Quantification of lectin receptors on B, T, T-gamma, and T-mu lymphocytes. II. PHA, SBA, Dolichos biflorus, and Tetragonolobus purpureus

Plasma membranes composition and structure of B, T, T gamma and T mu lymphocytes from peripheral blood have been studied using four 125I-labelled lectins. The results showed that B lymphocytes have a greater number of receptors for SBA and Dolichos biflorus lectins than T lymphocytes, whilst the opposite was observed for PHA-lectin. However, no significant differences between the two lymphocyte populations were found regarding the receptors for Tetragonolobus purpureus lectin. Among T lymphocyte subpopulations, SBA, Dolichos biflorus and tetragonolobus purpureus lectins appeared to have higher specificity for T gamma lymphocytes and PHA for T mu. PHA-lectin had greater affinity for B lymphocytes, while the rest showed higher affinity for T lymphocytes. The T gamma subpopulation had lower association constant for PHA-lectin than T mu lymphocytes, whilst the contrary was found to be true for the other three lectins.     

Quantification of lectin receptors on B, T, T-gamma and T-mu lymphocytes. I. Concanavalin A, Pisum sativum, Lens culinaris and wheat-germ agglutinin

The immune system is formed of different lymphocyte subpopulations, each one having a defined role to defend the organism. Their plasma membranes present differences in the glycoproteinic or/and glycolipidic composition, as detected with labelled 125I-lectins. B lymphocytes have a greater number of receptors for the Pisum sativum, Lens culinaris and WGA lectins than T lymphocytes. On the other hand, T lymphocytes bind a greater number of Concanavalin A molecules than B lymphocytes. WGA lectin appeared to be more specific for T mu subpopulation, while Con A and Pisum sativum lectins were bound preferentially to T gamma lymphocytes while no significant differences were observed between both subpopulations for Lens culinaris lectin. From the affinity of each lectin to each lymphocyte population it could be deduced that the receptor structure, conformation and arrangement on the membrane was optimal in B lymphocytes for Con A and WGA binding, and T lymphocytes for Lens culinaris and Pisum sativum binding.     

Comparative analysis of membrane glycoproteins of normal and chronic lymphatic leukemia B lymphocytes by lectins

Eight plant lectins were used to investigate membrane alterations in lymphocytes from patients with chronic lymphocytic leukemia (CLL). By rosetting with lectins attached to latex particles, the cell percentages with the abundance of each lectin receptor were compared in B normal and leukemic lymphocytes. Comparing these data with the number of lectin molecules bound to each cell and the affinity, which are values calculated with 125I-labeled lectins, it was possible to deduce differences in the composition of glycoproteins in B normal and B-CLL lymphocytes membrane. Compared to B normal, B-CLL lymphocytes had fewer receptors for WGA and more for Lens culinaris, SBA and Tetragonolobus purpureus lectins. Receptors for Concanavalin A, Pisum sativum, PHA and Tetragonolobus purpureus showed a higher affinity with B normal lymphocytes, while the other lectins assayed showed more affinity with B-CLL lymphocytes. So, it is possible to establish a comparative analysis about the plasma membrane glycoproteins in the B normal and CLL lymphocytes by lectin binding studies.     

Fractionation of lymphocytes using affinity chromatography with 9 lectins

Lymphocyte subclasses from normal peripheral blood have been fractionated by affinity chromatography with lectins. Concanavalin A (Con A), Lens culinaris lectin (LC), Pisum sativum lectin (PS), Phaseolus vulgaris lectin (PHA), Dolichos biflours lectin (DB), Glicine max lectin (SBA), Ricinus communis lectin (RCA II), Tetragonolobus purpureus lectin (TP) and Triticum vulgaris lectin (WGA), were coupled to Sepharose 6MB, and lymphocytes labelled with 125I were eluted through the chromatographic columns. The binding of lymphocytes to WGA and SBA lectins was 32% and 13% respectively. The binding to the other lectins tested were found to be between 32% and 13%. When solutions of increasing concentrations of specific sugar were added to the columns a progressive elution of bound lymphocytes was observed. These results indicate the existence of a large range of lymphocyte subclasses, with different binding capacity to lectins, which was a function of the receptor number or/and receptor affinity to each lectin. Furthermore, these two parameters were found to vary in each functional population. Even though all the lymphocytes had lectin receptors, T lymphocytes showed higher affinity for Con A, PHA and TP lectins, while B lymphocytes appeared to be more specific for LC, PS, SBA, DB, RCAII and WGA lectins.     

