Ultrastructural detection of lectin receptors by cytochemical affinity reaction using mannan-iron complex.

A two-step affinity reaction is described for electron microscopic demonstration of the Concanavalin A as well as the Lens culinaris lectin receptors by means of the yeast mannan-iron complex. First the tissue was incubated in the lectin. Afterwards the incubation in the yeast mannan-iron complex was performed and reaction takes place between the still free second sugar binding site of membrane bound lectin molecules and the polysaccharides. This membrane receptor-lectin-polysaccharide complex is revealed by the electron dense iron core of the yeast mannan-iron complex. The specificity of the reactions could be demonstrated by addition of the hapten or by incubation in the yeast mannan-iron complex only. The proposed technique has proved useful for demonstration of lectin receptors in the small intestine.     

Glycophorins of human erythroleukemic K562 cells

Glycophorins related to alpha glycophorin, of the human erythrocyte membrane, were isolated from human erythroleukemic K562 cells. The glycophorins were purified using sodium dodecyl sulfate (SDS)/trichloroacetic acid fractionation and Folch and hot phenol extractions. 0.1-0.2 micrograms was obtained/10(8) cells, or approximately a 15% yield. SDS-gel electrophoresis revealed a pattern similar to erythrocyte alpha glycophorin except for the slower mobility of the glycophorin monomer. Two populations of K562 glycophorins, present in nearly equivalent amounts, were distinguished by their binding to Lens culinaris lectin agarose. The two populations exhibited similar gel electrophoretic patterns except for the presence of delta-like glycophorin exclusively in the population that did not bind to L. culinaris lectin. Immunoblotting revealed a lack of reaction of the major alpha and delta-like glycophorin bands in all K562 glycophorins with M or N erythrocyte glycophorin-specific monoclonal antibodies. Only minor species of intermediate electrophoretic mobility in glycophorins not binding to L. culinaris showed a reaction with these antibodies. Both populations of glycophorins incorporated radiolabeled glucosamine, mannose, and fucose and contained O-glycosidically linked tri- and tetrasaccharides, present in a ratio of approximately 1:1 indicating a significant degree of hyposialylation when compared to erythrocyte alpha glycophorin. No precursor/product relationship was demonstrated between the major forms of two populations. K562 cell surface labeling with lactoperoxidase revealed that only the glycophorins that exhibited binding to L. culinaris were accessible to iodination and could be the only species expressed at the cell surface.     

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.     

Solubilization and enrichment of boar sperm membrane proteins

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.     

In vivo binding partners of the Lens culinaris lectin

The immobilized lectin from the lentil (Lens culinaris) specifically binds two fractions out of the L. culinaris seed globulins. Both fractions are displaced from the lectin at low pH values. In addition, fraction I fails to interact at high ionic strengths, and fraction II in the presence of glucose or other lectin-specific sugars. The behaviour in zonal isoelectric precipitation and electrophoretical patterns indicate that both fractions represent subpopulations of the storage proteins. The interaction as demonstrated by affinity chromatography is corroborated by nephelometry: If the dissolved proteins (lectin plus fraction I or fraction II) are mixed under proper conditions the solutions become turbid. An even more pronounced interaction is observed if the lectin is reacted with both fractions at the same time. Seed albumins able to interact with the immobilized lectin include the dissolved lectin and two glycosidases (alpha-mannosidase, alpha-galactosidase) all of which are located in the protein bodies. A third glycosidase (beta-galactosidase) from outside of the protein bodies does not bind to the lectin. The results are discussed in view of the possibility that lectins may serve as packaging aids for other proteins in the protein bodies.     

Proteins of Rapid Axonal Transport: Polypeptides Interacting with the Lectin from Lens Culinavis

Abstract: Rapidly transported fucose-labelled glycoproteins from axons of rabbit retinal ganglion cells were solubilized with nonionic detergents. The labelled glycoproteins carried hydrophobic sites as revealed by a reversible binding to a hydrophobic matrix. A major portion of the labelled glycoproteins carried α-D-mannopyranoside (or glucopyranoside) residues, as shown by a specific interaction with the lectin from Lens culinaris. Three major groups of labelled polypeptides capable of binding to the lectin are described.     

