Studies on phytohemagglutinins XXIII. A receptor which does not conform to the so called sugar specificity of the pea phytohemagglutinin

A receptor glycopeptide for hemagglutinin of the pea (Pisum sativum L.) was isolated from rabbit erythrocytes by the method originally applied for isolation of a receptor glycopeptide from human erythrocytes (Kubánek, J., Entlicher, G. and Kocourek, J. (1973) Biochim. Biophys. Acta 304, 93–102). The receptor isolated from rabbit erythrocytes contains galactose, mannose, N-acetylglucosamine, asparic acid, threonine, serine glutamic acid and glycine in approximate molar ratios of 2:1:3:3:1:1:1:1. It has a minimum molecular weight of about 2000. According to the sequence analysis the following structure has been proposed for the receptor:

Full-size image (1K)

About 90% of the receptor-site activity for the pea hemagglutinin is due to the presence of two galactose residues on non-reducing terminals of the saccharide chain in spite of the fact that the pea haemagglutinin is not inhibited by this sugar.     

Characterization of functional domains of the lymphocyte plasma membrane

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (fraction 1) eluted freely from the affinity column, the second (fraction 2) adhered specifically to concanavalin A-Sepharose. Previous analysis showed that both subfractions were right-side-out (Resch, K., Schneider, S. and Szamel, M. (1981) Anal. Biochem. 117, 282-292). The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. After enzymatic radioiodination of thymocytes, the relative distribution of labelled proteins and externally exposed phospholipids was very similar in isolated plasma membranes and in both membrane subfractions, indicating the plasma membrane nature of the subfractions separated by affinity chromatography on concanavalin A-Sepharose. This finding was further substantiated by the nearly identical specific activities of some membrane-bound enzymes, Mg2+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase. The specific activities of (Na+ + K+)-ATPase and of lysolecithin acyltransferase were several-fold enriched in fraction 2 compared to fraction 1, especially after rechromatography of fraction 1 on concanavalin A-Sepharose. Unseparated membrane vesicles contained two types of binding site for concanavalin A. In contrast, isolated subfractions showed a linear Scatchard plot; fraction 2 exhibited fewer binding sites for concanavalin A: the association constant was, however, 3.5-times higher than that measured in fraction 1. When plasma membranes isolated from concanavalin A-stimulated lymphocytes were separated by affinity chromatography, the yield of the two subfractions was similar to that of membranes from unstimulated lymphocytes. Upon stimulation with concanavalin A, Mg2+-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase were suppressed in their activities in both membrane subfractions. In contrast, the specific activities of (Na+ + K+)-ATPase and lysolecithin acyltransferase were enhanced preferentially in the adherent fraction (fraction 2). The data suggest the existence of domains in the plasma membrane of lymphocytes which are formed by a spatial and functional coupling of receptors with high affinity for concanavalin A, and certain membrane-bound enzymes, implicated in the initiation of lymphocyte activation.     

Isolation and identification of the major concanavalin A binding glycoproteins from murine-lymphocytes.

The major glycoproteins which bind concanavalin A have been isolated and identified from murine spleen cells, thymocytes,and purified thymus-derived (T) lymphocytes, and from the spleen cells of congenitally athymic (nude) mice. The cells were radiolabeled by lactoperoxidase catalyzed 125I iodination or by culturing the cells in media containing [3H]leucine or [3H]fucose. The cell membrane was solubilized with Nonidet P-40 and the concanavalin A binding proteins were isolated by affinity chromatography and analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The major proteins from various lymphocyte preparations were identified by immunoprecipitation with specific antisera. The molecules coded by the histocompatibility-2 complex acted as concanavalin A binding proteins H-2K and H-2D were isolated from T lymphocytes, thymocytes, and bone marrow derived (B) lymphocytes. The Ia antigens were identified from B lymphocytes and tentatively identified from T lymphocytes. In addition to these H-2 complex proteins, immunoglobulin M and D on B lymphocytes also bound concanavalin A binding. All these glycoproteins have previously been identified as cell surface molecules. The presence of certain minor unidentified concanavalin A binding proteins on lymphoid cells is indicated.     

