Immunochemical Analysis of Discoidins I and II at the Cell Surface in Wild Type and Aggregation-Defective Mutants of Dictyostelium discoideum

The endogenous lectins discoidins I and II are believed to be primary components of the morphogenetic cell cohesion system of D discoideum. We have developed two immunochemical methods to analyze the association of the discoidins with the cell surface. One method is a two-stage specific antibody binding assay in which intact cells are incubated on ice with rabbit serum (either control serum or antidiscoidin I and II), washed, then incubated with ¹²⁵I-Protein A. Specific antibody binding is defined as the difference between percent radioactivity bound with antidiscoidin versus control serum during the first stage. Substantial specific binding was observed with developed A3 cells but not with vegetative cells, and nearly all of the activity could be removed by pread-sorption of the antiserum with discoidin-Sepharose. As a complementary method, quantitative immunoadsorption analysis was performed in which we tested the ability of intact cells to remove antibodies reactive with purified ¹²⁵I-discoidin I or II. Developed cells, but not vegetative cells, were capable of adsorbing antibodies reactive with discoidin I as well as those reactive with discoidin II. This represents the first demonstration that both lectins are present on the surface of cohesive cells. These procedures, coupled with other methods to analyze soluble discoidin in cell extracts, were used to study discoidin expression in wild type cells and in two newly isolated aggregation-defective mutants. Strain EB-32 fails to aggregate and displays little or no discoidin in cell extracts or at the cell surface. On the other hand, strain EB-18 forms loose amorphous mounds, and expresses substantial quantities of the discoidins, both in cell extracts and at the cell surface. These mutants should prove valuable in studying the organization and regulation of discoidins I and II at the surface of aggregating cells.     

Monoclonal antibodies: use to detect developmentally regulated antigens on D. discoideum amebae

We have used monoclonal antibodies to detect developmentally regulated cell surface antigens on D. discoideum amebae. A study of an antigen detected using an antibody produced by a hybridoma line implicates a previously undescribed component in the process of cell aggregation. This antigen (consisting of a doublet of 69,000 and 73,000 molecular weight) is first detected during the early hours of cell starvation and is present until cells begin slug formation. The developmental appearance of the antigen is not controlled by cAMP pulses and is distinct from that of Contact A sites. Fab fragments directed against the antigen are potent inhibitors of aggregation but do not inhibit the differentiation of cells to aggregation competence.     

Immunological analysis of a glycoprotein (contact sites A) involved in intercellular adhesion of dictyostelium discoideum

We have prepared antisera in rabbits to the “contact sites A” glycoprotein (gp80) purified from Dictyostelium discoideum. IgG isolated from these anti-sera reacts with a number of different proteins in D discoideum lysates, as analyzed by immune precipitation and by antibody staining of gel electropherograms transferred to nitrocellulose. Blocking experiments indicate that this cross-reactivity reflects the presence of common antigeneic determinants on gp80 and other cellular proteins, rather than the presence of extraneous antibodies in the antisera. The spectrum of reactive proteins is different a: different stages of development. In particular, gp80 itself is synthesized only for a restricted period during the cell aggregation phase. The protein persists throughout development and can be detected in spores. Anti-gp80 Fab fragments bind to the surface of developing D discoideum cells and specifically block their developmentally regulated adhesion. After absorption with vegetative cells, the IgG stains only gp80 and (to a lesser extent) one other band in lysates of aggregation-competent cells. The absorbed antibodies also can block adhesion. Several proteins that appear late in development also arc stained by the absorbed IgG.     

Ligand induced changes in stability and distribution of acetylcholine receptors on surface membranes of muscle cells

Distribution and stability of acetylcholine receptor (AChR) in cultured muscle cells was studied following the binding to these cells of concanavalin A (Con A) and specific antibodies against the receptor molecules. Con A (10 μg/ml) significantly slowed the rate of receptor degradation. In contrast, the rate of AChR degradation was enhanced 4-fold in the presence of the antibodies while the monovalent antibody fragments (Fab) were without effect. Divalent antibodies induced formation of large clusters on the surface of the muscle cultures within 2 h of incubation at 37°C. Monovalent antibody fragments and Con A had no effect on receptor distribution. It is suggested that receptor aggregation and turnover can be modulated by specific ligands acting at the cell surface.     