Lectin binding to cystic stages of Taenia taeniaeformis

Studies of membrane glycoconjugates of Taenia taeniaeformis were initiated by assays of the lectin binding characteristics of 35-day-old cysticerci. Parasites fixed in glutaraldehyde were incubated with one of the following FITC-labelled lectins: Concanavalin A (Con A), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin (RCA), peanut agglutinin (PNA), fucose binding protein (FBP) and wheat germ agglutinin (WGA) and either their specific or a nonspecific sugar. Ultraviolet microscopy revealed that only Con A and LCA bound in large amounts to the surface of cysticerci. This binding was partly inhibited by the specific sugar, but the nonspecific sugar had little effect. The lectin not removed by either of the sugars may have been bound nonspecifically to the charged glycocalyx. Lectins were primarily bound on the anterior third of the parasite around the scolex invagination. Kinetic studies of lectin interactions were carried out with LCA and RCA by spectrophotofluorometric analysis of the amount bound specifically or nonspecifically over a range of lectin concentrations. Lens culinaris lectin binding was found to be specific and involve 2 receptors which showed large differences in their affinity for lectin and prevalence on the surface. Ricinus communis lectin did not bind specifically but nonspecific interactions were observed. Adherence of small numbers of host cells was shown to have no measurable effect on the lectin binding characteristics. The results suggest that the major surface carbohydrates exposed are D-mannose and/or D-glucose residues with the other sugar groups poorly represented. This relatively homogeneous surface may have implications for the antigenicity of the parasite in its host.     

T-cell immunodeficiency in mice receiving lectin and cyclophosphamide

Cyclophosphamide injections to mice following T cell mitogen (lectins from Lens culinaris and concanavalin A) were shown to suppress (20-40-fold) thymus-dependent response to SRBC. At the same time no damage-specific and polyclonal response to thymus-independent antigen and polyclonal activator of B cells--lipopolysaccharide--has been observed. Injections of lectin and cyclophosphamide to mice prevented the onset of DTH reaction to SRBC and induction of antigen-specific DTH suppressor cells. Thus, cyclophosphamide injection after T cell mitogen leads to T-cell anergy, with B-cell activity remaining unchanged.     

Alteration of the carbohydrate-binding specificity of the Bauhinia purpurea lectin through the preparation of a chimeric lectin.

A chimeric lectin gene was constructed by using a cDNA clone coding the Bauhinia purpurea lectin (BPA) in which a part of the metal-binding region was replaced by the corresponding region of the mannose-binding Lens culinaris lectin (LCA). The chimeric lectin expressed in Escherichia coli was found to bind alpha mannosyl-bovine serum albumin (BSA) and this binding was inhibited by mannose.     

Purification and properties of 5'-nucleotidase from lymphocyte plasma membranes.

5'-Nucleotidase is purified from lymphocyte plasma membranes by two affinity chromatographies. The first one, on Lens culinaris lectin-Sepharose 4B yields a fraction of twelve lectin-binding glycoproteins (lectin-receptor fraction). The second one on 5'-AMP-Sepharose 4B leads to pure enzyme. This enzyme is a glycoprotein with a molecular weight of 130 000; it gives a single band in polyacrylamide/dodecylsulfate electrophoresis and displays a very high specific activity (2500-3000 mumol Pih-1mg-1). Some properties of purified 5'-nucleotidase are similar to those of membrane-bound enzyme: substrate specificity, temperature dependence, effects of ions and SH-blocking reagents. Others are completely different for the two systems and these differences result from an interaction between the enzyme molecule and other Lens culinaris lectin binding proteins.     

A Computational analysis on Lectin and Histone H1 protein of different pulse species as well as comparative study with rice for balanced diet

The issue of balanced nutrition is of great concern to human. Meat and fish are the best sources of protein. The affordability of these resources for people in developing countries is less. Thus, there is an increasing interest in pulses and its derivates as an alternative to fish and meat. Lectin and histone H1 are the most common proteins in various pulses and our interest is in identifying the dominant essential amino acids in them for use as supplements. However, actin and lectin are common among Oryza Sativa and cicer arietinum. We describe the amount of lectin and histone H1 in cicer arietinum, Lens culinaris and Pisum sativum in a comparative manner. cicer arietinum was found to contain more essential amino acids than Lens culinaris and Pisum sativum. The secondary structures of lectin and histone H1 protein were analyzed to gain functional inferences in these species. The comparative study shows the relatively poor presence of the amino acid methionine in most pulses. However, Oryza Sativa was found to contain sufficient methionine. The study shows that pulses (especially cicer arietinum) were a suitable alternative source to meat and fish for Lectin and Histone H1 balance. Hence, pulses could be suggested with rice for balanced protein diet.     