Are cyclic nucleotides involved in the initiation of mitogenic activation of human lymphocytes?

In order to obtain more insight into the possible role of cyclic AMP or cyclic GMP in modulating the initial cellular processes following activation of lymphocytes, we measured the effects of the T-cell mitogen concanavalin A and other substances including hormones on the cyclic nucleotide levels in human peripheral blood lymphocytes. The enzyme activities of the corresponding nucleotide cyclases, adenylate cyclase and guanylate cyclase were measured in both isolated plasma membranes or the cytosol of resting or concanavalin A stimulated rabbit thymocytes. Concanavalin A in a mitogenic concentration of about 5-10 micrograms/ml caused small, but consistent increases in cAMP but no changes in cGMP levels during the first hour of activation. Concomitantly, the specific activity of plasma membrane-bound adenylate cyclase was always increased at least 1.5-fold 30 min after stimulation of rabbit thymocytes with concanavalin A, but no effect could be detected on the specific activities of plasma membrane-bound or soluble guanylate cyclase. At high, supraoptimal concentrations of concanavalin A (more than 20 micrograms/ml) cAMP levels dramatically increased in human lymphocytes within minutes, but cGMP levels again were unaffected. Forskolin and beta-adrenergic hormones elevated cAMP in human lymphocytes, whereas cGMP levels were increased by the addition of sodium nitroprusside or alpha-adrenergic hormones. Sodium nitroprusside, in concentrations which elevated cGMP in human lymphocytes, had no influence on the incorporation of [3H]uridine into RNA of resting or concanavalin A stimulated human lymphocytes. Addition of forskolin resulted in an increase of cAMP levels and a dose-dependent decrease of [3H]uridine incorporation into RNA of concanavalin A-stimulated lymphocytes with no effect on resting lymphocytes. The data suggest that cGMP does not play a role in the initial phase of mitogenic activation of lymphocytes, whereas cAMP may be involved in the blast transformation process as an inhibitory signal.     

An application of ellipsometry: Assessment of polysaccharide and glycoprotein interaction with lectin at a liquid/solid interface

Lectins were specifically adsorbed from solution onto metallized glass slides coated with polysacchride, glycopeptide and glycoprotein films. The degree of interaction was determined by measuring the thickness of the bound lectin layer with an ellipsometer after washing and drying the slide. The binding of concanavalin A (tetrameric) and succinyl concanavalin A (dimeric) to a yeast mannan film was studied as a function of lectin concentration, temperature, rinsing time and the extent of stirring of the slide. The maximum thickness of bound concanavalin A and succinyl concanavalin A was 11 and 3.8 nm, respectively. The method permitted the measurement of the association constants for both lectins (1.0 · 10⁷ M^?1 for concanavalin A, 2 · 10⁶ M^?1 for succinyl concanavalin A) and the detection of 0.6 pmol concanavalin A. The same sensitivity was observed with anti-mannan antibodies. The binding of both lectins was shown to be specific using sugar haptens. When compared with methyl α-D-mannoside, the affinity of concanavalin A for D-mannose and D-glucose was 14 and 3%, respectively. A film of mucin glycopeptide (universal adsorbent) interacted similarly with concanavalin A, Ricinus communis I, soya bean and wheat germ lectins. However, films of glycoproteins such as fetuin, ceruloplasmin and Aspergillus niger β-D-galactosidase interacted to different degrees with these lectins. The relative affinity of wheat germ agglutinin for $\text{N-}\text{acetyl}\text{-}\text{D}\text{-}\text{glucosamine}$ and for chitin-derived oligosaccharides was also determined. When films of sialoglyproteins were treated with neuraminidase, the thickness of the bound peanut agglutinin layer increased. Although this method cannot determine quantitatively the sugar composition of the film, it permits rapid estimation of the interaction of lectins with polysaccharides and glycoproteins, usingg little material.     