Detection of anomalies in cell-surface carbohydrates on thrombasthenic platelets using 125I-labeled lectins

The composition of carbohydrates on the surface of platelets from a patient with Glanzmann's thrombasthenia and from seven normal donors were determined and compared. To this end, binding studies were performed using nine different purified 125I-labeled lectins; Concanavalin A, P-Phytohaemagglutinin, Wheat Germ Agglutinin, Dolichos biflorus, Pisum sativum, Ricinus communis II Agglutinin, Tetragonolobus purpureus, Lens culinaris and Soybean Agglutinin. These studies show that thrombasthenic platelets bear significantly decreased numbers of receptors for Concanavalin A and Lens culinaris, both with a specificity for D-mannose, and Ricinus communis II, with specificity for D-galactose. There were no detectable differences in the numbers of other lectin receptors. These results provide further evidence of molecular defects in thrombasthenic platelets. Moreover, the use of 125I-labeled lectins, as shown here, provides a fast and reliable technique for identifying abnormalities in the carbohydrate composition on the surface of platelets in various thrombopathies.     

The carbohydrate components of corn prolamins]

This work was aimed to study the patterns of zein glycosylation. Zein proteins included 1-3% of sugars. The affinity of different lectins, such as concanavalin A (Con A), Lens culinaris lectin (LCL) and lectins of Arachis hypogaea (PNA), of Triticum vulgaris (WGA), of Dolichos biflorus (DBA), of Glycin max (SBA), of Lotus tetragonolobus (LTA), of Laburnum anagiroides (LAL), of Ricinus communis (RCA), of Phaseolus vulgaris (PHA) was used to analyze the glycosylation sites. All selected lectins interacted with zein proteins. It may serve a basis for determination of mannose, galactose, fucose and aminosugars. Some lectins were bound only by prolamines of some inbred lines, while others were connected with all lines.     

The binding of lectins to components of plasma membranes from porcine submaxillary lymph node lymphocytes

By sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis the plasma membranes from porcine lymphocytes contain at least 30--35 glycopolypeptides and one or more glycolipids to which one or more of 12 purified lectins bind. The specificities of binding generally followed the same pattern as those of the reaction of the lectin with intact pig lymphocytes. Some lectins (e.g., the isolectin pair, Agaricus bisporus lectins A and B and a group consisting of the Lens culinaris A and B isolectins and the closely related Pisum sativum lectins) bind to almost identical populations of plasma membrane components and compete with each other for all their binding sites. Others (e.g., Concanavalin A and the Lens culinaris-Pisum sativum group and a group consisting of phytohemagglutinin-L, Ricinus communis lectin-60 and Ricinus communis lectin-120 bind in a cross reactive manner to some common binding moieties but, in addition, to certain nonshared ones. Still others (e.g., soybean agglutinin, peanut agglutinin and wheat germ agglutinin) do not share any common binding moieties with the other lectins. The amount of lectin binding and the number of membrane components to which a lectin binds is directly related to the Ka of binding of the lectin to the intact lymphocyte. Those with high Ka (Cocanavalin A Lens culinaris lectins, Pisum sativum lectins, phytohemagglutinin-L), bind to 20-30 different components giving very complex binding patterns while those with lower Ka (Agaricus bisporus lectins, wheat germ agglutinin, peanut agglutinin, and soybean agglutinin) bind to 8--13 components with easily distinguishable patterns. Soybean agglutinin binds almost exclusively to a glycolipid fraction while for the others one or more glycopolypeptides served as the major lectin-binding molecule. The Ricinus lectins, two lymphocyte toxins, bind to essentially every plasma membrane component to which the mitogen phytohemagglutinin-L binds, in fact competing for most of those plasma membrane moieties which bind phytohemagglutinin-L.     

ESR and fluorescence studies on the adenine binding site of lectins using a spin-labeled analogue

