Lectin binding to cell surfaces: comparisons between normal and migrating corneal epithelium

Comparisons were made between cell surfaces of normal and migrating corneal epithelium of the rat by localizing and/or quantifying concanavalin A (Con A) and wheat germ agglutinin (WGA) binding. Our results indicate that apical cell surfaces of the leading edge of a migrating sheet of epithelium differ from those of normal epithelium and that the various cell layers within the stratified normal epithelium have different lectin-binding characteristics. Three methods of monitoring lectin binding to cell surfaces were employed. Based on ferritin-conjugated Con A, ferritin-conjugated WGA, and [3H]Con A binding, apical cell membranes of migrating epithelia bind more Con A and WGA than do apical membranes of superficial cells of normal stratified epithelia. With both fluorescein isothiocyanate (FITC)-Con A and -WGA, membranes of all the cells of the leading edge of the migrating sheet fluoresce intensely. FITC-Con A binding of normal stratified epithelium is relatively uniform through all cell layers with no discernible staining of the apical membrane of superficial cells. With FITC-WGA, however, fluorescence is present only on basal cell layers but not on superficial cells. These data demonstrate that apical cell surface sugars on a sheet of epithelium migrating to cover a wound differ from the apical cell surface sugars of normal epithelium. As indicated by FITC-WGA binding, cells of the migrating sheet have cell surface characteristics similar to basal cells of normal epithelia. Perhaps, upon wounding, the leading edge of the migrating sheet is derived from the basal cell population of the normal stratified epithelium, or perhaps there is an alteration in cell surface glycoproteins as the cells become migratory.     

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.     

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.     

Lectin receptor sites on rat liver cell nuclear membranes.

The presence and localization of lectin receptor sites on rat liver cell nuclear and other endomembranes was studied by light and electron microscopy using fluorescein and ferritin-coupled lectin conjugates. Isolated nuclei labelled with fluorescein-conjugated Concanavalin A (Con A) or wheat germ agglutinin (WGA) often showed membrane staining, which sometimes was especially bright on small stretches of the nuclear surface. Unlabelled nuclei and nuclei with a complete ring fluorescence were also seen. The nuclear fluorescence corresponded in intensity to that seen on the surface of isolated rat liver cells. Con A-ferritin particles were seldom detected on the cytoplasmic surface of the intact nuclear envelope. However, at places where the 2 leaflets of the envelope were widely separated or where the outer nuclear membrane was partly torn away, heavy labelling was seen on the cisternal surface of both the inner and outer nuclear membranes. Labelling with Con A-ferritin was also found on the cisternal side of rough endoplasmic reticulum present in the specimens. No labelling was seen on the cytoplasmic surface of mitochondrial outer membrane. The results demonstrate the presence of binding sites for Con A and WGA in nuclei and an asymmetric localization of these sites on the cisternal side of ribosome-carrying endomembranes in rat liver cells.     

Interactions of lectins with CHO cell surface membranes. II. Differential effects of local anesthetics on endocytosis of CON A and WGA binding sites

Using fibroblastic CHO cells, we have examined (1) the internalization and redistribution of surface binding sites for the lectins Concanavalin A and wheat germ agglutinin and (2) the sensitivity of these processes to putative inhibitors of cytoskeletal activity. Following initial exposure to fluorescein conjugated Con A (CAF) or WGA (WGAF) at 4° C, kinetic analysis of internalization and intracellular aggregation of lectin at 37°C indicated more rapid aggregate formation in the case of WGA than in the case of Con A. Treatment with tertiary amine local anesthetics (tetracaine, dibucaine, and xylocaine) or with a lysosomatrophic amine, m-dansyl cadaverine, blocked internalization of Con A but not of WGA. Similar differential effects on redistribution of Con A and WGA were not however observed with the antimicrotubule agents colchicine and nocodazole. Simultaneous treatment of cells with WGAF and with rhodamine labeled Con A (CAR) resulted in the accumulation of both labels in a central perinuclear aggregate; a similar experiment in the presence of local anesthetic resulted in a diffuse peripheral distribution of CAR and a central aggregate of WGAF. These results suggest (1) CHO cells possess at least two distinct pathways for lectin internalization and redistribution, which can be discriminated in terms of drug sensitivity; (2) CHO cells can sort out and independently internalize different populations and lectin binding sites; and (3) different pathways may be a manifestation of biochemically distinct linkages between cytoskeletal elements and various groups of surface glycoproteins. Present findings concur with our previous results concerning the mutual independence of the surface binding sites for Con A and WGA (Emerson and Juliano, 1982).     