Fractionation of membrane vesicles : I. A separation method for different populations of membrane vesicles of thymocytes by affinity chromatography on Con A-Sepharose

Affinity chromatography, a separation technique which makes use of biospecific properties, is well established for the separation of molecules in solution. We applied this method to the subfractionation of biomembranes. Using a microsomal fraction mainly consisting of plasma membrane from rabbit or calf thymocytes, 20–40% of the protein adhered specifically to concanavalin A-Sepharose, whereas the majority of the membrane vesicles were recovered from the effluent. The adherence involved the binding of Con A to membranes, as addition of the hapten sugar α-methyl-mannoside completely prevented separation. The fractions which bound to Con A-Sepharose could be eluted by combining mechanical forces with the addition of α-methyl-mannoside. All fractions exhibited the same vesicular appearance and were identical with respect to the phospholipid cholesterol ratio. The method proved to be highly reproducible and it offers a possible way for the subfractionation of membranes according to their biospecific structure.     

A novel early effect of concanavalin A on thymocytes.

Concanavalin A, added to freshly isolated rabbit thymocytes, markedly enhanced the extracellular appearance of non-immunoglobulin proteins. Time course studies revealed that the onset of enhancement occurred virtually without delay. The effect appeared to be restricted only to certain of the thymus-derived cells because thymocytes obtained from rabbits treated with hydrocortisone, as well as splenocytes derived from untreated rabbits essentially did not exhibit the enhancement. Stimulation by concanavalin A was specific in that pokeweed mitogen and lipopolysaccharide were without effect and also in that α-methyl-mannoside, but not galactose, abrogated the concanavalin A-mediated enhancement. Experiments with mouse thymocytes demonstrated that the cells which responded to concanavalin A were primarily cells that bear the θ-antigen on their surface (T-cells).     

Expression and phosphorylation of transferrin receptors in mitogen-activated peripheral blood lymphocytes

Expression of transferrin receptors of cultured human lymphocytes has been investigated by using monoclonal antibody (5E9) specific for human transferrin receptors. When isolated lymphocytes were cultured in a medium containing fetal calf serum, the biosynthesis of transferrin receptor was barely detectable. The addition of concanavalin A or human serum to the medium caused a slight stimulation of the biosynthesis. The addition of concanavalin A and human serum in combination caused the highest biosynthetic activity. Appearance of the receptor on the cell surface increased in parallel with the degree of the synthesis. Treatment of concanavalin A- and human serum-treated cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a marked stimulation of the phosphorylation of the receptor. Enhancement of phosphorylation occurred within 20 min after the addition of TPA. The density of the receptor on the cell surface slightly increased upon TPA treatment of cells, and the treatment was without effect on iron incorporation from transferrin into the cells. The density of newly synthesized receptor in TPA-treated cells was similar to that in non-treated cells. These results indicated that TPA treatment of mitogen-activated human lymphocytes stimulated the phosphorylation of transferrin receptors, but TPA had no effect on the expression of the receptors thereafter.     

Fractionation of membrane vesicles: I. A separation method for different populations of membrane vesicles of thymocytes by affinity chromatography on Con A-Sepharose

Affinity chromatography, a separation technique which makes use of biospecific properties, is well established for the separation of molecules in solution. We applied this method to the subfractionation of biomembranes. Using a microsomal fraction mainly consisting of plasma membrane from rabbit or calf thymocytes, 20–40% of the protein adhered specifically to concanavalin A-Sepharose, whereas the majority of the membrane vesicles were recovered from the effluent. The adherence involved the binding of Con A to membranes, as addition of the hapten sugar α-methyl-mannoside completely prevented separation. The fractions which bound to Con A-Sepharose could be eluted by combining mechanical forces with the addition of α-methyl-mannoside. All fractions exhibited the same vesicular appearance and were identical with respect to the phospholipid cholesterol ratio. The method proved to be highly reproducible and it offers a possible way for the subfractionation of membranes according to their biospecific structure.     

