The affinity of Concanavalin A and wheat germ agglutinin for components of the muscle spindle

Summary Sections through the soleus muscle of the rat were incubated with concanavalin A (Con A) or wheat germ agglutinin (WGA) conjugated to fluorescein isothiocyanate. Binding of these lectins to structures which comprise the muscle spindle was studied by fluorescence microscopy. The distribution of the lectins was heaviest in the outer capsule of the spindle and at the surface of intrafusal muscle fibres. The periaxial space in the equatorial region of spindles was unlabelled except in the immediate vicinity of the axial bundle. Binding by Con A was more extensive than by WGA, suggesting that more glucopyranoside units are accessible within the muscle spindle than are those of N-acetylglucosamine.     

Lectin histochemistry of the hyaline layer in the asteroid,pisaster ochraceus

Six different lectins were used to study the carbohydrate nature of the hyaline layer (HL), the external extracellular matrix of the starfish embryo. Thin sections of embryos fixed in the late gastrula stage were incubated with five fluoresceinated lectins: Con A, WGA, RCA, UEA-I, and SBA. All but UEA-I labelled the HL, suggesting that the following sugars are present: mannose and/or glucose, glcNAc and/or Neu5Ac, galactose, and galNAc. The different lectins produced variable degrees of labelling, with WGA, RCA, and SBA producing more intense labelling than Con A. Binding of lectins by the HL was studied at the ultrastructural level by exposing ultrathin sections to the following lectin-gold conjugates: Con A, WGA, PNA, SBA, and LFA. Lectin binding was observed over the various regions of the HL, recognized by Crawford and Abed (J. Morphol. 176:235–246, '86), i.e., the intervillus layer, the supporting layer and the coarse outer meshwork. Local differences in labelling patterns were observed among the various lectins, with SBA labelling all regions intensely, WGA and PNA labelling the supporting layer predominantly, and Con A labelling the HL only lightly. No labelling was observed with LFA. These lectin-labelling patterns in the HL demonstrate the presence of different glycoconjugates in different regions of the HL, suggesting that the layers differ biochemically. The existence of biochemical differences strengthens the idea that each layer may have different functions in the developing starfish embryo.     

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.     

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.     

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.     

Lectin histochemistry of human skeletal muscle

Biotinyl derivatives of seven plant lectins-concanavalin A (Con A), peanut agglutinin (PNA), Ricinus communis agglutinin I (RCA I), Ulex europeus agglutinin I (UEA I), soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), and wheat germ agglutinin (WGA)-were bound to cryostat sections of biopsied normal human muscle and visualized with avidin-horseradish peroxidase conjugates. A distinct staining pattern was observed with each lectin. The most general staining was observed with Con A, RCA I, and WGA, which permitted strong visualization of the plasmalemma-basement membrane unit, tubular profiles in the interior of muscle fibers, blood vessels, and connective tissue. PNA gave virtually no intracellular staining, while SBA and UEA I selectively stained blood vessels. DBA was unique in providing good visualization of myonuclei. In each case, lectin staining could be blocked by appropriate sugar inhibitors. Neuraminidase pretreatment of the cryostat sections altered the pattern of staining by all lectins except UEA I and Con A; staining with RCA I became stronger and that with WGA became less intense, while staining with PNA, SBA and DBA became stronger and more generalized, resembling that of RCA I. These effects of neuraminidase pretreatment are in conformity with the known structure of the oligosaccharide chains of membrane glycoproteins and specificities of the lectins involved.     

Lectin binding to neural crest cells. Changes of the cell surface during differentiation in vitro

To examine possible changes in cell surface carbohydrates, fluorescent lectins were applied at various times during differentiation of neural crest cells in vitro. The pattern and intensity of binding of several lectins changed as the crest cells developed into melanocytes and adrenergic cells. Considerable amounts of concanavalin A (Con A) and wheat germ agglutinin (WGA) bound to all unpigmented cells throughout the culture period. Melanocytes, however, bound much less of these lectins. Soy bean agglutinin (SBA), unlike Con A and WGA, only bound later in development to unpigmented cells at about the time when catecholamines were detected histochemically. Binding of SBA could be induced in younger cultures by pretreating the cells with neuraminidase. Melanocytes, however, did not bind detectable amounts of SBA even if treated with neuraminidase. The SBA-binding sites were often concentrated on cytoplasmic extensions and on contact points between neighboring cells, even when receptor mobility was restricted by prefixation of the cells or adsorption of lectin at 0 degrees C. All three lectins bound to cell processes resembling nerve fibers in particularly high amounts.     

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)     

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)     

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.     

Cell surface saccharide differences in drug-susceptible and drug-resistant strains of Trichomonas vaginalis.

