Comparative lectin-histochemistry on the pre-epithelial mucus layer in the distal colon of conventional and germ-free rats

The mucin composition of the rat distal colonic pre-epithelial mucus layer (PML) was studied by lectin histochemistry in conventional (CV), and germ-free (GF) rats to define effects exerted by the gut flora. No peanut agglutinin (PNA) binding was observed in the PML of GF rats, while the PML of their CV counterparts showed a considerable PNA linkage, indicating terminal Gal-beta1,3-GalNAc residues. Soybean agglutinin (SBA) and Helix pomatia agglutinin (HPA) stained the PML mucins in CV and in GF rats, indicating terminal GalNAc moieties. A quantitative difference in the Limax flavus agglutinin (LFA) binding capacity was found between CV and GF rats, indicating terminal sialic acid moieties: the staining intensity of bound LFA/ FiTC was higher in CV rats than in GF rats. No linkage of Datura stramonium agglutinin (DSA) and of wheat germ agglutinin (WGA) was found in the PML of GF rats, indicating the absence of terminal GlcNAc, while in CV rats, a clearly marked border was visible next to the luminal content as a "nipple edge" when stained with DSA or WGA. Canavalia ensiformis agglutinin (ConA), indicative for branched mannose, stained PML mucins and goblet cell mucins of GF rat distal colon. In CV rats, both locations were free of ConA binding sites. These results suggest degrading effects, exerted by the gut flora on the rat colonic pre-epithelial mucus layer.     

Anaplasma marginale, Eperythrozoon wenyoni: lectin reactions with bovine erythrocytes

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

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.     

Lectin receptors in the salivary glands of the rat during postnatal ontogenesis

Distribution of lectin-binding sites in rat submandibular and sublingual salivary glands during postnatal development has been investigated. Lectin preparations include con A, lentin lectin, castor beans agglutinin, peanut, soybean and Sophora japonica agglutinins, wheat germ agglutinin and lectin from the bark of Laburnum anagyroides. The direct and indirect peroxidase techniques are used. According to the similarities of histochemical patterns, all lectins are divided into four groups. Besides the general patterns of lectin binding sites, some details are noted. Lectins of peanut and Sophora japonica possess an extremely high affinity to mast cells, con A, lens lectin, castor beans and wheat germ agglutinins--to serous demilunes cells. Laburnum lectin--to salivary ducts epithelia in adult rat salivary glands. Lentin lectin, con A and Laburnum lectin preferentially stain cells with specific granularity in granular ducts at early stages of postnatal development. Considering the character of staining, we propose for further histochemical investigations of the salivary glands lentin lectin, peanut agglutinin, wheat germ agglutinin and Laburum anagyroides lectin.     

Binding of Shigella to rat and human intestinal mucin

Invasion of epithelial cells by Shigella is an early step in their pathogenesis. Adherence is generally presumed to be a prerequisite for invasion. This study examined the possibility of intestinal mucins serving as initial binding sites for clinical isolates of S. boydii and S. sonnei. The interactions of Shigella with rat and human small intestinal and colonic mucin were investigated. In solid phase binding assays, [³⁵S] labelled Shigella did not show any preferential binding to rat/human small intestinal mucin or to rat colonic mucin. On the other hand, Shigella bound specifically to human colonic mucin in a concentration-dependent manner. This specific binding to human colonic mucin was not by weak hydrophobic interactions and could not be attributed to the presence of contaminating glycolipids in the mucin preparation. The human colonic mucin receptor was sensitive to periodate treatment suggesting the involvement of the carbohydrate portion of the mucin. Reduction and alkylation of mucin enhanced adherence probably by exposing buried binding sites. The monosaccharides present in mucins were ineffective as hapten inhibitors as was the lectin wheat germ agglutinin suggesting that the mucin receptor is a more complex one. This study identifies, for the first time, the presence of a specific Shigella-binding site on the carbohydrate portion of human colonic mucin, which is not present in rat colonic mucin or in rat/human small intestinal mucin.     

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line. Analysis using double labeling with lectins

Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheatgerm agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(beta 1-3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosaccharide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.     

