Glycoproteins of the plasma membrane of dictyostelium discoideum during development

Differential binding of ¹²⁵I-Con A to whole cells throughout the life cycle of Dictyostelium discoideum indicates that the appearance of Con A binding proteins on the plasma membrane is under developmental regulation. Con A-Sepharose affinity chromatography of radio-iodinated plasma membrane preparations, followed by analysis with SDS-PAGE, revealed that there are at least 15 Con A binding proteins associated with the surface of 0 hr cells. Between 6 and 18 hr of development, the relative intensity of one of the bands, which corresponds to a protein of molecular weight of 150,000 daltons, increases dramatically.     

Transfer of concanavalin A between responding lymphocytes and syngeneic stimulating cells in cell-mediated mitogenic response.

We have studied the mechanism of the cell-mediated mitogenic response (CMMR), in which proliferative responses are generated in mouse T lymphocytes co-cultivated with syngeneic mitomycin C-treated spleen cells (Mito-SP), when either responder or stimulator cells are briefly pretreated with concanavalin A (Con A) under nonmitogenic conditions. We present evidence that an intact membrane of the stimulator cells is required in CMMR, since the response was abolished by fixation, or by freezing and thawing of stimulator cells. The fate of cell-bound Con A was studied by tracing I-Con A bound to either stimulator or responder. A lymphoblast that developed from untreated CRT stimulated by I-Con A-coated syngeneic Mito-Sp carried about 105 molecules of 125I-Con A. The amount of Con A released into the culture medium was not sufficient for inducing a mitogenic response by itself, nor to bind to cells at the level found in CMMR, suggesting that 125I-Con A was transferred directly from labeled cells to unlabeled cells. Transferred 125I-Con A found in lymphoblasts was undergraded intact Con A, as demonstrated by gel electrophoresis. Autoradiography allowed visualization of the movement of 125I-Con A from stimulator to responder cells within 60 min of cell contact.     

Kinetic aspects of hemoglobin.haptoglobin-receptor interaction in rat liver plasma membranes, isolated liver cells, and liver cells in primary culture

125I-Hemoglobin.haptoglobin injected intravenously into rats was incorporated into liver parenchymal cells as evidenced by a cell separation technique. A mixture of freshly isolated liver parenchymal and nonparenchymal cells failed to internalize and degrade the 125I-hemoglobin.haptoglobin added, although it retained the ability to bind the molecule. The liver parenchymal cells in primary culture also lacked the ability to degrade 125I-hemoglobin.haptoglobin, although they bound the molecule more extensively as compared with the freshly isolated liver cells. It was confirmed that the 125I-hemoglobin.haptoglobin which was bound to the freshly isolated liver parenchymal cells localized on the outer surface of liver plasma membranes. Scatchard plots revealed the existence of two binding sites for 125I-hemoglobin-haptoglobin on the isolated liver plasma membrane: an apparent high affinity binding site (Kd = 1.3 X 10(-7) M) and an apparent low affinity binding site (Kd = 4.0 X 10(-6) M) at 37 degrees C. In contrast, freshly isolated liver parenchymal cells had only an apparent low affinity binding site (Kd = 1.4 X 10(-6) M) at 37 degrees C. Impairment of the apparent high affinity binding site during the isolation procedure with collagenase seemed to be related to loss of the ability to internalize and degrade the 125I-hemoglobin.haptoglobin molecules into the freshly isolated liver parenchymal cells or liver parenchymal cells in primary culture.     

Schistosomula of Schistosoma mansoni clear concanavalin A from their surface by sloughing

The lectin concanavalin A (Con A) was used as a model probe to study the behavior of molecules bound to the surface of recently transformed schistosomula of Schistosoma mansoni. Con A binding was saturable (150- 180 pg/organism) and specifically competed by alpha-methyl mannoside. Both FITC-Con A and 125-I-Con A were lost from the surface of schistosomula with a halftime of 8-10 h in culture in defined medium. A comparable decrease in the binding of Con A to schistosomula cultured and then labeled with the lectin indicated that the labeling procedure itself was not inducing the observed change. Internalization of Con A was not seen by either fluorescence microscopy or electron microscope radioautography. In addition, 70-80% of the radioactivity lost from the parasite was recoverable by TCA precipitation from the culture medium as intact Con A (27,000 mol wt on SDS PAGE). Thus, the mechanism of clearance of bound Con A from the surface of cultured schistosomula is apparently by sloughing of Con A molecules intact into the culture media and not by endocytosis and degradation. Con A binding sites, visualized with hemocyanin by scanning electron microscopy, appeared homogeneously distributed over the surface of schistosomula when organisms were labeled at 4 degree C or after fixation with glutaraldehyde. However, Con A and hemocyanin formed aggregates on the surface of schistosomula when labeling was performed at 37 degrees C, which suggests that lectin binding sites have lateral mobility within the plane of the membrane. These aggregates are likely independent of metabolism by the parasite because aggregation also occurs on the surface of organisms killed with azide.     

