Comparison of the carbohydrate-binding specificities of seven N-acetyl-D-galactosamine-recognizing lectins

Seven plant lectins, Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia agglutinin (GSA, isolectin A4), Helix pomatia agglutinin (HPA), soybean (Glycine max) agglutinin (SBA), Salvia sclarea agglutinin (SSA), Vicia villosa agglutinin (VVA, isolectin B4) and Wistaria floribunda agglutinin (WFA), known to be specific for N-acetyl-D-galactosamine-(GalNAc) bearing glycoconjugates, have been compared by the binding of their radiolabelled derivatives, to eight well-characterized synthetic oligosaccharides immobilized via a spacer on an inert silica matrix (Synsorb). The eight oligosaccharides included the Forssman, the blood group A and the T antigens, as well as alpha GalNAc coupled directly to the support (Tn antigen) and also structures with GalNAc linked alpha or beta to positions 3 or 4 of an unsubstituted Gal. The binding studies clearly distinguished the lectins into alpha GalNAc-specific agglutinins like DBA, GSA and SSA, and lectins which recognize alpha- as well as beta-linked GalNAc residues like HPA, VVA, WFA and SBA. HPA was the only lectin which bound to the beta Gal1----3 alpha GalNAc-Synsorb adsorbent (T antigen) indicating that it also recognizes internal GalNAc residues. Among the alpha GalNAc-specific lectins, DBA strongly recognized blood group A structures while GSA displayed weaker recognition, and SSA bound only slightly to this affinity matrix. In addition, DBA and SSA were able to distinguish between GalNAc linked alpha 1----3 and GalNAc linked alpha 1----4, to the support, the latter being a much weaker ligand. These results were corroborated by the binding of the lectins to biological substrates as determined by their hemagglutination titers with native and enzyme-treated red blood cells carrying known GalNAc determinants, e.g. blood group A, and the Cad and Tn antigens. For SSA, the binding to the alpha GalNAc matrix was inhibited by a number of glycopeptides and glycoproteins confirming the strong preference of this lectin for alpha GalNAc-Ser/Thr-bearing glycoproteins.     

Trypanosoma rhodesiense bloodstream trypomastigote and culture procyclic cell surface carbohydrates

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

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

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

Lectins facilitate calcium-induced fusion of phospholipid vesicles containing glycosphingolipids

Ca2+-induced fusion of phospholipid vesicles containing globoside (GL-4) or disialoganglioside (GDla) is several-fold slower than the fusion of the pure phospholipid vesicles. Lectins specific for these glycosphingolipids, soybean agglutinin and wheat germ agglutinin, respectively, enhance the rate of fusion when added to the vesicle suspension before the introduction of Ca2+. The enhancement depends on the lectin concentration and the time of preincubation with the lectin. We propose that lectins facilitate membrane fusion by inducing intermembrane contact, which is the first step in the overall process of membrane fusion, or by laterally phase separating the inhibitory glycolipids.     

Alteration of cell surface carbohydrates associated with ordered and disordered proliferation of oral epithelia: a lectin histochemical study in oral leukoplakias, papillomas and carcinomas

Cell surface carbohydrates in healthy oral mucosa (n = 15), leukoplakias without (n = 48) and with (n = 62) dysplasia, oral papillomas (n = 6) and squamous cell carcinomas (SCCs) (n = 40) were examined using the lectins peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEA I), soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), and Griffonia simplicifolia agglutinin I (GS I-B4). Binding of these lectins in formalin-fixed, paraffin-embedded tissues was demonstrated using either the peroxidase-anti-peroxidase (PAP) method or the avidin-biotin method. Healthy oral epithelia revealed binding sites for these lectins mostly in the suprabasal keratinocytes with occasional PNA binding also in their basal cells. Unlike healthy mucosa, a number of leukoplakias without and with dysplasia revealed receptor sites for UEA I also in their basal layer. Only those keratinocytes undergoing squamoidal differentiation exhibited SBA binding. Staining patterns of UEA I and SBA did not vary significantly between either leukoplakias without and with dysplasia or papillomas and SCCs. Conversely, a reduction or lack of binding sites for PNA (Gal beta 1-3GalNAc), HPA (D-GalNAc alpha) and GS I-B4 (alpha D-Gal) was observed more frequently in leukoplakias with dysplasia and SCCs contrasting their counterparts lacking epithelial dysplasia. Cell surface glycosyl residues play an important role in the regulation of cell proliferation and epithelial growth. Aberrant glycosylation in oral dysplastic leukoplakias and carcinomas leading to the lack of the relevant terminal sugar residues from their cell surface carbohydrates is probably a major reason for the hyper-/disordered proliferation.     