The techniques of electron spin resonance (ESR) and fluorescence spectroscopy have been used to study the interaction of a spin-labeled analogue of adenine, N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine (I), with several plant lectins. While most adenine derivatives enhanced lectin-induced fluorescence of 1,8-anilinonaphthalenesulfonic acid by binding to a separate, adenine-specific site [Roberts, D.D., & Goldstein, I.J. (1982) J. Biol. Chem. 257, 11274-11277], the spin label I caused a decrease in this fluorescence with certain lectins. ESR showed the ligand to interact strongly with lectins from lima bean (Phaseolus lunatus), Dolichos biflorus, and Phaseolus vulgaris (PHA); however, no binding was observed with Griffonia simplicifolia isolectins A4 and B4, soybean agglutinin, or Amphicarpaea bracteata lectins. The spin label was highly immobilized by each of these proteins (2T magnitude of = 68 G). Apparent affinities of the spin label for the lectins decreased in the order lima bean lectin greater than PHA erythroagglutinin greater than PHA leukoagglutinin greater than D. biflorus. Spin-labeled adenine appeared to bind specifically to the adenine binding site of D. biflorus and PHA leukoagglutinin, as demonstrated by total abolition of the ESR spectrum of bound spin label by adenine. PHA erythroagglutinin and lima bean lectin bound the analogue with apparent dissociation constants of 5 X 10(-5) and 3.2 X 10(-5) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interferon induction in mouse spleen cells by mitogenic and nonmitogenic lectins

Twenty-two species of lectin were tested for their ability to induce interferon (IFN) in mouse spleen cells. Twenty-two species of lectins representing four groups, based on competition patterns with monosaccharides, were examined for their ability to induce IFN in cultured mouse spleen cells. The lectins, all belonging to the third group (concanavalin A, succinylated concanavalin A, Lens culinaris lectins type A and B, and poke weed mitogen) induced IFN mainly composed of IFN gamma. They were either T cell or T/B cell mitogens. Five nonmitogenic lectins, Lotus tetragonolobus seed lectin, crude and type II lectins of Ulex europeus, Bandeiraea simplicifolia type II and Salanum tuberosame lectins, and wheat germ agglutinin belonging to either the first or the third group, induced IFN beta. The production of IFN during stimulation IFN beta- and IFN gamma-inducing lectins followed different kinetic curves. WGA induced IFN in circulation when injected i.p. in mice, and a peak titer was found 2 hr after inoculation.     

Effects of lectins with different carbohydrate-binding specificities on hepatoma, choriocarcinoma, melanoma and osteosarcoma cell lines

The effects of lectins with different carbohydrate-binding specificities on human hepatoma (H3B), human choriocarcinoma (JAr), mouse melanoma (B16) and rat osteosarcoma (ROS) cell lines were investigated. Cell viability was estimated by uptake of crystal violet. Wheat germ lectin was the lectin with the most deleterious effect on the viability of H3B, JAr and ROS cell lines. The cytotoxicity of lectins with similar sugar-binding specificity to wheat germ lectin, including Maackia amurensis lectin and Solanum tuberosum lectin, was weaker than that of wheat germ lectin. N-acetylgalactosamine-and galactose-binding Tricholoma mongolicum lectin ranked third, after wheat germ lectin and Maackia amurensis lectin, with regard to its effect on H3B, and ranked, together with Maackia amurensis lectin, as the lectins with the second most pronounced effects on ROS. However, the cytotoxic effects of Tricholoma mongolicum lectin on JAr were much weaker than those of Maackia amurensis lectin, Solanum tuberosum lectin and Anguilla anguilla lectin. Artocarpus integrifolia lectin, Lens culinaris lectin and Anguilla anguilla lectin possessed milder cytotoxicity than the remaining lectins. which were approximately equipotent. The mannose-binding Narcissus pseudonarcissus and Lens culinaris lectins were only weakly cytotoxic, the exception being a stronger effect on H3B. The N-acetylgalactosamine-binding Glycine max lectin and methylgalactose-binding Artocarpus integrifolia lectin similarly exhibited low cytotoxicity. It can thus be concluded that in general the ranking was wheat germ lectin > Maackia amurensis lectin approximately Trichloma mongolicum lectins > other aforementioned lectins in cytotoxicity. A particular lectin may manifest more conspicuous toxicity on certain cell lines and less on others.     

Interaction with lectin of kappa opioid binding sites solubilized from human placenta

Kappa opioid binding sites from human placenta, prelabeled with 3H-etorphine and solubilized, were retained on wheat germ agglutinin (WGA) agarose and specifically eluted with N-acetylglucosamine. No significant retention was found with other immobilized lectins, including Concanavalin A (Con A), soybean seed lectin (SBA), Pisum sativum lectin (PsA), Lens culinaris Medik. lectin (LcA), and Lathyrus tingitanus lectin(LtA). About 23% of applied kappa sites were specifically eluted from WGA agarose, less than half of the proportion of rat brain opioid binding sites eluted from the same lectin (55%). Receptors from placental extracts were compared with those from other tissues enriched in either kappa or mu sites. The proportion of applied kappa sites from guinea pig cerebellum eluted specifically from WGA agarose was 36%, whereas elution of binding sites from rat thalamus and rabbit cerebellum (enriched in mu sites) was at a level of 55%. This difference in the level of retention on and elution from WGA may reflect differences in the sugar composition of the glycoproteins of the two types of receptors. Succinylation of WGA abolished its ability to retain opioid binding sites, consistent with involvement of sialic acid. However, currently available evidence suggests that differences in retention on WGA between kappa and mu sites may be due to differences in either sialic acid or N-acetylglucosamine content or both.     

