Serial lectin affinity chromatography with concanavalin A and wheat germ agglutinin demonstrates altered asparagine-linked sugar-chain structures of prostatic acid phosphatase in human prostate carcinoma

Differences between human prostate carcinoma (PCA, five cases) and benign prostatic hyperplasia (BPH, five cases) in asparagine-linked (Asn) sugar-chain structure of prostatic acid phosphatase (PAP) were investigated using lectin affinity chromatography with concanavalin A (Con A) and wheat germ agglutinin (WGA). PAP activities were significantly decreased in PCA-derived PAP, while no significant differences between the two PAP preparations were observed in the enzymatic properties (Michaelis–Menten value, optimal pH, thermal stability, and inhibition study). In these PAP preparations, all activities were found only in the fractions which bound strongly to the Con A column and were undetectable in the Con A unbound fractions and in the fractions which bound weakly to the Con A column. The relative amounts of PAP which bound strongly to the Con A column but passed through the WGA column, were significantly greater in BPH-derived PAP than in PCA-derived PAP. In contrast, the relative amounts of PAP which bound strongly to the Con A column and bound to the WGA column, were significantly greater in PCA-derived PAP than in BPH-derived PAP. The findings suggest that Asn-linked sugar-chain structures are altered during oncogenesis in human prostate and also suggest that studies of qualitative differences of sugar-chain structures of PAP might lead to a useful diagnostic tool for PCA.     

A histochemical study of the distribution of lectin binding sites in the developing oocytes of the lancelet Branchiostoma belcheri

The distribution of carbohydrate moieties in lancelet (Branchiostoma belcheri) oocytes has been studied at different stages of development, using a peroxidase-labeled lectin incubation technique, the PAS-reaction and Alcian Blue staining. Binding sites of 5 lectins, indicating the presence of different sugar moieties (Wheat germ agglutinin (WGA) for N-acetylglucosamine, Concanavalin A (Con A) for glucose/mannose, Helix pomatia agglutinin (HPA) for N-acetyl-D-galactosamine, Ricinus communis agglutinin (RCA-I) for galactose and Ulex europaeus agglutinin (UEA-I) for fucose), were identified and were shown to undergo considerable variation during oocyte development. In the previtellogenic stage, HPA, RCA-I and UEA-I were not identified on the oocyte surface, but WGA and Con A gave strongly positive reactions at this site. In the cytoplasm, 4 lectins (Con A, HPA, RCA-I and UEA-I) gave a weak or moderate reaction, and Con A was also observed in the perinuclear region. In vitellogenic oocytes, these 4 lectins were found to also bind to the nuclear envelope, karyoplasm and nucleolus, and, with the exception of Con A, could also be found in the nuclei of more mature stages. The cytoplasmic yolk granules and Golgi vesicles of the vitellogenic oocyte, were moderately positive for Con A, HPA, RCA-I and UEA-I, but HPA, RCA-I and UEA-I were only weakly bound at the oocyte surface. In mature oocytes, all 5 lectins bound moderately or strongly to yolk granules and cell surface. HPA, RCA-I and UEA-I bound moderately or strongly to various nuclear compartments. Thus, carbohydrate content varied with the development and maturation of the oocytes, and the PAS results were in agreement with the lectin-binding results. Charged carbohydrate residues were observed in the egg envelope and Golgi bodies.These results suggest that the appearence of Con A-, HPA-, RCA-I- and UEA-I-binding glycoconjugates in the nuclei of developing oocytes show a varying pattern indicating different phases of nuclear activity which correlate with different carbohydrate synthetic activities of the oocyte.     

Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides.