Adhesion of Phytophthora palmivora zoospores: detection and ultrastructural visualization of concanavalin A-receptor sites appearing during encystment.

The binding of concanavalin A (Con A) to the cell surface of zoospores and cysts of Phytophthora palmivora was studied by radiometry (125I-Con A), ultraviolet microscopy (fluorescein-Con A) and electron microscopy peroxidase-diaminobenzidine technique). Zoospores were found to secrete during the early stages of encystment a Con A-binding material susceptible to trypsin digestion. This glycoprotein is contained in the so-called peripheral vesicles and is probably responsible for the adhesion of the encysting zoospores to solid surfaces.     

Concanavalin A inhibition of alpha-bungarotoxin binding to a nonfusing muscle cell line.

Incubation of a nonfusing muscle cell line, BC3H1, with concanavalin A (Con A) results in a maximum decrease of 35% in the cell's ability to bind alpha-bungarotoxin (alpha-BuTx). The Con A-induced inhibition of 125I-alpha-BuTx binding is reversible and the degree of inhibition parallels the degree of saturation of Con A binding sites on the cell surface. The maximum level of Con A-induced inhibition of 125I-alpha-BuTx binding is not affected by increasing the time of incubation in Con A, using higher concentrations of Con A or by increasing the time of incubation in the presence of 125I-alpha-BuTx. In addition, all BC3H1 cells in culture are sensitive to the Con A-induced inhibition of 125I-alpha-BuTx binding. A comparison of the pseudo-first order rate constants for 125I-alpha-BuTx binding to untreated (8.6 x 10(4) M-1 S-1) and Con A-treated (5.4 x 10(4) M-1 S-1) BC3H1 cells, however, shows that those acetylcholine receptors in Con A-treated cells which bind 125I-alpha-BuTx do so with a lowered apparent affinity. Partial inhibition of toxin-binding capacity is not a consequence of two classes of acetylcholine receptors on the cell surface. Furthermore, individual receptors experience partial inhibition of their binding capacity by Con A, resulting in receptors with at least one binding site blocked and at least one site available for alpha-BuTx binding.     

Identification of cell surface receptors for murine macrophage inflammatory protein-1 alpha.

We have produced recombinant proteins for a cytokine, L2G25BP (macrophage inflammatory protein-1 alpha) (MIP-1 alpha). By using the recombinant protein (rMIP-1 alpha), receptors for MIP-1 alpha were identified on Con A-stimulated and unstimulated CTLL-R8, a T cell line, and LPS-stimulated RAW 264.7, a macrophage cell line. The 125I-rMIP-1 alpha binds to the receptor in a specific and saturable manner. Scatchard analysis indicated a single class of high affinity receptor, with a Kd of approximately 1.5 x 10(-9) M and approximately 1200 binding sites/Con A-stimulated CTLL-R8 cell and a Kd of 0.9 x 10(-9) M and approximately 380 binding sites/RAW 264.7 cell. 125I-rMIP-1 alpha binding was inhibited by unlabeled rMIP-1 alpha in a dose-dependent manner, but not by IL-1 alpha or IL-2. rMIP-1 alpha inhibited the proliferation of unstimulated CTLL-R8 cells. Rabbit anti-rMIP-1 alpha antibodies blocked the growth-inhibitory effect of the rMIP-1 alpha on CTLL-R8 cells.     

Production of monoclonal antibodies against a cell surface concanavalin a binding glycoprotein

Concanavalin A-binding (Con A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate-polyacrylamide gels than did the Tritonsoluble surface components, which were not retarded by the Con A-Sepharose column. [¹²⁵I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten α-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [¹²⁵I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [¹²⁵I]-labeled CHO surface component of ～265,000 daltons.     

Purification and partial characterization of a cell adhesion molecule (gp150) involved in postaggregation stage cell-cell binding in Dictyostelium discoideum.

