated by ConA. However, when hLPS was not added to buffer both SBA and PHA strongly agglutinated erythrocytes presensitized by hLPS. Also, the binding of ³H-labeled ConA to red cells was unaffected by presensitization of the cells with hLPS. It appears, therefore, that membrane-associated hLPS localizes in the ConA receptor regions of erythrocyte membranes and inhibits ConA erythroagglutination by disruption of receptor responses to bound ConA or alteration of the active subunit structure of bound ConA.     

Inhibition of conjugation in Tetrahymena pyriformis by concanavalin A : Binding of concanavalin A to material secreted during starvation and to washed cells

A group of glycoproteins which form an insoluble complex with concanavalin A (ConA) are secreted during starvation of the mating types strain, as well as by the non-mating types of Tetrahymena pyriformis. These glycoproteins were isolated by Sepharose-ConA, characterized, and their relevance to the conjugation process studied. The isolated ConA binding proteins (CBM) contain about 26% of their total weight, a phenol sulfuric acid-positive material, presumably carbohydrates, and exhibit about 5–8 major bands by gel electrophoresis analysis. The possibility that ConA inhibits the conjugation process by precipitating with this material was tested. Addition of isolated CBM restored conjugation previously inhibited by ConA. However, incubation of isolated CBM with either of the mating types or with both did not have any effect on the kinetics of the conjugation process. Antibodies prepared against CBM-secreted by both mating types did not prevent conjugation when added to the conjugation medium. The data suggest that CBM does not directly participate in the conjugation process. Inhibition of conjugation by ConA is probably due to its interaction with specific membrane-bound glycoproteins.     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

Binding of I-Concanavalin a and agglutination of embryonic neural retina cells : Age-dependent and experimental changes

Embryonic chick neural retina cells dissociated from retina tissue by treatment with EGTA (a calcium chelator) show an age-dependent decline in ability to agglutinate with concanavalin A (ConA). This developmental change in cell surface properties is not due to loss of ConA-binding sites, since mature retina cells can be rendered agglutinable by mild trypsinization. It is also not due to masking of ConA receptors, or to a decrease in their amount, since retina cells from late embryos (19 days) bind four times as much ¹²⁵I-ConA as cells from early embryos (8 days). Our findings lead us to suggest that, as the retina differentiates the lateral mobility of ConA receptors in the cell membrane decreases resulting in a reduction of cell agglutinability; trypsinization of late embryo retina cells increases the mobility of the receptors and thereby facilitates their clustering by the lectin into a configuration conducive to cell agglutination.The ability of late embryo (19 day) retina cells dispersed with EGTA to agglutinate with ConA could be increased by still other treatments: by pre-incubation of the cell suspension in Tyrode's balanced salt solution (1 h, 37 °C); and by brief pre-exposure to glutaraldehyde. These two treatments did not enhance cell agglutination with wheat germ agglutinin (WGA). Glutaraldehyde treatment of trypsinized cells made them agglutinable with ConA also at 4 °C; cells treated otherwise agglutinated only at higher temperature. Surface-saturation of monodispersed retina cells with ConA at 37 °C—but not at 4 °C—prevented their agglutination with this lectin, but not with WGA; this inhibition was reversible by methyl a-D-glucopyranoside (αMG).