The carbohydrate-binding properties of wheat-germ agglutinin (WGA) have been studied by using glycopeptides isolated from the cell surfaces of a cultured murine myeloid cell line (416B). The glycopeptides were passed through affinity columns of lentil lectin (LCA), concanavalin A (Con A) and WGA arranged in series so that material reaching the WGA column had failed to bind to LCA or Con A. WGA-binding glycopeptides were step-eluted with 0.01 M, 0.1 M and 0.5 M-N-acetylglucosamine (GlcNAc), to yield weak (WGA-W), intermediate (WGA-I) and strong (WGA-S) affinity fractions. WGA-W and WGA-I contained 'N'- and 'O'-linked oligosaccharides bound to separate polypeptides. WGA-S consisted almost entirely of N-linked components. Our analytical work was concentrated mainly on the N-linked fractions. In these carbohydrates WGA affinity was directly proportional to molecular size but inversely related to N-acetylneuraminic acid content. The binding of the weak-affinity fraction was dependent on N-acetylneuraminic acid, but the intermediate- and strong-binding species interacted with the lectin by N-acetylneuraminic acid-independent mechanisms. N-linked glycopeptides in each WGA-binding class were almost totally degraded to monosaccharides by the concerted action of the exoglycosidases neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase. Treatment with endo-beta-galactosidase caused partial depolymerization, yielding some disaccharides but also a heterogeneous population of partially degraded components. These findings suggest that WGA binds with high affinity to internal GlcNAc residues in large oligosaccharides containing repeat sequences of Gal beta(1----4)GlcNAc beta(1----3) (i.e. polylactosamine-type glycans). N-Acetylneuraminic acid is involved only in low-affinity interactions with WGA. WGA therefore displays an intricate pattern of saccharide specificities that can be profitably utilized for structural analysis of complex carbohydrates.     

Trypanosoma rhodesiense bloodstream trypomastigote and culture procyclic cell surface carbohydrates

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRAT at 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diamino-benzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A greater than PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WAG, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trypanosoma rhodesiense Bloodstream Trypomastigote and Culture Procyclic Cell Surface Carbohydrates1, 2, 3

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRATat 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diaminobenzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A < PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WGA, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M. The expression of lectin binding cell surface saccharides of T. rhodesiense WRATat 1 is related to the parasite stage. Sugars resembling α-D-mannose are on the surface of bloodstream trypomastigotes and culture procyclics; n-acetyl-D-galactosamine and D-galactose residues are on bloodstream forms; and n-acetyl-D-glucosamine-like sugars are on procyclic stages.     

Glycoconjugates of human sperm surface. A study with fluorescent lectin conjugates and lens culinaris agglutinin affinity chromatography

Distribution of glycocompounds in human spermatozoa was studied by using fluorescent lectin-conjugates. Con A bound predominantly to acrosomal and posterior head regions whereas RCA I bound to the acrosomal region of intact spermatozoa, stained in suspension. Other lectins used (LCA, WGA, SBA, PNA) stained the the entire sperm surface. In airdried sperm smears binding of both Con A and RCA I were identical with the staining pattern obtained with living cells whereas LCA, WGA, SBA and PNA now bound heavily into acrosomal region. As a similar staining pattern was obtained with permeabilized sperm cells, this staining is apparently due to binding to intracellular structures. The efficiency of Lens culinaris agglutinin affinity chromatography in purification of human sperm glycoproteins was tested after their external radiolabelling with the neuraminidase/galactose oxidase/sodium borohydride method. 22% of applicated radioactivity could be eluted from the column with the specific inhibitory saccharide, and most of the radiolabelled surface glycoproteins of the whole sperm lysate, were also present in the LCA affinity column eluate. LCA affinity chromatography seems thus be an effective method to enrich membrane glycoproteins of human spermatozoa.     

Hemocyte surface changes in the migratory grasshopper,Melanoplus sanguinipes in response to wounding and infection withBeauveria bassiana

Surface changes of hemocytes from the migratory grasshopper,Melanoplus sanguinipes (Fab.), following wounding and/or infection withBeauveria bassiana (Bals.) Vuillemin were assessed using fluorescein labelled wheat germ agglutinin (WGA) and concanavalin A (Con A). Binding was observed on outer wall of hemocytes when either Con A or WGA conjugates were tested. After infection there was an increase in the percentage of hemocytes which bound to WGA and which remained so for 24 h. However, after wounding this increase in binding subsided after only 1 h. Furthermore, it was found that the increase in WGA-binding occurred within the first 15 min of wounding or infection. In addition, ConA binding in males only increased 3-fold in fungus-injected insects within 1 h of treatment and remained high for 24 h. These results indicated the presence of mannose (and/or glucose) residues (Con A specificity) and N-acetyl-D-glucosamine residues (wheat germ agglutinin; WGA specificity) on outer wall surfaces of the hemocytes. The implications of these results in the immune response ofM. sanguinipes to infection withB. bassiana are discussed.     

