Plant lectins detect age and region specific differences in cell surface carbohydrates and cell reassociation behavior of embryonic mouse cerebellar cells

When plated at high cell density in a microwell culture system, freshly dissociated embryonic mouse cerebellar cells assemble into reproducible, 3-dimensional patterns. The addition of the dimeric lectin Succinyl Concanavalin A blocks reversibly the formation of the microwell pattern, suggesting that cell surface carbohydrates affect the reassociation behavior of embryonic mouse cerebellar cells. Agglutination studes of dissociated cell populations harvested from different regions of the embryonic brain reveal that different lectins agglutinate cell populations from different embryonic brain regions. Cells from E13 cerebellum are agglutinated with Concanavalin A, wheat germ agglutinin, Ricinus communis agglutinin, mol wt 60,000, Ricinus communis agglutinin, mol wt 120,000, and Lens culinaris, but not by soybean agglutinin or a fucose-binding protein. Cells from the midbrain are agglutinated only with Concanavalin A, Ricinus communis agglutinin, mol wt 60,000 and Ricinus communis agglutinin, mol wt 120,000; those from the cerebral cortex are agglutinated only with Lens culinaris; and those from the medulla are agglutinated only with Ricinus communis agglutinin, mol wt 60,000, and Ricinus communis agglutinin, mol wt 120,000. In addition, agglutination of cerebellar cells with Concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin is diminished over the course of development from embryonic day 13 to postnatal day 7. These studies suggest regional differences in the cell surfaces of the developling brain that are further modulated during the differentiation of the tissues. On a poly(D-lysine) treated substrate in microwell cultures, cell migration is unique to the cerebellum of the 4 brain regions studied. Surfaces treated with carbohydrate-derivatized poly(D-lysine) are currently being tested for their efficacy as substrates for differential cell migration.     

Changes in number, mobility, and topographical distribution of lectin receptors during maturation of chick erythroid cells

Plant lectins have been used to probe changes in cell surface characteristics that accompny differentiation in a complete series of chick erythroid cells. Dramatic differences in lectin receptor mobility were observed between the most immature cells of the series, the proerythroblasts, and cells at the next stage of maturation, the erythroblasts. Both concanavalin A and Ricinus communis agglutinin form caps on proerythroblasts, whereas they develop a patchy distribution on erythroblasts. Erythroid cells at later developmental stages show a homogeneous distribution of surface-bound R. communis agglutinin. Concanavalin A also shows a uniform distribution on the cell periphery, but appears to be concentrated in a ring above the perinuclear region of the cell. In addition to changes in mobility of lectin receptors, a large reduction (50-70%) in the number of lectin receptors per cell accompanies maturation of proerythroblasts to erythroblasts. Pretreatment of the cells with neuraminidase results in enhanced binding of R. communis agglutinin to proerythroblasts. The number of additional R. communis agglutinin receptors exposed by enzyme treatment remains relatively constant during subsequent cell maturation.     

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.     

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.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell.

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

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.     

Interaction of human erythrocyte Band 3 with Ricinus communis agglutinin and other lectins

Agglutination and competition studies suggest that human erythrocyte Band 3 can interact with both mannose/glucose- and galactose-specific lectins. Purified Band 3 reconstituted into lipid vesicles binds concanavalin A, but the nonspecific binding component, measured in the presence of alpha-methylmannoside, is very high. This glycoprotein also carries binding sites for the galactose-specific lectin Ricinus communis agglutinin. Binding was inhibited poorly by lactose, but much more effectively by desialylated fetuin glycopeptides, suggesting that the lectin recognizes a complex oligosaccharide sequence on Band 3. The glycoprotein bears two separate classes of binding sites for R. communis agglutinin. High-affinity binding sites exist which show strong positive cooperativity and correspond in number to the outward-facing Band 3 molecules. A low-affinity binding mode is abolished by 40% ethyleneglycol, suggesting the involvement of hydrophobic lectin-glycoprotein interactions. Studies on binding of R. communis agglutinin to human erythrocytes indicate positively cooperative binding to 7 X 10(5) very-high-affinity sites per cell, and lectin binding is completely inhibitable by lactose. Based on its binding characteristics in vesicles, it seems likely that Band 3 forms the major receptor for this lectin in human erythrocytes. Properties such as positive cooperativity thus appear to be a common feature of the interaction of Band 3 with a variety of lectins of different specificity, both in erythrocytes and lipid bilayers.     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae. A light- and electron-microscopic study

The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone which was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.     