Alteration of cell surface carbohydrates associated with ordered and disordered proliferation of oral epithelia: a lectin histochemical study in oral leukoplakias, papillomas and carcinomas

Abstract. Cell surface carbohydrates in healthy oral mucosa (n = 15), leukoplakias without ( n = 48) and with (n = 62) dysplasia, oral papillomas (n = 6) and squamous cell carcinomas (SCCs) (n – 40) were examined using the lectins peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEA I), soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), and Griffonia simplicifolia agglutinin I (GS I-B₄). Binding of these lectins in formalin-fixed, paraffin-embedded tissues was demonstrated using either the peroxidase-anti-peroxidase (PAP) method or the avidin-biotin method. Healthy oral epithelia revealed binding sites for these lectins mostly in the suprabasal keratinocytes with occasional PNA binding also in their basal cells. Unlike healthy mucosa, a number of leukoplakias without and with dysplasia revealed receptor sites for UEA I also in their basal layer. Only those keratinocytes undergoing squamoidal differentiation exhibited SBA binding. Staining patterns of UEA I and SBA did not vary significantly between either leukoplakias without and with dysplasia or papillomas and SCCs. Conversely, a reduction or lack of binding sites for PNA (Galβl-3GalNAc), HPA (D-GalNAcα) and GS I-B₄ (αD-Gal) was observed more frequently in leukoplakias with dysplasia and SCCs contrasting their counterparts lacking epithelial dysplasia.
Cell surface glycosyl residues play an important role in the regulation of cell proliferation and epithelial growth. Aberrant glycosylation in oral dysplastic leukoplakias and carcinomas leading to the lack of the relevant terminal sugar residues from their cell surface carbohydrates is probably a major reason for the hyper-/ disordered proliferation.     

The glycoprotein-bound large carbohydrates from embryonal carcinoma cells carry receptors for several lectins recognizing N-acetylgalactosamine and galactose

When various lectins were mixed with radioactively labeled embryoglycan (polylactosamine-type glycoprotein-bound carbohydrates from early embryonic cells) isolated from F9 embryonal carcinoma cells and the resulting complex was precipitated with ammonium sulfate, the glycan was found to react with the following lectins: Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), Sophora japonica agglutinin (SJA), and Ricinus communis agglutinin-1 (RCA-1). Furthermore, affinity chromatography on lectin-agarose revealed that receptors for Griffonia simplicifolia agglutinin-I (GS-I) were also carried by the glycan. Together with the previous finding that the glycan carries receptors for Dolichos biflorus agglutinin (DBA) and peanut agglutinin (PNA), the present result established that the glycan has receptors for a variety of lectins recognizing N-acetylgalactosamine and/or galactose in teratocarcinoma cells. Intact molecules carrying GS-1 receptors and SJA receptors were isolated from F9 cells and teratocarcinoma OTT6050 and were shown to be high-molecular weight glycoproteins similar to DBA receptors isolated from the same sources.     

Lectin affinity high-performance liquid chromatography. Interactions of N-glycanase-released oligosaccharides with Ricinus communis agglutinin I and Ricinus communis agglutinin II

The structural determinants required for interaction of oligosaccharides with Ricinus communis agglutinin I (RCAI) and Ricinus communis agglutinin II (RCAII) have been studied by lectin affinity high-performance liquid chromatography (HPLC). Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with columns of silica-bound RCAI and RCAII. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. RCAI binds oligosaccharides bearing terminal beta 1,4-linked Gal but not those containing terminal beta 1,4-linked GalNAc. In contrast, RCAII binds structures with either terminal beta 1,4-linked Gal or beta 1,4-linked GalNAc. Both lectins display a greater affinity for structures with terminal beta 1,4-rather than beta 1,3-linked Gal, although RCAII interacts more strongly than RCAI with oligosaccharides containing terminal beta 1,3-linked Gal. Whereas terminal alpha 2,6-linked sialic acid partially inhibits oligosaccharide-RCAI interaction, terminal alpha 2,3-linked sialic acid abolishes interaction with the lectin. In contrast, alpha 2,3- and alpha 2,6-linked sialic acid equally inhibit but do not abolish oligosaccharide interaction with RCAII. RCAI and RCAII discriminate between N-acetyllactosamine-type branches arising from different core Man residues of dibranched complex-type oligosaccharides; RCAI has a preference for the branch attached to the alpha 1,3-linked core Man and RCAII has a preference for the branch attached to the alpha 1,6-linked core Man. RCAII but not RCAI interacts with certain di- and tribranched oligosaccharides devoid of either Gal or GalNAc but bearing terminal GlcNAc, indicating an important role for GlcNAc in RCAII interaction. These findings suggest that N-acetyllactosamine is the primary feature required for oligosaccharide recognition by both RCAI and RCAII but that lectin interaction is strongly modulated by other structural features. Thus, the oligosaccharide specificities of RCAI and RCAII are distinct, depending on many different structural features including terminal sugar moieties, peripheral branching pattern, and sugar linkages.     