Alterations in glomerular lectin binding sites of human kidney as detected by fluorescence microscopy

Summary Different lectins were used to study frozen sections of kidney samples showing alterations in routine immunofluorescence studies.Arachis hypogaea agglutinin (peanut lectin, PNA), lacking binding sites in normal glomeruli, bound to the glomeruli in two of the five samples studied, giving a granular fluorescence pattern. Concomitantly with the appearance of PNA-binding, binding sites for wheat germ agglutinin (WGA) appeared to be lost at glomeruli. Furthermore, changes in the expression of glomerular binding sites forWistaria floribunda (WFA),Helix pomatia (HPA) andDolichos biflorus (DBA) agglutinins could be seen in the kidneys studied, whereas the binding sites forUlex europaeus agglutin (UEA I) in vascular endothelia seemed to be unaltered.The results show that kidney specimens presenting changes in routine immunofluorescence studies may also present altered binding for certain lectins. On this basis we propose that certain lectins may aid in characterizing these changes and are thus of potential use in studying diseased kidneys.     

Immunohistochemical localization of articular cartilage proteoglycan and link protein in situ using monoclonal antibodies and lectin-binding methods

Lectins have specificity for certain carbohydrate structures in macromolecules. Lectins are, therefore, useful histochemical tools for demonstrating the composition and localization of components of connective tissue matrices, such as articular cartilage. In order to assess the significance of observed lectin-binding patterns, experiments were performed in which monoclonal antibodies against chondroitin sulphate- and keratan sulphate-containing proteolgycans and link proteins were applied to sections of bovine articular cartilage after enzymatic digestion with chondroitinase ABC and keratanase. The following conclusions were made: (1) Binding of peanut agglutinin (PNA) in the interterritorial matrix predominantly indicates the presence of keratan sulphate, but may also detectO-linked oligosaccharides of proteoglycans. (2) In normal cartilage wheat germ agglutinin (WGA) binds nearly exclusively to keratan sulphate. In cartilage degraded with chondroitinase ABC and keratanase this lectin may also detect carbohydrates in link protein due to enhanced accessibility. Binding of WGA toO-linked oligosaccharides may eventually occur. (3) In enzymatically digested cartilage matrix, staining with soybean agglutinin (SBA) may be due to link protein, but not to chondroitin sulphate, because specific breakdown of the glycosaminoglycan chain is required for binding of SBA. (4)Ulex europaeus agglutinin I (UEA I) binding sites are only detectable in digested cartilage matrix.     

Properties of succinylated wheat-germ agglutinin.

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a pI of 4.0 +/- 0.2 while the native lectin is basic, pI of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 microgram/ml with both lectins and the concentrations of N-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins. The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins. The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.     

Binding site of the rat liver specific monoclonal antibody

A rat liver-specific antigen (RLSA) lost its binding ability to the corresponding monoclonal antibody after treatment with N-glycanase or sialidase, which suggested that the specific binding site might be in a portion of the sugar chain containing sialic acid. The specific antigen reacted with wheat germ agglutinin, lentil lectin, erythroagglutinating phytohemagglutinin and Ricinus communis agglutinin, but not with concanavalin A or peanut agglutinin. These results suggest that the specific antigen has asparagine-linked complex-type sugar chains which might be the binding sites of the monoclonal antibody.     

Trypanosoma brucei rhodesiense Bioodstream Forms: Surface Ricin-Binding Glycoproteins are Localized Exclusively in the Flagellar Pocket and the Flagellar Adhesion Zone

Specific binding of fluoresceinated succinyl-concanavalin A, wheat germ agglutinin, and ricin to untreated and trypsinized bloodstream forms of Trypanosoma brucei rhodesiense was quantitated by flow cytofluorimetry, and sites of lectin binding were identified by fluorescence microscopy. All three lectins only bound to the flagellar pocket of untreated parasites. When parasites were trypsinized to remove the variant surface glycoprotein coat, new lectin binding sites were exposed, and specific binding of all three lectins increased significantly. New specific binding sites for succinyl-concanavalin A and wheat germ agglutinin were present along both the free flagellum and flagellar adhesion zone and were uniformly distributed on the parasite surface. However, ricin did not bind uniformly on the surface and did not stain the free flagellum of trypsinized cells. Ricin only bound to the flagellar adhesion zone of trypsinized cells and of cells that had been treated with formaldehyde prior to staining. Electron microscopy of cells exposed to ricin-colloidal gold complexes revealed that that ricin binding was restricted to the anterior membrane of the flagellar pocket of untrypsinized cells and to this portion of the flagellar pocket and the cell body membrane in the flagellar adhesion zone of trypsinized cells. Evidence that these membranes constitute a functionally important membrane microdomain is reviewed.     

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.     