Characterization of zoospore and cyst surface structure in saprophytic and fish pathogenicSaprolegnia species (oomycete fungal protists)

Summary The importance of the surface structure and chemistry in zoospores and cysts of oomycetes is briefly reviewed and the organelle systems associated with encystment described. The surface structure and chemistry of primary and secondary zoospores and cysts ofSaprolegnia diclina (a representative saprophytic species) andS. parasitica (a representative salmonid fish pathogen) were explored using the lectins concanavilin A (Con A) and wheat germ agglutinin (WGA) and monoclonal antibodies (MAbs) raised against a mixed zoospore and cyst suspension ofS. parasitica. The binding of lectins and antibodies to spores was determined using immunofluorescence microscopy with fluorescein isothiocyanate-labelled probes and with electron microscopy with gold-conjugated probes applied to spore suspensions post-fixation. In both species Con A, which is specific for glucose and mannose sugars, bound to both the surface of primary and secondary zoospores (the surface glycocalyx) and their cyst coats and readily induced zoospore encystment. The binding to the cysts appeared to be mainly associated with the matrix material released from the primary and secondary encystment vesicles and which appeared to diminish with time. No binding to germ tube walls was observed with this lectin. The MAb labelling showed a generally similar binding pattern to the primary and secondary cysts to that observed with Con A, although the binding to zoospores was more variable. Primary zoospores bound the antibodies but secondary zoospores appeared less reactive. It is suggested that the MAbs share a common epitope with one or more of the Con A-binding components. In both species WGA, which is specific for amongst other things the sugar N-acetyl glucosamine, bound to localised apical patches on the primary zoospores. This lectin also binds to the ventral groove region of secondary zoospores ofS. diclina, which were induced to encyst by this lectin. In contrast secondary zoospores ofS. parasitica were not induced to encyst by the addition of WGA and showed a patchy dorsal binding with this lectin. WGA also binds to both the inner wall of discharged primary cysts and the young germ tube walls of both species. These observations are discussed both in relation to other oomycete spores and to their possible functional and ecological significance.Abbreviations BSA bovine serum albumin - Con A Concanavalin A - DBA Dolichos biflorus agglutinin - ELISA enzyme-linked immunosorbent assay - EM electron microscope - EV encystment vesicles - FCS foetal calf serum - FITC Fluorescein isothiocyanate - FV peripheral fibrillar vesicles - G+F 0.2% glutaraldehyde and 2.0% formaldehyde primary fixative solution - 2G 2% glutaraldehyde primary fixative - LM light microscopy - MAbs monoclonal antibodies - LPV large peripheral vesicles - PBS phosphate buffered saline - PCV flattened peripheral cisternae - PEV primary encystment vesicle - PIPES piperazine-N,N1-bis(2-ethane sulfonic acid) - PNA Ricinus communis agglutinin - RAM-FITC/Au_10–20 Fluorescein isothiocyanate/gold (10 or 20 nm) labelled rabbit anti-mouse immunoglobulin - RCA Ricinus communis agglutinin - SEM scanning electron micrograph - SBA soybean agglutinin - SEV secondary encystment vesicles - TEM transmission electron micrograph - UEA I Ulex europaeus agglutinin - WGA wheat germ agglutinin     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