A cell surface glycoprotein of apparent Mr 150,000 (gp150) has been implicated in mediating EDTA-resistant cell-cell adhesion in Dictyostelium discoideum. A simple purification scheme making use of high-performance liquid chromatography has been devised to purify gp150 to near homogeneity. Purified gp150 was capable of neutralizing the effect of a rabbit antiserum raised against gel-purified gp150, which was previously reported to be a potent inhibitor of cell-cell adhesion (Geltosky, J. E., Weseman, J., Bakke, A., and Lerner, R. A. (1979) Cell 18, 391-398). The binding of 125I-labeled gp150 to intact cells was both dose-dependent and saturable, demonstrating the presence of specific cell surface binding sites for gp150. When reassociation of postaggregation stage cells was carried out in the presence of soluble gp150, aggregate formation was strongly inhibited. In contrast, gp150 failed to exert any effect on cells at the aggregation stage. The inhibitory effect of gp150 was sensitive to protease treatment, suggesting that the protein moiety is crucial to gp150 function. These results, taken together, provide direct evidence that gp150 is a cell-cell adhesion molecule involved in cell-cell binding in the postaggregation stage of Dictyostelium development.     

Characterization of the C1q receptor on a human macrophage cell line, U937.

The binding of C1q to the human macrophage cell line U937 has been studied. Fluorescence microscopy with fluorescein-conjugated F(ab')2 anti-C1q antibody showed that 100% of the cell population is able to bind exogenous C1q. Monomeric C1q binding to U937 cells is very weak at normal ionic strength (I0.15) and was therefore investigated at I0.07, conditions which stabilize the binding. However, aggregation of C1q on dextran sulphate or a lipid A-rich lipopolysaccharide allowed a firm, binding at I0.15. Quantitative binding studies with monomeric 125I-C1q showed a concentration-dependent, saturable, specific and reversible binding involving specific membrane receptors. Scatchard plots of C1q binding indicated [1.6 +/- 0.7 (1 S.D.)] X 10(6) sites per cell with an equilibrium constant of (2.9 +/- 1.8) X 10(7) M-1 at I0.07. The location of the molecule region mediating C1q binding was established with collagen-like fragments prepared by partial pepsin digestion, confirming earlier results obtained by inhibition studies.     

Cyclic-AMP binding to the cell surface during development of Dictyostelium discoideum

Abstract. Cell aggregation in Dictyostelium discoideum is a chemotactic process mediated by cyclic adenosine monophosphate (CAMP), which is detected by cell surface receptors. The cAMP signal is degraded by cAMP phosphodiesterase. The possibility that cAMP signals are also used for cell communication in the multicellular stages was studied by determining whether the cAMP receptors, which are essential for signal transduction, continue to function in these stages. During slug migration, the number of binding sites per cell decreases to about 15% of the maximum level acquired during aggregation. At the onset of fruiting body formation, a three- to Four-Fold increase in cAMP binding activity occurs. This increase coincides with an increase in cAMP phosphodiesterase. Both phenomena suggest that cell-cell communication mediated by cAMP is used during culmination. During both slug migration and early culmination, the prestalk cells exhibit about twice as much binding activity as the prespore cells.     

Murine cell surface glycoproteins. Purification of the polymorphic Pgp-1 antigen and analysis of its expression on macrophages and other myeloid cells

We previously reported the initial characterization of a polymorphic major cell surface glycoprotein of about 80,000 daltons from mouse embryo 3T3 cells. This glycoprotein has now been purified 1800-fold to apparent homogeneity by monoclonal antibody affinity chromatography. The purified molecule retained the total antigenic activity of the cell, as determined by antibody binding assays. The quantity of the glycoprotein, 0.06% of the total protein of the crude cell extract, confirmed its presence as a major constituent of the cell plasma membrane. The monoclonal antibody was also used to detect related antigens in cells and tissues of C57BL/6J mice. The antigen was present in high concentration in macrophages and subpopulations of bone marrow and blood polymorphonuclear cells. Much lower concentrations of antigen were detected in spleen cells, thymocytes, and extracts of solid tissues. The apparent Mr of the target antigen of myeloid cells was 92,000. This molecule was a major surface constituent of myeloid cells with 10(6) antibody binding sites per cell containing 10% of total 125I incorporated by the lactoperoxidase procedure. The macrophage glycoprotein labeled on the cell surface with 125I was highly sensitive to trypsin, yielding an antigenically active soluble glycopolypeptide of about 65,000 daltons, that contained all of the incorporated 125I. A similar 65,000-dalton glycopeptide was released from 3T3 cells by trypsin cleavage. These data indicate that a major cell surface constituent of mouse myeloid cells is a 92,000-dalton glycoprotein closely related to the 80,000-dalton glycoprotein of mouse embryo 3T3 cells.     

Organization of teichoic acid in the cell wall of Bacillus subtilis.