Interaction with lectin of kappa opioid binding sites solubilized from human placenta

Kappa opioid binding sites from human placenta, prelabeled with 3H-etorphine and solubilized, were retained on wheat germ agglutinin (WGA) agarose and specifically eluted with N-acetylglucosamine. No significant retention was found with other immobilized lectins, including Concanavalin A (Con A), soybean seed lectin (SBA), Pisum sativum lectin (PsA), Lens culinaris Medik. lectin (LcA), and Lathyrus tingitanus lectin(LtA). About 23% of applied kappa sites were specifically eluted from WGA agarose, less than half of the proportion of rat brain opioid binding sites eluted from the same lectin (55%). Receptors from placental extracts were compared with those from other tissues enriched in either kappa or mu sites. The proportion of applied kappa sites from guinea pig cerebellum eluted specifically from WGA agarose was 36%, whereas elution of binding sites from rat thalamus and rabbit cerebellum (enriched in mu sites) was at a level of 55%. This difference in the level of retention on and elution from WGA may reflect differences in the sugar composition of the glycoproteins of the two types of receptors. Succinylation of WGA abolished its ability to retain opioid binding sites, consistent with involvement of sialic acid. However, currently available evidence suggests that differences in retention on WGA between kappa and mu sites may be due to differences in either sialic acid or N-acetylglucosamine content or both.     

Effect of thyroid hormones on the histotopography of lectin receptors in the rat salivary gland

Using lectin-peroxidase technique, the influence of hypo- and hyperthyroidism on histotopography of glycoconjugates has been investigated in rat submandibular gland. The following lectins were used: peanut agglutinin (PNA), wheat germ agglutinin (WGA), Laburnum anagyroides lectin (LAL) and concanavalin A (con A). It has been demonstrated that hyperthyroidism is accompanied by the loss of con A, WGA and LAL receptor sites. Hypothyrodism enhanced con A binding to granular duct cells with a parallel reduction in WGA and LAL binding to these or other duct cells. Hypothyroidism as well as hyperthyroidism markedly enhanced PNA binding to duct epitheliocytes with redistribution of these lectin binding sites from the luminal surface of salivary ducts into the cytoplasm of duct cells. Possible interpretations of the observed phenomena are discussed.     

Lectin binding sites related with rat ascites hepatoma cell adhesion

In order to elucidate the correlation between cell surface lectin binding sites and the degree of cell adhesiveness, quantitative lectin binding assays were performed using three types of rat ascites hepatoma cell lines (free cell, mixed cell, and island-forming cell types). The lectin binding site patterns showed no remarkable differences among the intact tumor cell lines, but treatment of the cells with L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-trypsin or neuraminidase induced remarkable differences in the modulation of the number of lectin binding sites. TPCK-trypsin treatment caused a marked decrease in the number of peanut agglutinin binding sites on the island-forming and mixed cell types, concomitant with disaggregation of the cells, showing that trypsin sensitive binding sites are involved in the cell-cell adhesion. Neuraminidase treatment caused a decrease in wheat germ agglutinin binding sites and an increase in castor bean agglutinin binding sites, and these effects were greater for the free cell type. These results indicated that alpha-sialyl-beta-D-galactosyl residues are more abundant on the cell surface of the free cell type than the other cell types. Therefore, it was suggested that electrostatic repulsion due to negative charges of the cell surface sialic acid contributes to the low cell adhesiveness of the free cell type.     

Interaction of lectins with human platelets. Effects on platelet stimulation by thrombin and ristocetin.

Wheat germ agglutinin induced aggregation and secretion of fresh platelets. Aggregation, but not secretion of serotonin by platelets in plasma, by the lectin was inhibited by 5 mM EDTA. Further, the lectin-induced stimulation of fresh platelets was blocked by prostaglandin E1. Thus, this lectin stimulates platelets by a mechanism which closely mimics thrombin activation and is independent of intercellular crosslinking. Lentil lectin did not stimulate platelets. Each platelet contained about 6 . 10(-5) binding sites for the lectins with an apparent dissociation constant of 3.0 . 10(-7) M. Wheat germ agglutinin, which binds mainly to glycoprotein I (Mr 150 000), increased the subsequent binding of thrombin to fixed platelets while lentil lectin was without effect. It appears that thrombin and wheat germ agglutinin bind to independent but interacting sites. Wheat germ agglutinin, but neither thrombin nor lentil lectin, inhibited the agglutination of platelets by ristocetin. Further, rat platelets were not aggregated by either ristocetin or wheat germ agglutinin. It appears that the interaction sites of ristocetin and wheat germ agglutinin on platelets are overlapping.     