Differences in lectin binding in squamous metaplasia induced by benzo(a)pyrene and vitamin A deficiency in hamster tracheal explants

Summary Epidermoid metaplasia in the hamster trachea can be produced by treatment with benzo(a)pyrene (BP) or vitamin A deficiency. To elucidate distinguishing features of the two types of lesions, lectin binding to tissue sections of tracheal explants exhibiting metaplastic lesions was assessed. In squamous metaplasia induced by vitamin A deficiency, Dolichos biflorus agglutinin (DBA), wheat germ agglutinin (WGA), and peanut agglutinin (PNA) showed faint (+) to moderate (++) binding in both basal and suprabasal cells; Concanavalin A (Con A) showed moderate binding (++) to suprabasal cells and no binding in basal cells. In the BP-induced lesions, PNA and WGA bound intensely (++++, +++, respectively) in basal cells and faintly (+) to moderately (++) in suprabasal cells. The staining seemed to be predominant at the periphery of the cells. Further, the intensity of PNA and WGA staining increased significantly after the neuraminidase treatment. DBA and Con A showed faint (+) to moderate (++) binding in the BP-induced metaplasia. The results show that in BP-induced metaplasia, cells in the basal region show preferential binding of PNA and WGA. This research was supported by grant RO1-HL32308 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

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)     

Cross-linking of membrane glycoconjugates is not a sufficient condition for neural induction by concanavalin A

Tetravalent native concanavalin A (Con A) has a neural inducing effect on amphibian presumptive ectoderm. The divalent dimeric form of this lectin, succinylated Con A (Succ-Con A), is devoid of neuralizing action on this target tissue in Pleurodeles waltlii. These results suggested that cross-linking of Con A receptors on the cell membrane (which is not provoked by the divalent lectin) might be required for neural induction. To test this possibility, Succ-Con A binding sites were experimentally cross-linked after binding of Succ-Con A to the target cell surface, using anti-Con A antibodies. The combination of these two agents mimics the cross-linking of Con A. The results showed that cross-linking alone, either by treatment with Succ-Con A and anti-Con A antibodies, or with the lectins WGA and PHA, which also cross-link cell surface binding sites, was not able to induce neuralization. This suggested that the inductive action of Con A cannot be explained in terms of receptor cross-linking.     

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 binding to injured corneal endothelium mimics patterns observed during development

Summary 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. The 16-day fetal tissue is mitotically active, does not exhibit a well defined monolayer, and demonstrates weak fluorescence binding for WGA, Con A and RCA. Conversely, SBA binding is readily detected on many cell surfaces. By 19 days in utero, the endothelial monolayers becomes organized and cell proliferation greatly diminishes. WGA, Con A and RCA now exhibit binding similar to that seen in the adult tissue. SBA binding is not detected at this time. Thus, changes in lectin binding during wound repair of the adult rat corneal endothelium mimic changes in lectin binding seen during the development of the tissue.Supported by grant EY-06435 from The National Institutes of Health     

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.     

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.     

Lectin-bearing liposomes: differential binding to normal and to transformed mouse fibroblasts

The binding of covalent conjugates of concanavalin A (Con A) or wheat germ agglutinin (WGA) and liposomes (lectin-liposomes) to the surface of normal and transformed mouse fibroblasts was studied. Quantitation of the binding was performed by means of microfluorometry and radioactive lipid label counting using both sparse and dense cell cultures. It was found that 2.5-3 times more lectin-conjugated liposomes are bound to L or SV3T3 cells than to the mouse embryo fibroblasts and 3T3 cells in a broad concentration range. The binding of Con A- and WGA-liposomes was inhibited up to 70% in the presence of the corresponding carbohydrate inhibitors. A decreased binding of lectin-liposomes to cells was also observed when cells were pretreated with the free lectin. Trypsinization of the cells resulted in an increase in the Con A-liposomes binding to normal fibroblasts. When free fluorescent Con A or WGA was used in binding studies no profound differences in the binding of lectin to normal or transformed cells were detected. The relation of the lectin-liposome/cell to cell/cell interactions is discussed.     

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)     

Epidermal growth factor receptors on PC12 cells: Alteration of binding properties by lectins

The PC12 cell line displays cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). It has been previously shown that the lectin wheat germ agglutinin (WGA) alters the properties of NGF receptors on these cells. We now report that preincubations with either WGA or concanavalin A (Con A) decrease the binding of ¹²⁵I-EGF to PC12 cells by greater than 50%. The inhibition of binding occurred at 37°C and 4°C and could be blocked or reversed by the addition of sugars which bind specifically to WGA or Con A. Scatchard analysis revealed that these lectins decreased binding primarily by lowering the affinity of the receptor and to a lesser extent by decreasing receptor number. Succinylalion of Con A (sCon A) produced a derivative that was less effective than the native lectin in decreasing EGF binding; however, addition of an antibody against Con A restored the ability of sCon A to decrease binding. Similar to results obtained with ¹²⁵I-NGF binding, WGA but not Con A was found to increase, by scveralfold; the proportion of ¹²⁵I-EGF binding that is resistant to solubilization by Triton X-100 detergent. A potential association of the EGF receptor with cytoskeletal elements is discussed which could account for such results.     