The phytohemagglutinin, concanavalin A (Con A), interacts specifically and reversibly with the polyglucosyl glycerol phosphate teichoic acid of Bacillus subtilis 168 cell walls. Advantage has been taken of this interaction to examine the organization of the surface teichoic acid at the ultrastructural level. Con A-treated whole cells and cell walls contain an irregular, fluffy layer 25 to 60 nm thick which is absent in untreated or alpha-methyl glucoside-treated preparations. This discontinuous layer is present only on the outer profile of Con-A-treated cell walls. The surface teichoic acid is proposed to be oriented perpendicular to the long axis of the cell. Fixation and embedment for electron microscopy result in condensation of this layer which then contributes to the stainable portion of the wall. Con A treatment binds adjacent teichoic acid molecules in their native configuration producing the irregular, fluffy layer visualized.     

Concanavalin A Acts as a Factor in Establishing the Dorso-Ventral Gradient in the Ventral Mesoderm of Newt Gastrula Embryos1

Con A induced dorsal differentiation in the ventral mesoderm of Cynops gastrula embryo. This process apparently requires a certain amount of Con A to be internalized as supported by the following evidence: 1) Oligomannose-type oligosaccharide, a potent inhibitor of Con A, considerably inhibited dorsalization of ventral mesoderm by Con A. The incorporation of ¹²⁵I-Con A into the ventral mesoderm was greatly inhibited by this sugar. 2) Sepharose-immobilized Con A did not dorsalize the ventral mesoderm. Con A-induced dorsalization was found to be concentration-dependent. Microautoradiograms of ¹²⁵I-Con A-treated ventral mesoderm suggest that the target site (some receptor molecules) of Con A exists inside the cell. Con A is the first pure substance reported to mimic the two properties of the organizer—neural induction of the competent ectoderm and dorsalization of the ventral mesoderm. In neural induction, Con A acts on the cell surface, while Con A apparently needs to be internalized to trigger dorsal differentiation. Interestingly, Con A-dorsalized ventral mesoderm acquired the neural inducing function of the organizer within the early phase of dorsalization.     

von Willebrand factor. A protein which binds at the cell surface interface between platelets

von Willebrand factor (VWF) functions in platelet aggregation, a form of cellular interaction. In vitro analysis of platelet aggregation, as measured by the platelet aggregometer, requires addition of a promoter such as the glycopeptide antibiotic ristocetin. Native multimeric VWF (Mr = 1-20 X 10(6)) can be reduced with sulfhydryl reagents to a monomeric state (Mr = 2 X 10(5)). In this study, the binding of bovine VWF and ristocetin to bovine platelets was investigated using fluorescence anisotropy of derivatized monomer protein and ristocetin and also by radioisotope methods using 125I-labeled monomer and native protein. Ristocetin bound to bovine platelets but not to VWF. VWF binding to formaldehyde-fixed platelets was dependent on the presence of a promoter such as ristocetin. The monomer and multimer VWF bound equally well in the presence of low ristocetin concentrations. Under these conditions, plots of VWF binding versus platelet concentration were sigmoidal, indicating positive cooperativity with respect to platelets. At higher (100 micrograms/ml) ristocetin concentrations, the binding curve was no longer sigmoidal. Ristocetin promoted the formation of small platelet aggregates, an effect that was amplified by the presence of VWF. In fact, all conditions which resulted in monomer or multimer VWF binding to platelets also caused formation of platelet aggregates observed by light microscopy. These combined results were consistent with VWF binding only to the interface between proximal platelets. High affinity binding could be provided by the presence of two cell surfaces and the resulting multiple binding interactions. Polycations, such as poly(L-lysine) and Polybrene, also promoted the formation of platelet aggregates and facilitated the binding of VWF to platelets. Physiological platelet activators such as thrombin, ADP, and collagen also facilitated VWF binding to native platelets and caused platelet aggregation. It appears possible that any process which causes the surface membranes of platelets to become spatially close will allow expression of VWF activity.     

Retention of intact concanavalin A and Lens culinaris lectin in transformed lymphoblasts during mitogenic response.

The fate of cell bound mitogens during mitogenic response of mouse cortisone resistant thymocytes (CRT) was studied using ¹²⁵I-concanavalin A (Con A) and ¹²⁵I-Lens culinaris lectin (LcH). A majority of autoradiographic grains derived from ¹²⁵I-lectins bound to a CRT population were distributed in a single broad peak. Pulse labeling of CRT with ¹²⁵I-Con A or ¹²⁵I-LcH in the initial 60 min of incubation followed by 48 hr of culture with unlabeled mitogens revealed that transformed lymphoblasts carried over half of cell-bound ¹²⁵I-mitogens. ¹²⁵I-Con A and ¹²⁵I-LcH found in the lymphoblasts in the above pulse experiments were electrophoretically identical to the native mitogens. A significant loss of cell-bound ¹²⁵I-mitogens was observed only after extensive cell division.     