Reactivity of five N-acetylgalactosamine-recognizing lectins with preimplantation embryos, early postimplantation embryos, and teratocarcinoma cells of the mouse

The expression of receptors for N-acetylgalactosamine-recognizing lectins, namely Helix pomatia agglutinin (HPA), Sophora japonica agglutinin (SJA), Bauhinia purpurea agglutinin (BPA), Vicia villosa agglutinin (VVA), and Wistaria floribunda agglutinin (WFA) was studied in early mouse embryos and teratocarcinoma cells. Each of these lectins as well as Dolichos biflorus agglutinin (DBA) bound differently to early embryonic cells, with the exception of VVA and WFA which showed indistinguishable reactivities. SJA reacted intensely with visceral endoderm, but hardly at all with parietal and primitive endoderm. Therefore, SJA will be useful for analyzing the mechanism of visceral-endoderm formation. Furthermore, the inner cell mass (ICM) of early blastocysts reacted intensely with DBA, while the ICM of late blastocysts reacted only faintly with this lectin. Primary endoderm derived from the ICM reacted faintly with SJA, HPA, and DBA, and these reactivities increased again during the differentiation of the endoderm. Therefore, these three lectins could be used in the analysis of early stages during the differentiation of endoderm from the ICM. The results illustrate the highly complex nature of developmentally regulated alterations of cell-surface carbohydrates during the early stages of embryogenesis.     

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.     

Dolichos biflorus agglutinin (DBA) reacts selectively with mast cells in human connective tissues

Dolichos biflorus agglutinin (DBA) binds to N-acetyl-D-galactosamine (GalNAc) residues in glycoconjugates and agglutinates erythrocytes carrying blood group antigen A. In cryostat sections of various tissues from blood group-specified humans, fluorochrome-coupled DBA bound preferentially to fusiform connective tissue cells and to certain epithelial cells. The connective tissue cells were identified as mast cells by their typical metachromasia in consecutive staining with toluidine blue. Double labeling with DBA and conjugated avidin revealed two distinct populations of mast cells. In several tissues the DBA-reactive cells likewise displayed uniform avidin reactivity. In intestinal mucosa, however, morphologically distinct DBA-binding mast cells were found, which were labeled with the avidin conjugates only in specially fixed paraffin sections. DBA did not bind to vascular endothelial cells, which could be identified by double staining with antibodies to factor VIII-related antigen. Labeling with Helix pomatia agglutinin (HPA), another blood group A-reactive lectin, resulted in distinct blood group-dependent fluorescence of the endothelia. Sophora japonica agglutinin (SJA), a blood group B-reactive lectin, labeled vascular endothelial cells in tissues from blood group A, AB, and B donors. HPA and SJA reacted with small mast cells in the gastrointestinal mucosa but failed to label large mast cells in any of the tissues. These results indicate that the blood group reactivity of lectins, as determined by erythroagglutination, is not necessarily consistent with their reactivity with blood group determinants in tissue sections. Moreover, DBA conjugates appear to be a reliable probe for detection of mast cells in various human connective tissues.     

Localization of penultimate carbohydrate residues in zona pellucida and acrosomes by means of lectin cytochemistry and enzymatic treatments

Lectins from peanuts (PNA) and soy beans (SBA) bind terminal residues of galactose (Gal) and N-acetyl-galactosamine (GalNAc) respectively. Galactose oxidase oxidizes the hydroxyl group at C-6 of terminal Gal and GalNAc blocking the binding of PNA and SBA. Binding of these lectins to sugar residues is also severely limited by the existence of terminal residues of sialic acid. In the present study, lectin cytochemistry in combination with enzymatic treatments and quantitative analysis has been applied at light and electron microscopical levels to develop a simple methodology allowing the in situ discrimination between penultimate and terminal Gal/GalNAc residues. The areas selected for the demonstration of the method included rat zona pellucida and acrosomes of rat spermatids, which contain abundant glycoproteins with terminal Gal/GalNAc residues. Zona pellucida was labelled by LFA, PNA and SBA. After galactose oxidase treatment, terminal Gal/GalNAc residues are oxidized, and reactivity to PNA/SBA is abolished. The sequential application of galactose oxidase, neuraminidase and PNA/ SBA has the following effects: (i) oxidation of terminal Gal/GalNAc residues; (ii) elimination of terminal sialic acid residues rendering accessible to the lectins preterminal Gal/GalNAc residues; and (iii) binding of the lectins to the sugar residues. Acrosomes were reactive to PNA and SBA. No LFA reactivity was detected, thus indicating the absence of terminal sialic acid residues. Therefore, no labelling was observed after both galactose oxidase--PNA/SBA and galactose oxidase--neuraminidase--PNA/SBA sequences. In conclusion, the combined application of galactose oxidase, neuraminidase and PNA/SBA cytochemistry is a useful technique for the demonstration of penultimate carbohydrate residues with affinity for these lectins. This revised version was published online in November 2006 with corrections to the Cover Date.     