The identification of membrane glycoconjugates in Leishmania species

The membrane glycoconjugates of 8 different species of Leishmania were compared by lectin blotting. Five different lectins with various sugar specificities were examined: concanavalin A, Lens culinaris, Ricinus communis, soybean agglutinin, and peanut agglutinin. Concanavalin A and Lens culinaris reacted with every Leishmania tested. The patterns observed for these 2 lectins, as well as the various species of parasites, were different. However, a common 41,000-52,000 and a 160,000-185,000 Mr component was present in almost all the parasite isolates examined. Ricinus communis only recognized a nondiscrete galactose-containing glycoconjugate similar to Leishmania-excreted factor. Soybean and peanut agglutinins reacted with a few low molecular weight parasite components. Soybean agglutinin reacted with all the Leishmania species tested, whereas peanut lectin only recognized 3 isolates. The latter lectin bound to discrete components migrating with the dye front and with Mr's of 35,000 and 52,000. Increased glycosylation was noted on avirulent L. major promastigotes and was associated with the appearance of several new peanut agglutinin-binding glycoproteins.     

Synergistic effects of lectins in the interaction of thrombin with human platelets

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.     

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.     

Redistribution of membrane-bound and cytosolic action in rabbit polymorphonuclear leucocytes during phagocytosis.

In the analysis of highly purified surface membrane from both resting and phagocytosing neutrophils an increase in the surface membrane associated actin has been demonstrated. This change at the cell periphery is associated with a coincident increase in the F-actin content of the cells following stimulation of the cells by exposure to opsonized Oil Red O droplets. The actin which is newly associated with the surface membrane of the phagocytosing cells was more susceptible to removal by detergent than the membrane-associated actin in resting cells and it was also noted that the F-actin associated with phagosomes was readily disrupted by detergent. A redistribution of the surface membrane glycoprotein 5'-nucleotidase was observed during phagocytosis, but no change in distribution of a 125I-labelled Lens culinaris lectin was observed during the entire phagocytic process.     

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.     

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.     

Glycosylation pattern in the appendix testis in children with cryptorchidism

In humans, at about week 6, sex cords develop within the forming testes. Testes normally descend to the scrotum; cryptorchidism occurs when one or two testes do not descend to scrotum and in some case are accompanied by the appendix testis. The appendix testis is a small sessile or polypoid structure located at the antero superior pole of the testis, adjacent to the head of the epididymis. Glycans can be involved in development of the appendix testis and cryptorchidism. In this work, lectin histochemistry was used to evaluate glycans expression in appendix testis in children with cryptorchidism. Our results showed that lectin from Lens culinaris, Ulex europaeus I., Canavalia ensiformis, Artocarpus integrifolia, Glycine max, and Griffonia simplicifolia recognizes epithelial and estromal cells. Not interaction was observed with lectin from Amaranthus leucocarpus, while lectin from Dolichus biflorus lectin only recognizes epithelial cells. Our results suggest that O-glycans linked in some glycoproteins represent important elements in appendix testis development.     

Glycodendritic structures based on Boltorn hyperbranched polymers and their interactions with Lens culinaris lectin

Multivalent scaffolds bearing carbohydrates have been prepared to mediate biological processes where carbohydrates are involved. These systems consist of dendritic structures based on Boltorn H20 and H30 hyperbranched polymers to which carbohydrates are linked through a convenient spacer. Mannose has been chosen as a sugar unit to test the viability of this strategy. These glycodendritic compounds have been prepared in a few steps with good yields, showing a high solubility in physiological media and low toxicity. The binding of these dendritic polymers to the mannose-binding lectin Lens culinaris (LCA) was studied using STD-NMR experiments and quantitative precipitation assays. The results demonstrate the existence of a clear interaction between the mannose derivative systems and the Lens lectin where the dendritic scaffold does not have an important role in mannose binding but supplies the necessary multivalence for lectin cluster formation. These glycodendritic structures are able to interact with a receptor, and therefore they can be considered as promising tools for biological studies.     

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.