Surface carbohydrates of drug-resistant and drug-susceptible strains of Trichomonas vaginalis were analysed using lectins. The presence of D-GalNAc, D-Gal and mannose-like residues was detected in T. vaginalis. Marked differences in exposed surface carbohydrates were documented, e.g. wheat germ agglutinin (WGA) selectively agglutinated the drug-susceptible strain whereas drug-resistant parasites reacted preferentially with concanavalin A (Con A). In drug-resistant, but not in drug-susceptible strains, trypsinization induced the appearance of soybean agglutinin. Binding studies using fluorescein-labelled WGA and Con A essentially confirmed the agglutination experiments. Both the intense cell agglutination and the fluorescent WGA-binding displayed by a drug-susceptible strain, were completely nullified by neuraminidase treatment, suggesting the presence of an exposed sialic acid moiety on the T. vaginalis surface.     

Effect of lectins on the transport of food ingredients in Caco-2 cell cultures

We investigated the effect of several lectins, such as soy bean lectin (SBA), concanavalin A (Con A), and wheat germ agglutinin (WGA), on the transport of some food ingredients (isoflavones, quercetin glycosides, carnosine/anserine) across Caco-2 cell monolayers. After incubation of food ingredients (0.03 approximately 2 mmol/L) in the presence or absence of lectins (1 approximately 180 microg/ml) on the apical side, aliquots were taken from the apical and basolateral solution, and were subjected to HPLC analysis. We also examined the effect of lectins on the permeability of the tight junction by measuring the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayer. Isoflavones, which was not transported to the basolateral solution without lectins, could be transported in the presence of lectins, whereas their aglycones were detected at the same levels with or without the lectin treatment. The transport of quercetin glycosides also increased in the presence of lectins, however, that of peptides was not affected by the lectins. Con A and WGA, but SBA, decreased the TER value, indicating that Con A and WGA increased the transport via paracellular pathway, whereas SBA did via a different pathway.     

Fluorophore-conjugated lectin labeling of the cell surface of isolated male and female gametes,central cells and synergids before and after fertilization in maize

Three fluorescein isothiocyanate (FITC)-conjugated lectins, Canavalia ensiformis agglutinin (Con A), Triticum vulgaris agglutinin (WGA) and Phaseolus vulgaris erythroagglutinin (PHA-E), were used as probes to localize sugar moieties of glycoconjugates on the cell surface of isolated maize sperm, egg, central, antipodal cells, synergids, and in vitro- and in vivo-fertilized zygotes. Fluorescence signals on the surface of the cells were due to specific binding. Calcium was necessary for WGA and PHA-E binding and enhanced Con A labeling. Differences in glycoconjugate composition of the membranes of gametes and other embryo sac component cells were found. FITC-Con A strongly labeled egg and central cells, but labeled sperm only weakly. FITC-WGA binding sites were detected on egg, but not sperm cells. Con A and WGA binding sites were equally distributed around egg and central cell protoplasts. FITC-PHA-E binding sites were not found on sperm and egg cells before fertilization. Binding sites of these lectins were located on synergids, especially on their filiform apparatus. Interestingly, WGA binding to egg cells was enhanced after fertilization, whereas PHA-E binding to egg cell membranes could only be detected after fertilization. These results suggest the occurrence of fertilization-induced changes in glycoconjugate composition of the maize egg cell membrane. An increase in the number of WGA and PHA-E binding sites was also observed on newly formed cell walls of cultured two-celled embryos derived from in vitro-produced zygotes.     

Trypanosoma (Nannomonas) congolense: analysis by fluorescein-conjugated plant lectins of surface saccharides of cloned variant antigen types differing in infectivity for mice

Surface saccharides of 4 cloned VATs (variant antigen types) of Trypanosoma (Nannomonas) congolense, AmNats (Amherst Nannomonas antigen types) 1.1, 1.2, 2.1, and 3.1, derived from 3 different stocks, were compared by fluorescein-conjugated, plant lectins using a quantitative fluorescence method. It was ascertained by the ID63 assay that the 4 AmNats differed in their infectivity for mice. The lectins employed for AmNats 1.1, 2.1, and 3.1 were concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), garden pea agglutinin (GPA), and gorse seed (Ulex europaeus) agglutinin (UEA). In view of the results obtained with these 3 AmNats, only Con A, WGA, and GPA were used with AmNat 1.2, which was isolated after the lectin analyses of the other cloned VATs were completed. On the basis of experimental results, we concluded that the amounts of saccharide residues binding the several lectins differed among the 4 AmNats. In each instance, the reaction specificity was controlled by inclusion of an appropriate sugar in the incubation mixture. Although the actual numbers of various specific lectin-binding sites differed among the AmNats 1.1, 2.1, and 3.1, all of them were found to have the following sugars on their surfaces: alpha-D-mannose, N-acetyl-D-glucosamine, D-galactose, alpha-D-glucose, and alpha-L-fucose. AmNat 1.2 treated with Con A, WGA, and GPA only had the first 2 sugars named above and alpha-D-glucose residues. The results of the ID63 assay indicated AmNats 1.1 and 2.1 to be significantly more infective for mice than AmNats 1.2 and 3.1. The lectin analysis revealed that the 2, more infective, cloned VATs incubated with Con A or WGA emitted significantly (approximately 39% to approximately 62%) more fluorescence than the less infective ones. Thus there were significantly more numerous Con A and WGA binding sites on the more infective AmNats. The situation was reversed with regard to GPA. Upon treatment with this lectin, fluorescence emitted by AmNats 1.1 and 2.1 was significantly (approximately 56% to approximately 81%) lower than that recorded for the less infective AmNats 1.2 and 3.1. In light of our results, infectivity of T. congolense cloned VATs was correlated with the presence of higher numbers of alpha-D-mannose and N-acetyl-D-glucosamine residues and of lower numbers of alpha-D-glucose residues on the surface of the bloodstream trypanosomes. There appeared to be no correlation between infectivity and the numbers of D-galactose and alpha-L-fucose residues present on these parasites.     