Colonic mucins in ulcerative colitis: evidence for loss of sulfation

Colonic tissue obtained at surgery from control individuals and patients with ulcerative colitis was used to isolate mucins and to prepare mucin glycopolypeptides by pronase digestion. These were compared with mucins labelled with [³⁵S] sulfate and [³H]-glucosamine after organ culture tissue samples from the same patients. A significant loss of mucin sulfation was detected in the colitis patients by both metabolic labelling and chemical analysis of the glycopolypeptides. A change in the size distribution of purified mucin oligosaccharides fractionated on BioGel P6 after release by -elimination was seen in both radiolabelled and non-labelled colitis mucins compared with controls. Amino acid analysis of the glycopolypeptides showed a close similarity to the expected ratio of serine:threonine:proline for MUC2 and did not vary between control and colitis groups. Analysis of the mucins confirmed >90% purity in the labelling experiments, characteristic behaviour on density gradient centrifugation and agarose gel electrophoresis in control and ulcerative colitis groups and differences in sulfation and turnover at various sites in the normal colon.Abbreviations WGA wheat germ agglutinin - UC ulcerative colitis - HRP horseradish peroxidase     

Lectin-receptor interactions in liposomes.

The major sialoglycoprotein of mammalian erythrocytes has been incorporated into phosphatidylcholine membranes to generate a model system, glycoprotein-liposomes. Electron microscopic examination revealed these structures to be vesicles, approximately 300 A in diameter. An aqueous compartment inside the glycoprotein-liposomes has been identified by trapped volume studies with [14C]sucrose. These glycoprotein-liposomes were found to interact with the lectins, wheat germ agglutinin, and phytohemagglutinin, to form aggregates of mainly unfused vesicles. The aggregation process has been studied by electron microscopy, 90 degrees light scattering, and differential ultracentrifugation analysis. Hapten inhibitors of the lectins were found to inhibit the lectin-induced aggregation of the glycoprotein-liposomes. Binding of 125I-labeled wheat germ agglutinin to glycoprotein-liposomes was studied by differential ultracentrifugation. Hapten inhibitors of wheat germ agglutinin were also found to inhbit the binding of 125I-labled wheat germ agglutinin to the glycoprotein-liposomes. The characteristics of the lectin interactions with glycoprotein-liposomes appeared to be phenomenologically similar to lectin-cell interactions.     

Trypanosoma brucei rhodesiense bloodstream forms: surface ricin-binding glycoproteins are localized exclusively in the flagellar pocket and the flagellar adhesion zone

Specific binding of fluoresceinated succinyl-concanavalin A, wheat germ agglutinin, and ricin to untreated and trypsinized bloodstream forms of Trypanosoma brucei rhodesiense was quantitated by flow cytofluorimetry, and sites of lectin binding were identified by fluorescence microscopy. All three lectins only bound to the flagellar pocket of untreated parasites. When parasites were trypsinized to remove the variant surface glycoprotein coat, new lectin binding sites were exposed, and specific binding of all three lectins increased significantly. New specific binding sites for succinyl-concanavalin A and wheat germ agglutinin were present along both the free flagellum and flagellar adhesion zone and were uniformly distributed on the parasite surface. However, ricin did not bind uniformly on the surface and did not stain the free flagellum of trypsinized cells. Ricin only bound to the flagellar adhesion zone of trypsinized cells and of cells that had been treated with formaldehyde prior to staining. Electron microscopy of cells exposed to ricin-colloidal gold complexes revealed that that ricin binding was restricted to the anterior membrane of the flagellar pocket of untrypsinized cells and to this portion of the flagellar pocket and the cell body membrane in the flagellar adhesion zone of trypsinized cells. Evidence that these membranes constitute a functionally important membrane microdomain is reviewed.     

Lectin binding to the porcine and human ileal receptor of intrinsic factor-cobalamin

The purified porcine recpptor for the intrinsic factor-cobalamin complex bound to concanavalin A, lentil lectin and wheat germ lectin covalently coupled to Sepharose and was eluted with the corresponding soluble sugars. In contrast, human intrinsic factor bound efficiently to concanavalin A, to some extent to lentil lectin, but only slightly to wheat germ agglutinin. The binding of IF-Cbl to the receptor was inhibited when the receptor was pre-incubated with soluble wheat germ aglutinin, with an inhibition constant estimated to be 1.9 mol/l. After transfer of the purified receptor from SDS-PAGE to Immobilon, ligand blotting of the purified receptor with iodinated lectin showed that concanavalin A and lentil lectin bound to three (75, 56 and 43 kDa) components but that wheat germ agglutinin bound only to the 75 kDa component. These results showed that the subunit of the receptor could bind to wheat germ agglutinin, resulting in an inhibition of its binding with intrinsic factor. Both binding sites of intrinsic factor and of wheat germ agglutinin could be located near to each other.