Purification of the major outer membrane protein of Azospirillum brasilense, its affinity to plant roots, and its involvement in cell aggregation

The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.     

Identification of carbohydrate moieties involved in EDTA-stable or EDTA-sensitive cell contact of Dictyostelium discoideum

Antisera against purified contact site A glycoprotein, with an apparent molecular weight of 80 X 10(3) (80 kDa), from Dictyostelium discoideum were raised by using Freund's adjuvant (antiserum-A) and by using Alu-Gel-S (antiserum-B) as immunoadjuvants. They were converted into Fab fragments for the cell agglutination assay. Fab fragments of antiserum-B inhibited only EDTA-stable cell contact, whereas Fab fragments of antiserum-A (Fab-A) inhibited EDTA-sensitive cell contact as well as EDTA-stable cell contact. We prepared several cell types in order to identify target antigens for the adhesion-blocking Fab-A in EDTA-sensitive cell contact or EDTA-stable cell contact. One of these cell types produced contact site A without N-glycosidically-linked carbohydrate chains. It is known that contact site A contains two kinds of N-glycosidically-linked carbohydrate chains (carbohydrates I and II, Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2653-2670). When growth-phase cells were treated with tunicamycin (TM) at a final concentration of 2 micrograms/ml in nutrient medium (TM-pretreated cells), the cells produced contact site A without N-glycosidically-linked carbohydrate chains (53 kDa) at the normal developmental stage. These cells lacked EDTA-sensitive cell contact as well as EDTA-stable cell contact. The neutralization of the adhesion-blocking Fab-A was done by using particulate fractions from each cell type. The blocking activity in EDTA-stable cell contact was neutralized by the cell type with carbohydrate II. Taking these results into consideration, EDTA-stable cell contact may be formed by the interaction between protein moieties of contact site A and carbohydrate II. Concerning EDTA-sensitive cell contact, the blocking activity was neutralized by each cell type irrespective of TM treatment. This suggests that O-glycosidically-linked carbohydrate chains play a role in EDTA-sensitive cell contact. Moreover, the biological activity in EDTA-sensitive cell contact of TM-pretreated cells suggests that N-glycosidically-linked carbohydrate chains may also be involved in this contact.     

Identification of glycoprotein IV (CD36) as a primary receptor for platelet-collagen adhesion

The role of glycoprotein IV (GPIV) in platelet activation processes has been examined by several different approaches: (i) Fab fragments of a monospecific polyclonal antibody to purified platelet GPIV (approximately 20 micrograms/ml) completely inhibited platelet shape change, aggregation, and secretion induced by collagen. Aggregation and secretion by ADP (but not shape change) and by epinephrine were also inhibited, but there was no effect on platelet activation induced by thrombin, arachidonate, or ionophore A23187. (ii) Purified GPIV was able to compete completely with membrane-bound GPIV to inhibit platelet activation induced by collagen, including shape change, but not in activation induced by any of the other platelet agonists. 50% inhibition of collagen-induced activation and secretion were obtained at GPIV concentrations of approximately 10 nM (1 micrograms/ml). (iii) Purified GPIV bound rapidly and reversibly to collagen Type I fibrils, and binding was not inhibited by adhesive proteins such as denatured collagen, fibronectin, fibrinogen, or von Willebrand factor. The direct binding of purified GPIV to collagen Type I fibrils fit best to a single site model with Kd 0.34 +/- 0.10 nM. (iv) Using a microtiter assay, platelet adhesion to collagen was shown to be inhibited by Fab fragments of monospecific polyclonal anti-GPIV antibodies, but adhesion to other adhesive proteins was unaffected. (v) When anti-GPIV was added at various times during adhesion the time dependence of inhibition was seen to be biphasic. Anti-GP antibody was able to reverse adhesion that occurred within the first 5-8 min and to inhibit adhesion occurring thereafter. These results demonstrate that GPIV mediates the early stages of platelet recognition by and attachment to collagen but that there may be a second GPIV-independent mechanism that mediates the subsequent anchorage of these adherent platelets.     