Studies on the effective size of phospholipid headgroups in bilayer vesicles using lectin-glycolipid interaction as a steric probe

An experimental approach is described which provides information about the relative, effective size of phospholipid headgroups in bilayer vesicles. It is based on determination of the binding of lectins (Ricinus communis agglutinin or concanavalin A) to synthetic glycolipids inserted in such vesicles, using a vesicle agglutination assay. It is shown that the ability of a glycolipid containing a shorter (4-member) spacer arm to bind the appropriate lectin is highly sensitive to the headgroup structure of the surrounding phospholipid in mixed glycolipid-phospholipid vesicles. Furthermore, when the phospholipid was phosphatidate a change in protonation or in monovalent counter-ion species (Li+, NH+4, N(CH3)+4 or Na+) significantly influenced lectin binding. The interference with lectin binding described above was reduced when the glycolipid spacer arm was extended from a 4- to a 6-member length. Furthermore, the sensitivity to phospholipid headgroup structure or to changes in the ionic environment was completely eliminated when the glycolipid contained a longer (10- or 12-member) spacer arm between the hydrophobic part and the lectin-binding group. It is concluded that the modulation of lectin binding in the former case is due to steric inhibition determined by the effective (hydrated) size of the various phospholipid headgroups.     

Lectin Analysis of Surface Saccharides in Two Trichomonas vaginalis Strains Differing in Pathogenicity*

SYNOPSIS. Surface saccharides in 2 Trichomonas vaginalis strains, the moderately pathogenic, JH34A, and the mild, JH162A, were analyzed with the aid of plant lectins. Concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA), castor bean agglutinin (CBA), and lectin from the garden pea (GPA) were employed in agglutination tests and in treatment of ultrathin sections for electron microscopy according to the horseradish peroxidase-3,3′-diaminobenzidine method. With Con A and WGA, small quantitative differences were noted between the 2 strains in the results of agglutination and in the reaction-product deposits observed by electron microscopy. Distribution of the binding sites for the 2 lectins was also somewhat different in the JH34A and JH162A trichomonads. In general, the reactions with the more pathogenic strain were slightly stronger. Although the reactions with SBA and CBA lectins were weaker than those with Con A or WGA, they provided the means for qualitative differentiation between the 2 trichomonad strains. SBA alone agglutinated the JH34A strain and formed demonstrable deposits on the cell surfaces. On the other hand, only CBA reacted with JH162A flagellates. The garden pea lectin failed to bind to the surface of either strain. On the basis of results obtained with the control preparations incubated in the presence of specific inhibitors, it was concluded that both strains had α-methyl-D-mannoside and/or α-methyl-D-mannoside-like as well as N-acetyl-D-glucosamine residues on their surfaces. In addition, JH34A strain had D-lactose-containing residues while JH162A trichomonads had residues with D-galactose. Neither strain appeared to possess residues containing N-acetyl-D-galactosamine.     

Biochemical and structural analysis of Helix pomatia agglutinin. A hexameric lectin with a novel fold

Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin found in the albumen gland of the roman snail. As a constituent of perivitelline fluid, HPA protects fertilized eggs from bacteria and is part of the innate immunity system of the snail. The peptide sequence deduced from gene cloning demonstrates that HPA belongs to a family of carbohydrate-binding proteins recently identified in several invertebrates. This domain is also present in discoidin from the slime mold Dictyostelium discoideum. Investigation of the lectin specificity was performed with the use of glycan arrays, demonstrating that several GalNAc-containing oligosaccharides are bound and rationalizing the use of this lectin as a cancer marker. Titration microcalorimetry performed on the interaction between HPA and GalNAc indicates an affinity in the 10(-4) M range with an enthalpy-driven binding mechanism. The crystal structure of HPA demonstrates the occurrence of a new beta-sandwich lectin fold. The hexameric quaternary state was never observed previously for a lectin. The high resolution structure complex of HPA with GalNAc characterizes a new carbohydrate binding site and rationalizes the observed preference for alphaGalNAc-containing oligosaccharides.     