Embryo Cell Surfaces—Lectin Binding and Cell Proliferation

The interaction between chick embryo fibroblasts and various lectins has been studied at different stages of embryo development. There is evidence that Robinia lectin, Dolichos lectin, and Conca navalin A decrease cell number and proportion of cells incorporating [³H] thymidine in case of 8and 10day-old chick embryo fibroblasts, whereas they stimulated the proliferation of 16-dayold embryo cells. No effect was noticed in 12-day cells.
These results suggest that some cell surface changes occur during embryo development. The site number of Dolichos lectin remains the same during embryo development, and the affinity constant decreases. The site number of Robinia lectin and Concanavalin A decreases from the 8^th to the 12^th day of development, and slowly increases on the 16–day cells, the affinity constant remaining rather constant.
The results indicate that the age–dependent effect of lectin on embryo cells could not be directly related to the number of lectin–binding sites. Competitive binding experiments revealed that Dolichos receptor sites were distincts from binding sites of Robinia lectin and Concanavalin A, and Robina receptor sites distinct from those of Concanavalin A.
Lectin effects on embryo fibroblasts were very specific as determined by inhibitory assays.     

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.     

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.     

Leishmania braziliensis: localization of glycoproteins in promastigotes

Two species of glycoproteins from Leishmania braziliensis promastigotes of apparent molecular weights of 53,000 (glycoprotein 53) and 47,000 (glycoprotein 47) were localized. Four lectins with different sugar specificities bound to the blotting sheet to which the electrophoretically separated materials were transferred. Concanavalin A and Ricinus communis agglutinin bound to the band of glycoprotein 53 and the lectin from Dolichos biflorus bound to the band of glycoprotein 47. Wheat germ agglutinin bound to the bands of both glycoproteins. Histochemical examinations using fluorescence labeled lectins demonstrated that the glycoproteins 53 and 47 were located on the cell surface and in the cytoplasm of promastigotes, respectively. The results are consistent with the result of agglutination test.     

Ultrastructural localization of lectin receptors on the luminal and abluminal aspects of brain micro-blood vessels

Lectin- or glycoprotein-colloidal gold complexes were used for detection of specific monosaccharide residues in mouse brain micro-blood vessels (MBVs). The lectins tested recognize the following residues: beta-D-galactosyl (Ricinus communis agglutinin-120, RCA-1), alpha-N-acetylgalactosaminyl (Helix pomatia agglutinin, HPA), alpha-D-mannosyl and alpha-D-glucosyl (Concanavalin A, Con A), sialoglycoconjugates (Limax flavus agglutinin, LFA), N-acetylglucosaminyl and sialyl (wheat germ agglutinin, WGA), and alpha-L-fucosyl (Ulex europeus agglutinin, UEA-1). Use of these lectin-gold complexes and ultrathin sections of Lowicryl K4M-embedded tissue makes it possible to gain insights into localization of lectin receptors in the entire cross-section of MBV walls. Receptors for all lectins, except UEA-1, were found on both luminal and abluminal fronts of the endothelial cells (ECs). Differential labeling of luminal and abluminal fronts of ECs with some lectins (Con A, HPL) is considered to reflect the polarity of the endothelium. Some differences noted in the distribution of lectin receptors in the wall of representatives of three types of MBVs (capillaries, arterioles, and venules) are thought to be associated with different functions performed by the above-mentioned segments of the microvasculature in maintenance of the blood-brain barrier.     

Quantitative variations of three different lectin receptors as a function of establishment and metabolism of normal and leukaemic human cell lines.

Lectin binding on the cell surface was measured by the method of Kornfeld [16] using three tritiated lectins: Robinia pseudo acacia, Concanavalin A and Ricinus. It has been shown that the number of binding sites for Robinia and Con A decreases after the establishment of a leukaemic cell line, whereas the affinity constant increases for Robinia. The relationship between lectin binding and cell growth was carried out on 5 cell lines, 3 of leukaemic and 2 of normal origin. In all cases the maximum number of sites was determined at the time of doubling for the cell population; the affinity constant reciprocally decreases at this moment. For Robinia, a difference was found in both cell growth and the number of sites between an Epstein-Barr virus (EBV) negative leukaemic cell line and the other EBV positive cell lines. Lectins offer a quantitative method for measuring membrane structure variations during and after establishment of cell lines.     

Postnatal development of lectin binding sites in the rat ventral prostate

Summary Five Fluorescein-isothiocyanate (FITC)-labelled lectins were used to study the postnatal development of carbohydrate constituents in the rat ventral prostate: Concanavalin A (Con A), wheat germ agglutinin (WGA), peanut agglutinin (PNA),Dolichos biflorus agglutinin (DBA) andRicinus communis agglutinin I (RCA-I) With all the lectins, tested, except RCA-I, specific binding sites could be shown for every stage of differentiation in the glandular epithelium. Binding sites for Con A, WGA, PNA and DBA were found from day 10 to 13 post partum onwards. Each lectin showed a characteristic localization. Binding sites for the lectins used changed to different extents during the following two weeks. After the 24th day post partum no further changes in the lectin binding pattern could be found. The development of the lectin binding properties showed that the changes in carbohydrate-containing constituents of the prostate correlate with the beginning of prostatic secretion and to prostatic epithelial differentiation. In the periacinar stroma the development of the lectin binding pattern was similar to that in the glandular epithelium. The changes of stromal binding sites for Con A and WGA during epithelial differentiation may reflect the changes of epithelial-stromal interactions in the prostate.     