Search for the presence of lectin-binding sites on Toxoplasma gondii

Evidence for the presence of carbohydrate on the surface membrane of Toxoplasma gondii trophozoites and on the cell wall of toxoplasma brain cysts was sought by fluorescent lectin staining. Using FITC-conjugated preparations of Concanavalin A (Con A), wheat germ agglutinin (WGA), or soy bean agglutinin (SBA), we have failed to obtain evidence for the binding of these lectins on the surface of T. gondii trophozoites. In contrast, the three test lectins bound effectively and specifically to the wall of toxoplasma brain cysts. Prefixation of cysts with glutaraldehyde or brief trypsinization of cysts did not affect the intensity of cyst wall fluorescence when stained with FITC-conjugated Con A, SBA, or WGA. The results are interpreted to indicate that whereas exposed Con A, SBA, and WGA binding sites are associated with the wall of toxoplasma brain cysts, such lectin-binding saccharide residues are not present on the surface of trophozoites in exposed or reactive form.     

Colloid droplets in the magnocellular secretory neurons of the reptilian hypothalamus: An immunocytochemical and lectin-histochemical study

Summary The immunocytochemical and lectin-binding properties of the magnocellular neurosecretory neurons in the hypothalamus of 2 reptilian species, the snake Natrix maura and the lizard Liolaemus cyanogaster, were investigated. Particular attention was paid to the secretory droplets present in these neurons. Antisera against bovine neurophysins I+II, arginine-vasotocin, and mesotocin were used. The following lectins were applied: concanavalin A (Con A), wheat-germ agglutinin (WGA), and Limax flavus agglutinin (LFA). Adjacent 1-m-thick methacrylate sections were used to investigate the same secretory neuron and the same colloid droplets with all three antisera and all three lectins. Several sections were treated with trypsin and urea before immunostaining or lectin binding. Con A bound to both vasotocin- and mesotocin-immunoreactive neurons, WGA exclusively to vasotocin neurons; neither of these neurons reacted with LFA. The colloid droplets were present in vasotocin neurons but absent in the mesotocin neurons. These secretory droplets showed an affinity for Con A but not for WGA, and reacted with antisera against neurophysins and vasotocin. In Natrix maura, the colloid droplets became reactive with Con A and the antisera used only after pretreatment of the sections with trypsin and urea. Within the hypothalamo-neurohypophyseal system, antiserum against vasotocin and WGA revealed the same fiber bundles. It is concluded (i) that in reptiles the vasotocin-neurophysin precursor is glycosylated, (ii) that vasotocin neurons have the exclusive capacity to form colloid droplets, and (iii) that these droplets are an intracisternal (RER) storage form of the vasotocin-neurophysin precursor.This work was partially supported by Grants BOJA 27/9/88 from the Dirección General de Universidades e Investigación de Junta de Andalucía and DGICYT PB87-0710 from the Comisión Interministerial de Ciencia y Tecnología, Madrid, to P.F.-LL.; and Grant 89-01 from the Dirección de Investigaciones, Universidad Austral de Chile, to E.M.R.     

Lectin binding of subunits of placental and serum oxytocinase after electrophoresis and transblotting to nitrocellulose

1. Oxytocinase enzymes were purified from maternal serum and human placenta, run by SDS-PAGE and transferred onto nitrocellulose. Both enzymes were homogeneous in protein staining with Mr of 145,000. 2. Both serum and placental oxytocinases bound concanavalin A (Con A), limax flavus agglutinin (LFA) and wheat germ agglutinin (WGA). The WGA-binding of the placental enzyme was more strongly inhibited by 0.2 M N-acetylglucosamine than that of the serum enzyme which may indicate a higher sialic acid content in the serum enzyme. 3. Neuraminidase treatment did not affect the binding of Con A but decreased the binding of WGA to serum and placental enzymes. Serum enzyme showed a pl 4.7 on isoelectric focusing.     