Evolution of developmental cyclic adenosine monophosphate signaling in the Dictyostelia from an amoebozoan stress response

The Dictyostelid social amoebas represent one of nature's several inventions of multicellularity. Though normally feeding as single cells, nutrient stress triggers the collection of amoebas into colonies that form delicately shaped fruiting structures in which the cells differentiate into spores and up to three cell types to support the spore mass. Cyclic adenosine monophosphate (cAMP) plays a very dominant role in controlling morphogenesis and cell differentiation in the model species Dictyostelium discoideum. As a secreted chemoattractant cAMP coordinates cell movement during aggregation and fruiting body morphogenesis. Secreted cAMP also controls gene expression at different developmental stages, while intracellular cAMP is extensively used to transduce the effect of other stimuli that control the developmental program. In this review, I present an overview of the different roles of cAMP in the model D. discoideum and I summarize studies aimed to resolve how these roles emerged during Dictyostelid evolution.     

野生仙人掌多糖对肺鳞癌细胞SK-MES-1 生长的抑制作用

目的:探讨仙人掌多糖对体外肺鳞癌细胞(SK-MES-1)生长抑制作用。方法:体外培养SK-MES-1细胞,四甲基偶氮唑盐(MTT)法观察野生仙人掌多糖对其生长的抑制,计算最低抑制浓度及抑制率;观察不同浓度多糖对细胞形态的影响;SDS-PAGE凝胶电泳简析不同组别蛋白质表达的差异。结果:野生仙人掌多糖24 h和48 h对肿瘤细胞的最低抑制浓度和抑制率分别为0.0625 mg/ml.34.06%和0.0625 mg/ml 35.37%;不同组间蛋白质表达有差异。结论:野生仙人掌多糖对SK-MES-1肺鳞癌细胞有抑制作用。     

Inhibition of N-linked glycosylation in Dictyostetium discoideum: Effects of aggregate formation

The aggregation program of Dictyostelium discoideum is extremely sensitive to the effects of tunicamycin when the drug is added to cells during the first few hours of starvation. Inhibition of development is observed with concentrations as low as 0.5 micrograms/ml, which cause only a 25%-30% inhibition of general N-linked glycosylation. However, 0.5 micrograms/ml tunicamycin can result in the total inhibition of N-linked glycosylation of specific, developmentally regulated, proteins, as exemplified by the glycoprotein 117 antigen. If added after the first hours of starvation, tunicamycin cannot inhibit aggregation even when present at 10 micrograms/ml, which maximally inhibits N-linked glycosylation. cAMP pulses can override the inhibitory effects of tunicamycin on cell aggregation. The data support the hypothesis that there is an early developmental pathway that is dependent on the N-linked glycosylation of one, or a small set of developmentally regulated proteins and that this pathway may involve the biogenesis of the chemotactic signalling system. In addition, the data raise questions as to the role of N-linked oligosaccharides in cell cohesion.     

Modification of fibrin network ultrastructure by Fab fragments specific for different domain of fibrinogen

Kinetics of inhibition of fibrin monomer polymerization produced by Fab fragments prepared from immunochemically purified monospecific antibodies to the surface epitopes of different domains of fibrinogen molecule has been correlated with electron microscopic observations of resulting specimens. Fab fragments prepared from anti FgD antisera were the most efficient inhibitors of thrombin-catalysed conversion of fibrinogen to fibrin; polymerization of fibrin monomers as detected spectrophotometrically was abolished at 2:1 molar ratio of anti FgD Fab fragments to fibra monomer. These Fab fragments acting as a steric hindrance of polymerization sites inhibited the first stage of fibrin monomer aggregation. Interaction of Fab fragments derived from antibodies specific for alpha 239-476 with corresponding segment of fibrinogen molecule resulted in a weak inhibition of fibrin monomer polymerization. However, fibrin obtained in the presence of these Fab fragments was significantly modified and showed no periodicity. This observation may suggest that anti alpha 239-476 Fab impaired the course of the second stage of fibrin monomer polymerization, i.e. lateral association of fibrin fibrils.     