Assessing acrosomal status of bovine sperm using fluoresceinated lectins

Binding of 12 lectins to bull sperm was analyzed to select a lectin that bound preferentially to the acrosomal region. Peanut agglutinin (PNA) and Pisum sativum agglutinin (PSA) were suitably specific for intracellular, acrosome-associated glycoconjugates. Peanut agglutinin exhibited almost no detectable binding to sperm surface receptors, but intense binding to the area of the acrosome anterior to the equatorial segment. In contrast, PSA bound intensely to anterior and equatorial acrosomal regions, and weakly to the other regions of the sperm. Acrosomal labeling by both lectins decreased when sperm were induced to acrosome-react with calcium ionophore. To determine if these lectins could be used to assess acrosomal status, we compared the percentage of acrosome-reacted sperm that were detected by staining with naphthol yellow and erythrosin B with the percentage that were detected by lectin labeling. The incidence of reacted sperm detected by PSA labeling was not significantly different from that detected by naphthol yellow/ erythrosin B (P = 0.46). The incidence of reacted sperm detected by PNA was correlated with the incidence detected by naphthol yellow/erythrosin B, but was significantly lower (P = 0.003). We conclude that labeling permeabilized sperm with fluoresceinated PSA can serve as a rapid assay for acrosomal status.     

Regulatory serum lipoproteins: regulation of lymphocyte stimulation by a species of low density lipoprotein.

Low density lipoproteins (LDL) containing apolipoprotein B were separated from 15 fresh normal human serum pools by three independent isolation methods including sequential ultracentrifugal flotation, affinity chromatography, and polyanion precipitation. A discrete subpopulation of LDL (LDL-In) was isolated which possessed comparable inhibitory activity for PHA, PWM, and allogenic cell stimulated human lymphocytes in vitro at concentrations of 1 to 10 mug protein/1 x 10(5) lymphocytes/0.25 ml culture. LDL-In was characterized by a mean buoyant density of 1.055 g/ml in KBr, a m.w. of 2 to 3 x 10(6) daltons and a composition of 20 to 25% protein and 75 to 80% lipid with beta electrophoretic mobility. The biologic activity of LDL-In was non-cytotoxic, independent of mitogen concentration, and dependent upon the concentration of serum in the culture assay. The effect was temporally dependent requiring approximately 24 hr for induction of a stable suppressed state. Suppression was reversible with shorter periods of exposure to LDL-In. LDL-In did not inhibit lymphocytes at periods greater than 19 hr after stimulation, suggesting that LDL-In may influence metabolis events associated with the inductive phase of lymphocyte activation by lectins and allogeneic cells. LDL-In was clearly distinguishable from T lymphocyte E rosette inhibitory factor since it did not influence E rosette function of lymphocytes. The physicochemical and biologic properties of LDL-In clearly distinguish this reguloratory lipoprotein from previously described immunoregulatory factors.     

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line. Analysis using double labeling with lectins

Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheatgerm agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(beta 1-3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosaccharide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.     

Further characterization of monoclonal antibody 6,F-8 reacting with Lathyrus, Lens and Pisum lectins

The murine monoclonal antibody (MoAB) 6,F-8 made against the glucose/mannose-specific Lathyrus odoratus mitogen has previously been shown to react with Lens culinaris and Pisum sativum lectins, but not with the lectin from Vicia faba [(1988) Biol. Chem. Hoppe-Seyler 369, 365-370]. The reactivity against seven other completely sequenced Lathyrus lectins has now been tested after separation of the subunits by SDS-polyacrylamide gel electrophoresis and electroblotting to nitrocellulose filters. Two of these lectins reacted with the antibody. Comparison of the amino acid sequences of the examined lectins and the predicted hydrophilic, flexible and accessible regions of Pisum sativum suggest that valine-147 is involved in antibody binding.     

Receptors for IgM on certain human B lymphocytes.