Distribution of cell surface saccharides on pancreatic cells. II. Lectin-labeling patterns on mature guinea pig and rat pancreatic cells

The surface saccharide composition of collagenase-dispersed pancreatic cells from adult guinea pig and rat glands was examined by using eight lectins and their ferritin conjugates: Concanavalin A (ConA); Lens culinaris (LCL); Lotus tetragonolobus (LTL); Ricinus communis agglutinins I and II (RCA I, RCA II); Soybean agglutinin (SBA); Ulex europeus lectin (UEL); and wheat germ agglutinin (WGA). Binding studies of iodinated lectins and lectin-ferritin conjugates both revealed one population of saturable, high-affinity receptor sites on the total cell population (approximately 95% acinar cells). Electron microscopy, however, revealed differences in lectin-ferritin binding to the plasmalemma of acinar, centroacinar, and endocrine cells. Whereas acinar cells bound heavily all lectin conjugates, endocrine and centroacinar cells were densely labeled only by ConA, LCL, WGA, and RCA I, and possessed few receptors for LTL, UEL, and SBA. Endocrine and centroacinar cells could be differentiated from each other by using RCA II, which binds to centroacinar cells but not to endocrine cells. Some RCA II receptors appeared to be glycolipids because they were extracted by ethanol and chloroform-methanol in contrast to WGA receptors which resisted solvent treatment but were partly removed by papain digestion. RCA I receptors were affected by neither treatment. The apparent absence of receptors for SBA on endocrine and centroacinar cells, and for RCA II on endocrine cells, was reversed by neuraminidase digestion, which suggested masking of lectin receptors by sialic acid. The absence of LTL and UEL receptors on endocrine and centroacinar cells was not reversed by neuraminidase. We suggest that the differential lectin-binding patterns observed on acinar, centroacinar, and endocrine cells from the adult pancreas surface-carbohydrate-developmental programs expressed during morphogenesis and cytodifferentiation of the gland.     

Distribution of glycoconjugates in normal human skin using biotinyl lectins and avidin-horseradish peroxidase

Lectin binding patterns in normal human skin were studied using five different biotinyl lectins and avidin-horseradish peroxidase. The staining pattern was specific for each lectin. In the epidermis, peanut agglutinin (PNA) and soybean agglutinin (SBA) preferentially stained the cell membranes of keratinocytes in the spinous and granular cell layers, indicating changes in the saccharide residues during keratinocyte differentiation. In the secretory segment of an eccrine sweat gland, the superficial cells gave a strong granular staining with Ricinus communis agglutinin (RCA). Dolichos biflorus agglutinin (DBA) and SBA, on the other hand, strongly stained the basal cells. With these lectins, two types of cells in the secretory segment were clearly distinguished. These results show that (1) PNA and SBA binding sites increase during the course of keratinocyte differentiation, and (2) RCA, DBA, and SBA are good markers to distinguish two types of cells in the secretory segment of an eccrine sweat gland.     

Anaplasma marginale, Eperythrozoon wenyoni: lectin reactions with bovine erythrocytes

Normal bovine erythrocytes were agglutinated with four of five lectins specific for different oligosaccharides. The order of reactivity was wheat germ greater than ricin greater than soybean greater than peanut. Concanavalin A did not agglutinate normal bovine erythrocytes. After neuraminidase treatment of normal bovine erythrocytes, each lectin agglutinated the cells with decreased concentrations of lectin, verifying that partial removal of sialic acid exposes more of each lectin's binding sites or alters the binding site such that fewer molecules of lectin are required to initiate agglutination. A change in agglutination of erythrocytes using soybean agglutinin and peanut agglutinin occurred when cells were obtained from cattle infected with Eperythrozoon wenyoni. The results suggested that an alteration in erythrocyte membranes occurred as a result of this infection as manifested by the increased recognition of both the soybean agglutinin and peanut agglutinin receptor carbohydrates. A similar effect was indicated with erythrocytes obtained during an acute Anaplasma marginale infection; however, an ensuing reticulocytosis masked the effect, requiring the use of fluoresceinated lectins to verify that increased binding of each lectin occurred with infected cells when compared to normal cells.     

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.     

Changes in lectin-induced deoxyribonucleic acid synthesis in cultures of chick-embryo fibroblasts at various stages of development

Concanavalin A and Robinia pseudoacacia lectin decreased [(3)H]thymidine incorporation into acid-insoluble material of fibroblasts cultured from 6-10-day chick embryos. In contrast, these lectins stimulated [(3)H]thymidine incorporation in cells from 16-day embryos. These effects are due to neither [(3)H]thymidine permeability modification nor toxicity of the lectins. The specificity of lectin action was proved by blocking experiments with alpha-methyl mannopyranoside and with anti-(Robinia lectin) serum.