Membrane sites regulating developmental gene expression in Dictyostelium discoideum

Postaggregative gene expression in Dictyostelium discoideum requires cell contact. Polyspecific monovalent antibodies (Fab) prepared from sera raised against membranes of aggregation- and postaggregation-stage cells were used to probe the cell interactions that induce rapid postaggregative synthesis of UDP-glucose pyrophosphorylase. When cells of strain V12M2 were dissociated after 8 hr of development and replated in the presence of immune Fab, both reaggregation and pyrophosphorylase synthesis were blocked. Fab neutralized by incubation with EDTA-high salt extracts of cells developed for 3 hr blocked pyrophosphorylase synthesis but not reaggregation. Therefore, some cell-surface components that regulate pyrophosphorylase synthesis (called E sites) are antigenically distinct from those required for reaggregation. The Fab provides a means to assay E sites during their purification. Addition of 10(-3) M cyclic AMP or cyclic GMP enabled the cells to bypass the blocking of E sites by Fab; pyrophosphorylase was synthesized in the absence of reaggregation. We hypothesize that E sites function by raising the level of intracellular cyclic AMP.     

Monoclonal antibodies block cell-cell adhesion in Dictyostelium discoideum

Of 39 monoclonal antibodies that bind the cell surface of aggregating Dictyostelium discoideum, 4 block 76-98% of cell-cell adhesion measured in an in vitro assay. The active antibodies all bind in the range of 10(6) antigenic sites/cell surface and react with more than one material on nitrocellulose blots prepared after polyacrylamide gel electrophoresis of whole aggregating cells in sodium dodecyl sulfate. Active antibodies can by grouped into two classes, each with two very similar members. Class I binds several molecules that are prominent in aggregating cells but scarce or undetectable in vegetative cells, blocks cell adhesion only in the presence of EDTA, and has no detectable effect on cell morphology. Class II binds a wide range of molecules present in both vegetative and aggregating cells, inhibits adhesion as well in the absence as in the presence of EDTA, and reversibly alters cell shape.     

Differential Cell Cohesiveness Expressed by Prespore and Prestalk Cells of Dictyostelium discoideum

Pseudoplasmodia of Dictyostelium discoideum at the culmination stage were separated into two cell populations by sedimentation in a discontinuous renografin gradient. The two lighter fractions (I and II) had enzymatic activities characteristic of the anterior prestalk cells, while the heaviest fraction (III) showed enzyme activities characteristic of the posterior prespore cells. Cell-cell adhesion among prespore cells is much more resistant to EDTA dissociation than 10-h cells and prestalk cells. Fab fragments prepared from antibodies directed against a specific cell surface glycoprotein gp150 were more effective in dissociating prespore cells than prestalk cells. In addition, prespore cells contained an approximately 2-fold higher concentration of the endogenous carbohydrate binding protein discoidin-I than prestalk cells. These differences may account for the differential cohesiveness of these two cell populations and provide a basis for cell recognition and cell sorting at the slug stage.     

Functional cooperation between the neural adhesion molecules L1 and N- CAM is carbohydrate dependent

The neural cell adhesion molecules L1 and N-CAM have been suggested to interact functionally by formation of a complex between the two molecules (Kadmon, G., A. Kowitz, P. Altevogt, and M. Schachner. 1990. J. Cell Biol. 110:193-208). To determine the molecular mechanisms underlying this functional cooperation, we have studied the contribution of carbohydrates to the association of the two molecules at the cell surface. Aggregation or adhesion between L1- and N-CAM-positive neuroblastoma N2A cells was reduced when the synthesis of complex and/or hybrid glycans was modified by castanospermine. Fab fragments of polyclonal antibodies to L1 inhibited aggregation and adhesion of castanospermine-treated cells almost completely, whereas untreated cells were inhibited by approximately 50%. Fab fragments of polyclonal antibodies to N-CAM did not interfere with the interaction between castanospermine-treated cells, whereas they inhibited aggregation or adhesion of untreated cells by approximately 50%. These findings indicate that cell interactions depending both on L1 and N-CAM ("assisted homophilic" binding) can be reduced to an L1-dominated interaction ("homophilic binding"). Treatment of cells with the carbohydrate synthesis inhibitor swainsonine did not modify cell aggregation in the absence or presence of antibodies compared with untreated cells, indicating that castanospermine-sensitive, but swainsonine-insensitive glycans are involved. To investigate whether the appropriate carbohydrate composition is required for an association of L1 and N-CAM in the surface membrane (cis-interaction) or between L1 on one side and L1 and N-CAM on the other side of interacting partner cells (trans-interaction), an L1-positive lymphoid tumor cell line was coaggregated with and adhered to neuroblastoma cells in the various combinations of castanospermine-treated and untreated cells. The results show that it is the cis-interaction between L1 and N-CAM that depends on the appropriate carbohydrate structures.     