A receptor for IgM was demonstrated on the surface of human B lymphocytes by using a rosette technique with ox erythrocytes coated with rabbit IgM antibody (EAM). Lymphocytes forming rosettes with EAM did not bind sheep red cells, had membrane Ia-like antigens and, in some instances, surface immunoglobulin. The specificity of EAM rosettes was confirmed by inhibition experiments with purified human Ig. IgM but not IgG molecules inhibited the rosette reaction. In addition, inhibition of EAM rosettes with IgM fragments showed that the receptor has affinity for a part of the molecule located in the Fc portion. By analogy with the receptors previously found on certain human T cells, receptors for IgM were not detected on freshly isolated B cells, but were expressed after overnight culture in IgM-free media. Studies on different human lymphoid tissues showed that IgM receptors are expressed on a limited percentage of both circulating and noncirculating B cells. In addition to normal B cells, the malignant B cells of a majority of cases of chronic lymphocytic leukemia expressed the receptors for IgM.     

A versatile immunoadsorbent capable of binding lectins of various specificities and its use for the separation of cell populations

A procedure for cell fractionation using lectin-affinity chromatography is described. It consists of a single affinity adsorbent, hog gastric mucin blood group A+H substance covalently coupled to Sephadex or Sepharose, to which lectins of various specificities can bind. The complex formed, lectin in equilibrium hog A+H substance-Sephadex, then serves as an affinity probe for isolating and fractionating cells. The lectins from Ulex europaeus, Lotus tetragonolobus, Helix pomatia, Dolichos biflorus, and Phaseolus lunatus were used with the same blood group substance as adsorbent. The affinity columns retained erythrocytes with blood group specificity for the adsorbed lectin and thus fractionate cells in mixtures. Cells as well as lectins are eluted by specific sugar inhibitors. Mixtures of two kinds of cells can be separated when the proportion of the adsorbed cells is not too low.     

Effect of lectins on induction of apoptosis in the populations of mammalian cells in vitro

The cytotoxic action of lectins different in origin and carboxyl specificity has been studied. It has been shown that all types of lectins at high concentrations (20 mkg/ml) were able to induce apoptosis in the in vitro populations of Chinese hamster cells two days after the treatment. In the case of Persa fluviatilis lectin this effect was detected immediately after the treatment and two days later as well. It was shown that Sambucus nigra lectin did not influence the frequency of apoptosis in the culture of human cells in contrast to the Lens culinaris and P. fluviatilis lectins. The tendency of stimulation of human cell proliferation under exposure to P. fluviatilis lectin at low concentration (0.2 microg/ml) has been registered.     

Studies on platelet surface carbohydrates in normal and uraemic platelets using 125I-labelled lectins

Binding studies with six different purified 125I-labelled lectins, concanavalin A (con A), wheat germ agglutinin (WGA), Ricinus communis agglutinin II (RCA II), Dolichos biflorus (DB), Tetranolobus purpureus (TP) and P-phyto-hemagglutinin (P-PHA), were used to investigate the surface topography of carbohydrates in platelets from uraemic and normal subjects. Compared with normal the uraemic platelets, bear significantly decreased (more than 2.5-fold) numbers of receptors for P-PHA (N-acetyl D-galactosamine specificity) and Con A (specificity glucose, mannose). The number of WGA, RCA, II, DB and TP receptors in uraemic platelets did not differ from the number in normal platelets. Binding studies with 125I-labelled lectins provide further evidence of molecular defects in uraemic platelets. Moreover, this method might provide a fast and reliable technique for identifying abnormalities in the surface topography of carbohydrates on platelets in several pathological states.     

In vitro human reactivity to staphylococcal phage lysate.

The cell-mediated reactivity of normal individuals to staphylococcal phage lysate (SPL) were tested in vitro in the lymphocyte stimulation (LS) and leukocyte migration inhibition (LMI) assays. There were 95% positive responses in LS (stimulation ratio larger than or equal to 3 with p less than 0.01) and 67% positive responses in LMI (migration index less than or equal to 0.80). Enriched subpopulations of T and B lymphocytes were prepared with rosette formation and density gradient centrifugation. SPL stimulated lymphoproliferative responses in both T and B cell subpopulations whereas phytohemagglutinin (PHA) stimulated only the T cell subpopulation. Cord blood leukocytes were tested in the LS assay and 41% gave positive responses to SPL, 81% to PHA, and 17% with SLO. SPL appears to be a useful reagent for the in vitro study of cell-mediated reactivity, and may provide somewhat different information from that obtained with other mitogens or antigens.