Concanavalin A induced glucocorticoid resistance in rat thymocytes in relation to glucose metabolism and glucocorticoid receptors.

Concanavalin A stimulates glucose uptake in isolated rat thymocytes, at concentrations as low as 1.0 μg/ml. The magnitude of the response is dosedependent with maximal glucose uptake (greater than 100% over control) observed over the 50–500 μg/ml range. The response to concanavalin A occurs within 5 minutes and is maximal by 1 hour. Exposure of thymocytes to physiological concentrations of cortisol has been shown previously to lead to a 15–30% reduction in glucose uptake within 20 minutes (1). Cortisol added simultaneously to or after concanavalin A has no effect on glucose uptake, but when it is added before concanavalin A, a glucocorticoid response is observed even in the face of concanavalin A-induced glucose uptake. Unlike the cortisol effect, the concanavalin A-induced increase in thymocyte glucose uptake is not inhibited by either cycloheximide or cordycepin. The antagonism between cortisol and concanavalin A may in part exist at the specific glucocorticoid receptor level, since exposure of cells to concanavalin A rapidly and significantly reduces the number of specific, saturable glucocorticoid binding sites in isolated thymocytes.     

Concanavalin A receptor(s) possibly interacts with at least two kinds of GTP-binding proteins in murine thymocytes

A part of the GTP gamma S-binding activity in murine thymocyte membranes was found to have affinity to a concanavalin A (Con A)-Sepharose column. The material was identified as Gi (inhibitory GTP-binding protein) on the basis of the molecular weight and by islet activating protein-dependent ADP-ribosylation and anti-alpha i (alpha subunit of Gi) immunoblotting. However, when the membranes prepared from Con A-stimulated thymocytes were used, no GTP gamma S-binding activity was detected in the Con A-bound fraction, suggesting that Gi physically and specifically associated with Con A acceptors dissociates upon Con A stimulation. Furthermore, another GTP gamma S-binding protein (25 kDa), which is quite similar to a novel phosphoinositide-specific phospholipase C (PI-PLC)-associated G protein in calf thymocytes (Wang, P., Toyoshima, S., & Osawa, T. (1988) J. Biochem. 103, 137-142), was detected among the Con A-Sepharose-bound proteins with the chemical cross-linking technique. When the 40 kDa and 25 kDa G proteins associated with Con A receptor(s) were isolated and their direct effects on the activity of partially purified PI-PLC as to phosphatidylinositol 4,5-bisphosphate hydrolysis were examined, the 25 kDa G protein was found to enhance the PI-PLC activity more effectively. On the other hand, pretreatment of cells with islet-activating protein completely abolished the inhibitory effect of Con A on the prostaglandin E1 and isoproterenol-induced increases of cellular cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of IgM-binding factors by murine thymocytes

Normal mouse thymocytes activated with concanavalin A (Con A) released soluble factors which selectively inhibited rosette formation between human peripheral blood lymphocytes (PBL) and ox erythrocytes (E_O) sensitized with rabbit IgM antibodies. The factors were removed by absorption with mouse IgM-coupled Sepharose, and were recovered from the beads by elution at acid pH. They neither bound to mouse IgG-Sepharose nor inhibited rosette formation of PBL with E_o sensitized with rabbit IgG antibodies. Mouse IgM enhanced the formation of IgM-binding factors by Con A-activated thymocytes. Unstimulated normal mouse thymocytes also released IgM-binding factors upon incubation with mouse IgM. The molecular weights of IgM-binding factors were approximately 90–110 and 35–50 kDa as estimated by gel filtration. Each species of IgM-binding factors markedly suppressed the IgM-plaque-forming cell (PFC) response of sheep red blood cell-primed spleen cells and slightly suppressed the IgG PFC response.     