Cell-cycle regulation of center initiation in Dictyostelium discoideum

The center-initiating behavior of Dictyostelium discoideum amoebae in various cell-cycle phases was investigated. Small populations of synchronized AX-2 cells were seeded 1 in 1000 into cultures of a nonsignaling mutant (NP160) incapable of initiating centers. The ability of the wild-type AX-2 cells to initiate centers among mutant amoebae displayed cell-cycle regulation. Approximately 50% of a population of S-phase cells initiated centers while only 7.5% of a population of late G2-phase cells resulted in center formation. The timing of center formation also varied with cycle position. Synchronous cultures containing only AX-2 S-phase amoebae (no NP160) displayed the initial signs of aggregation after 4.5 hr of starvation and streaming into the aggregate was complete after 6 hr. In contrast, cultures of late G2-phase amoebae initiated aggregation centers after 5.5 hr of starvation and did not complete streaming until 7.5 hr. In addition, the number of aggregates formed by these synchronous cultures of AX-2 cells also varied with cycle position. In general, these results suggest a cell-cycle modulation of the autonomous signaling responsible for center initiation.     

Cell adhesion molecules: detection with univalent second antibody

Identification of cell surface molecules that play a role in cell-cell adhesion (here called cell adhesion molecules) has been achieved by demonstrating the inhibitory effect of univalent antibodies that bind these molecules in an in vitro assay of cell-cell adhesion. A more convenient reagent, intact (divalent) antibody, has been avoided because it might agglutinate the cells rather than blocking cell-cell adhesion. In this report, we show that intact rabbit immunoglobulin directed against certain cell surface molecules of Dictyostelium discoideum blocks cell-cell adhesion when the in vitro assay is performed in the presence of univalent goat anti-rabbit antibody. Under appropriate experimental conditions, the univalent second antibody blocks agglutination induced by the rabbit antibody without significantly interfering with its effect on cell-cell adhesion. This method promises to be useful for screening monoclonal antibodies raised against potential cell adhesion molecules because: (a) it allows for the screening of large numbers of antibody samples without preparation of univalent fragments; and (b) it requires much less antibody because of the greater affinity of divalent antibodies for antigens.     

Role of class I and class II antigens in the allogenic stimulation: class I and class II recognition in allogenic stimulation; blocking of MLR by monoclonal antibodies and F(ab')2 fragments

Purified monomorphic monoclonal antibodies against Class I and Class II antigens in the inhibition of in vitro allogenic response were assayed. As expected, anti-Class II antibodies are highly inhibitory when used in concentrations greater than 5 micrograms/ml in MLRI and 50 micrograms/ml in MLRII. Surprisingly, anti-Class I monoclonal antibodies are as effective as anti-Class II in inhibiting primary MLR although they have no effect in MLRII. These results were confirmed by using F(ab')2 fragments. The inhibitory effect of anti-Class I has been shown to occur at the stimulator cell level. It is proposed that the allogenic stimulation is elicited after Class I and Class II recognition although only Class II differences are responsible for the proliferative response.     

Myelin-associated glycoprotein, a member of the L2/HNK-1 family of neural cell adhesion molecules, is involved in neuron-oligodendrocyte and oligodendrocyte-oligodendrocyte interaction

A monoclonal antibody to the myelin-associated glycoprotein (MAG) was prepared and characterized to probe for the involvement of MAG in cell surface interactions among neural cells in vitro. The antibody reacts specifically with oligodendrocyte cell surface and myelin-rich brain regions as expected from previous investigations. Not all O4 antigen- positive oligodendrocytes express MAG in vitro. Fab fragments of the antibody interfere with neuron to oligodendrocyte and oligodendrocyte to oligodendrocyte adhesion, but not with oligodendrocyte to astrocyte adhesion. MAG-containing liposomes bind to the cell surfaces of the appropriate target cells by a mechanism that is specifically inhibitable by Fab fragments of monoclonal MAG antibodies, demonstrating that MAG is a neural cell adhesion molecule.