Properties of thyroglobulins from normal thyroid and thyroid tumor on a concanavalin A-sepharose column

Affinity chromatography on a concanavalin A (con A)-Sepharose column is a potentially useful for the isolation of whole thyroglobulin (Tg) at least from normal thyroid tissue. In addition to being a simple procedure for the isolation of Tg, large amounts of Tg can be applied to the column and recovered in good yield with a buffer containing MeG. In gradient elution with buffer containing increasing amounts of MeG, a single but broad peak was obtained, without separation into subfractions. However, a hemagglutination-inhibition test showed that the Tg preparation eluted early from the column had less affinity for con A than the Tg preparation eluted later, suggesting a heterogeneous distribution of carbohydrate moieties among Tg preparations. When human Tg from thyroid tumor was applied to the column, tumor Tg partly passed through the column without being adsorbed. This unadsorbed Tg showed a very low affinity for lectins, con A and wheat germ agglutinin (WGA), as determined by a double diffusion reaction in agar gel. In contrast to this fraction, the Tg adsorbed on the con A-gel column showed a very strong affinity for WGA, differing from Tg of normal thyroid tissue. Therefore, tumor Tg preparation appears to have an abnormally modified carbohydrate structure, at least in part. The higher affinity for WGA (with a specificity for N-acetylglucosamine) seen in adsorbed Tg could be due to a larger amount of GlcNAc residues which bind irregularly in the carbohydrate moiety of tumor Tg.     

Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man(9 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.     

Carbohydrate exposure of human promyelocytic HL60 cells and histiocytic U937 cells during phagocytic differentiation assessed with fluoresceinated lectins

The display of carbohydrate structures was measured in promyelocytic HL60 cells and in histiocytic U937 cells induced to differentiate to phagocytic cellsin vitro during three to seven days of cultivation in the presence of dimethylsulfoxide (DMSO). It was assessed by micro-or spectrofluorometric quantification of the binding of fluorescent lectins. Changes in the cell size and the association and uptake of IgG-or complementopsonized yeast cells (Saccharomyces cerevisiae) were used as signs of phagocyte differentiation.The binding of wheat germ agglutinin (WGA), concanavalin A (Con A),Ricinus communis agglutinin-I (RCA-I) andUlex europaeus agglutinin-I (UEA-I) varied due to the presence of DMSO during cultivation, and without DMSO also on the number of days in culture and the type of cell.Abbreviations DMSO dimethylsulfoxide - PMA phorbol 12-myristate 13-acetate - KRG Krebs-Ringer phosphate buffer with glucose - WGA wheat germ agglutinin - Con A concanavalin A - RCA-I Ricinus communis agglutinin-I - UEA-I Ulex europaeus agglutinin-I     

An ultrastructural search for lectin-binding sites on surfaces of spinach leaf organelles

Organelles isolated from leaves of spinach (Spinacia oleracea L.) were prefixed in glutaraldehyde and then incubated with ferritin conjugates of four lectins — Concanavalin A (Con A), Ricinus communis L. agglutinin, MW 120,000 (RCA), soybean agglutinin (SBA), and wheat germ agglutinin (WGA) — in order to probe their cytoplasmic surfaces for saccharide residues. In each case the major leaf organelles, including microbodies, mitochondria and chloroplast derivatives, failed to exhibit labeling when examined with the electron microscope. Tobacco (Nicotiana tabacum L.) leaf protoplasts, incubated simultaneously with and under identical conditions to the spinach organelles, showed specific labeling of their plasma membranes with all four lectin conjugates, thus establishing the efficacy of the procedure for demonstrating the presence of binding sites when they exist. Further attempts to show binding of one of the lectins, Con A, by labeling with fluorescein-Con A and by organelle agglutination, yielded results consistent with the absence of ultrastructural labeling. It is concluded that no saccharide residues recognized by the four lectins are present on the cytoplasmic surfaces of organelles and that those residues reported to be constituents of intracellular membranes, therefore, are most likely exposed on the luminal (extracytoplasmic) surfaces.Abbreviations Con A Concanavalin A - RCA Ricinus communis agglutinin, MW 120,000 - SBA soybean agglutinin - WGA wheat germ agglutinin     

Lectin binding to newt epidermis: fluorescent localization and effects on motility