Urtica dioica agglutinin, a new mitogen for murine T lymphocytes: unaltered interleukin-1 production but late interleukin 2-mediated proliferation

Urtica dioica agglutinin, a small-molecular-weight lectin purified from stinging nettle rhizomes, induces murine cell proliferation. U. dioica agglutinin is a specific T-cell mitogen for both thymocytes and spleen T lymphocytes; its mitogenic properties are strictly dependent on the presence of accessory cells. The kinetics of proliferation are markedly different from those of the classical T-cell mitogen concanavalin A, with a 2 to 3-day delay for both splenic and thymic populations and a rate of DNA synthesis twofold lower than that observed with concanavalin A. The late T-lymphocyte proliferation induced by U. dioica agglutinin correlates well with (i) the observed late interleukin-2 production and interleukin-2 receptor expression, and (ii) the long-lasting cyclosporin A-sensitive early activation period. In contrast, the production of interleukin-1 is not different, both in terms of concentration and kinetics, from that observed with concanavalin A.     

Monoclonal antibodies to ThB detect close linkage ofLy-6 and a gene regulating ThB expression

We have generated three hybridomas producing rat monoclonal antibodies to a surface antigen, ThB, that is shared by murine B lymphocytes and approximately 50 percent of murine thymocytes. These antibodies, produced by immunizations with MOPC-104E cells, appear to recognize the same antigen that was previously detected by rabbit and goat antisera to MOPC-104E cells (Yutoku et al. 1974, Yutoku et al. 1976).Using these antibodies, we have studied a genetic polymorphism that is associated with the level of ThB expression on B lymphocytes but not with the antigen's expression on thymocytes. We present evidence that this trait is controlled by one gene,Thb, which we find to be very closely linked to the gene or genes controlling the Ly-6, Ly-8, DAG, and Ala 1 antigen(s). While the latter four antigens were described as markers on mature T (or activated T and B) lymphocytes, ThB is restricted to immature thymocytes and all B cells. ThB is not expressed on kidney, although some investigators (McKenzie et al. 1977 a, Halloran et al. 1978) report Ly-6 expression on that tissue. SJL/J, C57BL/10JHz, DBA/2J, and AKR/J are among the mouse strains carrying theThb ^h allele, while BALB/cN, CBA/J, C3H.SW/SnHz, and A/J carry theThb ^l allele. The ThB antigen has not yet been identified as a glycoprotein after cell-surface iodination, NP-40 solubilization, and immunoprecipitation.This work was supported in part by grants from the National Institutes of Health (AI-08917, CA-04681, GM-17367).     

Concanavalin A stimulation modifies the lipid and protein structure or rabbit thymocyte plasma membranes. A laser raman study.

(1) We have compared the laser Raman spectra of isolated plasma membranes from resting rabbit thymocytes and cells mitogenically stimulated with concanavalin A. (2) Major alterations in the CH stretching, CH deformation and CC stretching regions indicate a different lipid architecture in the membranes from activated cells. (3) Spectral changes in the Amide I and II regions, by reference to the spectra of model compounds indicate greater protein amidation in the membranes from stimulated cells.     

Interleukin 1 and protein kinase C activator are dissimilar in their effects on Il-2 receptor expression and Il-2 secretion by T lymphocytes

T lymphocytes respond to mitogenic stimulation by expressing the receptor for interleukin 2 (Il-2) and secreting Il-2; once the receptor is expressed, Il-2 induces these cells to proliferation. In the present report using mouse T lymphocytes, thymocytes, and the lymphoma cell line EL4, we studied receptor expression and Il-2 secretion as early parameters for T-lymphocyte activation in response to ionomycin, concanavalin A (Con A), 12-O-tetradecanoyl-phorbol 13-acetate (TPA), and interleukin 1 (Il-1). Il-1 is required for mitogenic response of lymphocyte preparations that are rigorously depleted of macrophages. On its own, Il-1 had very little effect on Il-2 secretion and Il-2 receptor expression by T lymphocytes. TPA strongly synergized with ionomycin both for Il-2 secretion and for Il-2 receptor expression whereas Il-1 did not. Il-1 required the simultaneous presence of ionomycin and TPA to have any demonstrable effect on T lymphocytes from spleen and on thymocytes. However, on EL4 cells which were also partially responsive to TPA alone, Il-1 showed strong synergy with TPA to induce Il-2 secretion and Il-2 receptor expression. The effect of Il-1 on EL4 cells was dose dependent where increasingly higher concentrations of Il-1 in the presence of a fixed concentration of TPA caused higher percentage of EL4 cells to become Il-2 receptor positive. The present results suggest that Il-1 does not cause its effect on T lymphocytes via the same mechanism of protein kinase C activation that has been proposed for TPA.     