Electron microscope study of the binding of Con A-gold to superficial and inner ectoderm layers ofXenopus laevis and its relation to the neural-inducing activity of this lectin

Summary Isolated competent amphibian ectoderm differentiates into neural (archencephalic) structures when treated with the plant lectin concanavalin A (Con A). While the inner ectoderm layer ofXenopus laevis forms brain structures after incubation with Con A, the outer ectoderm layer differentiates into ciliated epidermis only. This difference can be correlated with the pattern of Con A bound to the plasma membrane. With gold-labelled Con A it could be shown by transmission electron microscopy (TEM) that the outer ectoderm binds substantially less lectin than the inner layer. Furthermore we observed characteristic differences at the apical and basal surfaces of the cells of the same layer, i.e. on the apical cell surface of the superficial layer almost no Con A-gold could be found. In contrast, we observed a lot of gold particles on the basal cell side of the superficial layer. However, the number on both surfaces (apical and basal side of the cell) of the inner ectoderm layer was essentially higher, which could explain its biological reaction to the Con A stimulus and the differentiation into neural structures. The data presented in this paper indicate that early and late gastrula ectoderm bind similar amounts of Con A and support the view that the decrease in competence is not correlated with a loss of receptors for inducing factors. Furthermore, we describe the binding and the internalization of Con A via receptor-mediated endocytosis and the further fate of the Con A-gold-receptor complex inside the target cell.     

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.     

Distribution of concanavalin A binding sites on the surface of dissociated rat submandibular gland acinar cells.

The submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation of divalent cations and mechanical force. A suspension of single cells was obtained in low yield by centrifugation in a Ficoll-containing medium. Immediately after dissociation and after a culture period of 16-18 hr the dissociated cells were tested for agglutinability by concanavalin A (Con A). Using ferritin (tfer)-conjugated Con A the lectin binding by the isolated acinar cells was also studied. The dissociated cells were agglutinated by low concentrations of Con A and bound Fer-Con A molecules on their entire surface without any indication of polarization of the cell membrane. There was a considerable cell to cell variation in the amount of Fer-Con A binding which was, in general, sparse and patchy. The contact surfaces between agglutinated cells revealed a dense binding of Fer-Con A molecules irrespective of the types of cells participating in the agglutination reaction. Cells cultured for 16-18 hr were no longer agglutinated by Con A. As compared to the freshly dissociated cells the cultured acinar cells revealed a more uniform and denser binding of Fer-Con A molecules. Furthermore, there were more lectin molecules bound to the cell surface corresponding to the basal part of the cell, where the nucleus and most of the rough surface endoplasmic reticulum were located, than to the apical cell surface. It is suggested that the higher density of lectin-binding sites on the cell surface in the vicinity of the cisternae of the rough endoplasmic reticulum indicates insertion sites of newly synthesized membrane glycoproteins.     

Lectin binding by eosinophils

Lectins which identify carbohydrates and glycoproteins have been used to characterize specific components of the surface of guinea pig peritoneal exudate eosinophils. Agglutination of eosinophils purified by discontinuous metrizamide gradients was scored microscopically. Wheat germ agglutinin (WGA) was most effective (0.05 micrograms/ml). However, higher concentrations of soybean lectin and concanavalin A (Con A) were also effective. No differences in lectin binding were noted between eosinophils harvested from uninfected animals, Trichinella spiralis-infected animals, or animals receiving weekly intraperitoneal injections of polymyxin B. Neuraminidase pretreatment to remove surface sialic acid reduced agglutination by WGA. Eosinophils did not adhere to WGA-coated Sepharose beads; however, they did adhere to Con A-coated beads. Pretreatment with neuraminidase did not affect the adherence of eosinophils to plastic surfaces, suggesting that sialic acid does not play an important role in adherence. Formation of lectin-inhibitor complexes within the incubation mixture complicated interpretation of studies of binding to plastic surfaces. These studies demonstrate that lectin binding sites are present on the surface of eosinophils. Lectin-type binding may be important in interactions between eosinophils and noningestible parasites.     

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.