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.     

Insulin-mimetic actions of wheat germ agglutinin and concanavalin A on specific mRNA levels

Insulin has pleiotropic effects on sensitive cells, including the regulation of specific mRNA accumulation initiated by the binding of insulin to its plasma membrane receptor. Lectins, such as wheat germ agglutinin (WGA) and concanavalin A (Con A), are known to be insulin mimetic. It is thought that WGA and Con A interact with the insulin receptor or associated membrane glycoproteins which, when activated, lead to insulin-mimetic responses. We attempted to determine whether WGA and Con A could induce the accumulation of a specific messenger RNA (p33-mRNA). Insulin treatment of H4IIE (H4) hepatoma cells increased the concentration of p33-mRNA within 30 min after addition, with a maximum effect of 10- to 15-fold. WGA and Con A also exhibited time- and dose-dependent stimulatory effects on p33-mRNA accumulation with maximal effects of 30- to 40-fold. The effect of insulin was maximal by 1 h and plateaued thereafter, whereas lectins had maximal effects at 2 h after addition to cell cultures. Insulin, WGA, and Con A did not significantly alter the stability (half-life) of p33-mRNA. The addition of RNA synthesis inhibitors blocked the ability of insulin, WGA, and Con A to induce the amount of p33-mRNA. These data suggest that lectins, as well as insulin, induce the synthesis of p33-mRNA in acutely treated H4 hepatoma cells.     

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes

Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.     

Wheat germ agglutinin and concanavalin A inhibit the response of human fibroblasts to peptide growth factors by a post-receptor mechanism

The effect of plant lectins on amino acid uptake and DNA synthesis in cultured human skin fibroblasts stimulated by various peptide mitogens was studied. Wheat germ agglutinin (WGA), at a concentration of 5 micrograms/ml, which by itself had little effect on 3H-aminoisobutyric acid (AIB) uptake, markedly inhibited stimulation of 3H-AIB uptake by somatomedin-C, insulin, epidermal growth factor (EGF) and platelet-derived growth factor. This inhibition could not be overcome by increasing the concentration of peptide added. Neither WGA nor concanavalin A (Con A) significantly affected basal 3H-thymidine incorporation. However both lectins, at concentrations of 5-20 micrograms/ml, decreased EGF- and insulin-stimulated DNA synthesis while succinyl Con A, a divalent lectin derivative, did not. The inhibitory effects of lectins on mitogenic stimulation were reversed by alpha-methyl mannose (Con A) or N-acetylglucosamine (WGA), and were not due to a reduction in the binding of growth factors to their receptors. It is concluded that certain lectins noncompetitively inhibit the response of human fibroblasts to multiple peptide mitogens at the post-receptor level, possibly by interfering with lateral mobility and aggregation of mitogen-receptor complexes.     

Evaluation of fluorescently labeled lectins for noninvasive localization of extracellular polymeric substances in Sphingomonas biofilms

Three strains of Sphingomonas were grown as biofilms and tested for binding of five fluorescently labeled lectins (Con A-type IV-TRITC or -Cy5, Pha-E-TRITC, PNA-TRITC, UEA 1-TRITC, and WGA-Texas red). Only ConA and WGA were significantly bound by the biofilms. Binding of the five lectins to artificial biofilms made of the commercially available Sphingomonas extracellular polysaccharides was similar to binding to living biofilms. Staining of the living and artificial biofilms by ConA might be explained as binding of the lectin to the terminal mannosyl and terminal glucosyl residues in the polysaccharides secreted by Sphingomonas as well as to the terminal mannosyl residue in glycosphingolipids. Staining of the biofilms by WGA could only be explained as binding to the Sphingomonas glycosphingolipid membrane, binding to the cell wall, or nonspecific binding. Glycoconjugation of ConA and WGA with the target sugars glucose and N-acetylglucosamine, respectively, was used as a method for evaluation of the specificity of the lectins towards Sphingomonas biofilms and Sphingomonas polysaccharides. Our results show that the binding of lectins to biofilms does not necessarily prove the presence of specific target sugars in the extracellular polymeric substances (EPS) in biofilms. The lectins may bind to non-EPS targets or adhere nonspecifically to components of the biofilm matrix.     

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.