The ability of seven lectins to bind to newt epidermal cells and influence their motility was examined. Of the seven fluoresceinated lectins applied to frozen sections containing intact newt skin and migrating epidermis (wound epithelium), only Con A (concanavalin A), WGA (wheat germ agglutinin), and PNA (peanut agglutinin) produced detectable epidermal fluorescence. Con A and WGA each heavily labeled all layers of intact epidermis, but PNA bound only to the more superficial layers. In contrast to a single population of labeled cells in migrating epidermal sheets after treatment with Con A, there were both labeled and unlabeled cells after exposure to either WGA or PNA. The wound bed was labeled by both Con A and WGA, but not by PNA. DBA (Dolichos bifloris agglutinin), RCA I (Ricinus communis agglutinin), and UEA (Ulex europaeus agglutinin), did not produce significant fluorescence with either migrating or intact epidermis. In general, inhibitory effects on epidermal motility correlated with the binding studies. Thus, Con A, WGA, and PNA, the lectins which clearly bound to the epidermis, all produced a concentration-dependent depression in the rate of epidermal wound closure. RCA was somewhat paradoxical in that it was moderately inhibitory despite showing essentially no binding. The effects of SBA and UEA were equivocal. DBA had no effect. These results indicate that the inhibition of motility produced by Con A that we have described previously is not peculiar to this mannose-binding lectin, but is shared by at least one lectin with an affinity for D-GlcNAc (WGA), and one with an affinity for B-D-Gal(1-3)-D-GalNAc (PNA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the cell surface of zoospores and cysts of the fungus Phytophthora cinnamomi: nature of the surface saccharides as determined by quantitative lectin binding studies

The nature of the surface saccharides of zoospores, "partially encysted zoospores" and cysts of the root-rotting fungus Phytophthora cinnamomi, has been examined by quantitative lectin binding studies. Zoospores bound concanavalin A (Con A), but did not bind any of a variety of other lectins tested. In contrast, both cysts and "partially encysted zoospores" bound soybean agglutinin (SBA) as well as Con A. This indicates that accessible alpha-D-glucosyl/alpha-D-mannosyl-containing glycoconjugates predominate at the zoospore surface, whereas both alpha-D-glucosyl/alpha-D-mannosyl and galactosyl and/or N-acetyl-D-galactosaminosyl residues are accessible at the surface of cysts and "partially encysted zoospores." Neither Ulex europeus lectin nor wheat germ agglutinin (WGA) bound to any of the three cell preparations, indicating the absence of accessible alpha-L-fucosyl and N-acetyl-D-glucosaminosyl residues.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Lectin binding to injured corneal endothelium mimics patterns observed during development

Fluorochrome conjugated lectins were used to observe cell surface changes in the corneal endothelium during wound repair in the adult rat and during normal fetal development. Fluorescence microscopy of non-injured adult corneal endothelia incubated in wheat-germ agglutinin (WGA), Concanavalin A (Con A), and Ricinus communis agglutinin I (RCA), revealed that these lectins bound to cell surfaces. Conversely, binding was not observed for either Griffonia simplicifolia I (GS-I), soybean agglutinin (SBA) or Ulex europaeus agglutinin (UEA). Twenty-four hours after a circular freeze injury, endothelial cells surrounding the wound demonstrated decreased binding for WGA and Con A, whereas, RCA binding appeared reduced but centrally clustered on the apical cell surface. Furthermore, SBA now bound to endothelial cells adjacent to the wound area, but not to cells near the tissue periphery. Neither GS-I nor UEA exhibited any binding to injured tissue. By 48 h post-injury, the wound area repopulates and endothelial cells begin reestablishing the monolayer. These cells now exhibit increased binding for WGA, especially along regions of cell-to-cell contact, whereas, Con A, RCA and SBA binding patterns remain unchanged. Seventy-two hours after injury, the monolayer is well organized with WGA, Con A and RCA binding patterns becoming similar to those observed for non-injured tissue. However, at this time, SBA binding decreases dramatically. By 1 week post-injury, binding patterns for WGA, ConA and RCA closely resemble their non-injured counterparts while SBA continues to demonstrate low levels of binding. In early stages of its development, the endothelium actively proliferates and morphologically resembles adult tissue during wound repair.(ABSTRACT TRUNCATED AT 250 WORDS)