Phospholipid metabolism of stimulated lymphocytes: Comparison of the activation of acyl-CoA: Lysolecithin acyltransferase with the binding of concanavalin A to thymocytes

Calf thymocytes were isolated and incubated with concanavalin A. The effect of the mitogen on the enzyme activity of membrane-bound lysolecithin acyltransferase (acyl-CoA: $\text{1-acylglycero-3-phosphorylcholine}\text{-O-}\text{acyltransferase}$, EC 2.3.1.23) was determined as also the binding of ¹²⁵I-labelled concanavalin A to intact cells and isolated membranes.The lysolecithin acyltransferase was found to be activated three times in microsomal membranes. The activation occurred directly after binding of concanavalin A and was temperature independent, since similar activities were found in cells treated with concanavalin A at 0 and 37 °C.The acyltransferase activation using increasing concentrations of concanavalin A revealed a different behaviour, as compared to the binding of concanavalin A. While the binding of concanavalin A to intact cells expressed a normal hyperbolic saturation function the activation process of the acyltransferase described a sigmoidal relationship. Corespondingly, the interaction coefficients for both functions were different (Sips coefficient for binding = 1.0 and Hill coefficient of the enzyme activation = 1.8).These results indicate that the acyltransferase activation is due to a cooperative interaction between the ligand-receptor complex and the enzyme.     

Early plasma-membrane-potential changes during stimulation of lymphocytes by concanavalin A

1. We have monitored the plasma-membrane potential of lymphocytes by measuring the accumulation of the lipophilic cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). 2. The mitogen concanavalin A causes a decrease in TPMP+ accumulation by pig lymphocytes corresponding to a 3 mV depolarization with 2 1/2 min. Concanavalin A does not alter 86Rb+ uptake in the first 30 min. 3. In contrast concanavalin A increased TPMP+ accumulation and the rate of Rb+ uptake in mouse thymocytes. This is consistent with a previous proposal that the mitogen induces a hyperpolarization of mouse thymocytes as a result of stimulation of a Ca2+-dependent K+ channel. 4. Studies with the calcium ionophore A23187 and quinine (an inhibitor of the Ca2+-dependent K+ channel) suggest that the channel is partially closed in mouse resting thymocytes but is almost fully active in pig resting cells. Thus concanavalin A hyperpolarizes mouse thymocytes by activating the Ca2+-dependent K+ channel but cannot do so in pig lymphocytes because the channel is already maximally activated. 5. The 3mV depolarization of pig cells cannot be explained by a decrease in electrogenic K+ permeability.     

Shedding of gangliosides from calf thymocytes

In the course of our work on calf thymus gangliosides [Dyatlovitskaya, Zablotskaya, Azizov & Bergelson (1980) Eur. J. Biochem. 110, 475-483] we studied the gangliosides exfoliated from the cell surface of thymocytes. It was shown that calf thymocytes shed gangliosides both in vivo and in vitro. Various gangliosides were found to be present in high amounts both in extracellular plasma membrane vesicles and in the 64000 X g supernatant. The extracellular membrane fragments were comparatively higher in disialosyllactosylceramide and the 64000 X g supernatant was higher in sialosyllactosylceramide than the cells. Comparison of the ganglioside composition of extracellular membrane fragments, thymocytes and lymphocytes led us to suggest that the shedding of gangliosides from the surface of thymocytes may be involved in the transformation of immunologically incompetent cortical thymocytes into immunocompetent virgin T-cells.