Evidence for the glycoprotein nature of radish beta-fructosidase.

Concanavalin A (Con A) was utilized free, bound to Sepharose 4 B or cross-linked to glutaraldehyde to investigate the possibility of binding this lectin to radish beta-fructosidase (E.C.3.2.1.26). The choice of cross-linked Con A as affinoadsorbent is discussed and standard conditions for binding are defined. Specificity of precipitation of this enzyme by the lectin was especially investigated. Thus, the possibility of binding was tested in the presence of high ionic strength, ethylene glycol, alpha-methyl mannoside, alpha-methyl glucoside and during periodate oxidation of the enzyme. Based on the interactions observed between beta-fructosidase and Con A under these conditions it is concluded that the saccharide binding site of the lectin is primarily involved with a secondary contribution from the hydrophobic site. The specificity of binding and the complete precipitation of beta-fructosidase activity by the insolubilized lectin imply that all beta-fructosidase activity measured in Raphanus sativus seedling extracts is linked to (a) glycoprotein form(s) of this enzyme.     

Ferrocyanide enhancement of concanavalin A-ferritin and cationized ferritin staining blood cell surface glycoconjugates

Synopsis Ferrocyanide was used to enhance cationized ferritin and concanavalin A-ferritin (Con A-ferritin) staining of surface glycoconjugates of peripheral blood and bone marrow cells from rabbits and humans. The glutaraldehyde-fixed cells were stained with Con A-ferritin or cationized ferritin and then exposed to a ferrocyanide solution. The resulting cuboidal and irregular stain deposits averaged 50 nm in diameter when viewed with the transmission (TEM) and scanning electron microscope (SEM). Rabbit blood cells demonstrated more Con A binding sites than human blood cells and the decrease in binding sites observed with maturation of human granulocytic and erythrocytic cells was not evident in rabbit cells. Differences in binding of cationized ferritin to rabbit and human cell surfaces were less prominent than that observed for Con A. These results extend previous studies of blood cell surface glycoconjugates and demonstrate that ferrocyanide enhancement significantly facilitates SEM evaluation of Con A-ferritin and cationized ferritin bound to cell surfaces.     

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 1. Molecular characterization of the binding site

Using mono[125I]iodinated vasoactive intestinal peptide (125I-VIP), a very high number of specific binding sites for VIP were identified at the surface of the human melanoma cell line IGR39. The Scatchard analysis of competitive displacement experiments between native VIP and 125I-VIP was consistent with the existence of two classes of VIP-binding sites. IGR39 cells possess 0.54 x 10(6) high-affinity sites with a dissociation constant (Kd) of 0.66 nM and 1.3 x 10(6) sites of moderate affinity with a Kd of 4.7 nM. Pharmacological studies indicated that the order of potency in inhibiting 125I-VIP binding of the VIP/secretin family peptides was VIP much greater than peptide histidine methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin. Glucagon has no effect on the binding of the labelled peptide. By means of photoaffinity labelling a polypeptide of Mr 63,000 was characterized. The labelling of this species was completely abolished by native VIP. The order of potency of VIP-related peptides in inhibiting 125I-VIP cross-linking to its receptor was the same as in the competition experiments. The glycoprotein nature of the VIP-binding sites of IGR39 cells has been investigated by affinity chromatography on wheat-germ-agglutinin-Sepharose.     

Identification of a cell surface glycoprotein involved in cell aggregation in D. discoideum.

Analysis of the composition of cell surface-associated glycoproteins of D. discoideum by lactoper-oxidase-catalyzed radioiodination, followed by isolation by Con A-Sepharose chromatography, revealed that the developmentally regulated cell surface expression of a certain glycoprotein (gp150) parallels the onset of mutual cellular cohesiveness (Geltosky, Siu and Lerner, 1976). We have purified gp150 and raised specific antibodies to it. Through utilization of the specific antibody and a fluorescence-activated cell sorter, the expression of gp150 on the cell surface has been studied. Starting from a low level in noncohesive (vegetative) cells, there is a rapid accumulation of gp150 on the surfaces of aggregating cells. A peak level of expression is achieved by 10 hr and maintained at least until the steps of terminal differentiation. Most significantly, monovalent Fa'b derived from anti-gp150, when added to aggregation-competent cells, blocks the cells' ability to reaggregate. Fab's derived from antisera with different specificities were ineffective inhibitors of cell aggregation. These results suggest that gp150 serves an intimate role in cell adhesion.