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

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

A differential response elicited in macrophages on interaction with lectins

The interaction of several lectins, both native and chemically modified, with mouse peritoneal macrophages was studied. Surface distribution and interiorization of the lectins was assessed quantitatively using their radioactively-labeled derivatives, and qualitatively by employing fluorescein-labeled lectins. On the basis of their effect on the macrophages, the lectins tested fall into two classes: lectins that induce vacuole formation in the cells (concanavalin A (ConA), wax bean agglutinin (WBA) and wheat germ agglutinin (WGA)) and lectins that in their native form do not induce vacuolation (soybean agglutinin (SBA), peanut agglutinin (PNA) and the lectin from Lotus tetragonolobus (LT)). Neuraminidase treatment of the cells did not change their response to the lectins, though in the case of SBA and PNA binding was observed only with neuraminidase-treated macrophages. Incubation of the latter cells with SBA and subsequently with ConA resulted in significantly higher vacuolation than that observed with ConA alone. Glutaraldehyde-crosslinked polymers of SBA and of PNA, which are multivalent with respect to sugar binding, induced vacuolation in neuraminidase-treated macrophages. On the other hand, succinylation of ConA, which reduces the number of sugar binding sites per mole from four to two, abolished its ability to induce vacuole formation. These data suggest that multivalency of lectins and probably also their size are important factors in inducing vacuolation, by causing extensive crosslinkage of membrane receptors which is prerequisite for triggering of vacuole formation. Quantitative binding and internalization data indicate that vacuole formation is not directly related to the number of lectin receptors on the macrophages nor to the extent of their internalization.     

Effect of lectins on the invasion of Ichthyophthirius theront to channel catfish tissue.

This study determined the effects of lectin binding to theronts of Ichthyophthirius multifiliis on theront immobilization, invasion, trophont development and survival in channel catfish Ictalurus punctatus excised fins in vitro. Soybean agglutinin (SBA), lentil agglutinin (LCA), gorse agglutinin (UEA-I) and wheat germ agglutinin (WGA) were used to treat theronts. Percentages of theronts immobilized by 4 lectins ranged from 12.0 to 19.4% at a concentration of 1000 microg ml(-1). These lectins bound more than half of the theronts at a concentration of 50 microg ml(-1). More theronts were labeled by SBA and WGA than by lectin LCA at concentrations of 50 and 100 microg ml(-1), respectively. The binding of these lectins to theronts indicated that monosaccharides (D-galactose, L-fucose, D-mannose and D-glucose) and amino sugar derivatives (N-acetylgalactosamine and N-acetylglucosamine) were present on the surface of theronts. Invasion was reduced significantly for theronts treated with LCA, UEA-I and WGA. No difference in invasion was found between control and SBA bound theronts (p > 0.05). The binding of lectin LCA, UEA-I and WGA to theronts significantly reduced the development of trophonts (p < 0.05). The mean volumes of trophonts labeled with these 3 lectins were smaller than volumes in control trophonts from 8 to 48 h after exposure. Survival was lower in trophonts labeled with lectins than in control trophonts at 48 h after exposure.     

Activities of lectins and their immobilized derivatives in detergent solutions. Implications on the use of lectin affinity chromatography for the purification of membrane glycoproteins.

The effects of several commonly used detergents on the saccharide-binding activities of lectins were investigated using lectin-mediated agglutination of formalin-fixed erythrocytes and affinity chromatography of glycoproteins on columns of lectins immobilized on polyacrylic hydrazide-Sepharose. In the hemagglutination assays, Ricinus communis I (RCA1) and II (RCAII), concanavalin A (Con A), and the agglutinins from peanut (PNA), soybean (SBA), wheat germ (WGA), and Limulus polyphemus (LPA) were tested with several concentrations of switterionic, cationic, anionic, and nonionic detergents. It was found that increasing detergent concentrations eventually affected hemagglutination titers in both test and control samples, and the highest detergent concentrations not affecting lectin hemagglutinating activities were determined. The effects of detergents on specific binding of [3H]fetuin and asialo[3H]fetuin to and elution from columns of immobilized lectins were less severe when compared with lectins in solution, suggesting that the lectins are stabilized by covalent attachment to agarose beads. Nonionic detergents did not affect the binding efficiency of the immobilized lectins tested at concentrations used for membrane solubilization while cationic and zwitterionic detergents caused significant inhibition of Con A- and SBA-Sepharose activities. In sodium deoxycholate (greater than 1%) only RCAI-Sepharose retained its activity, whereas the activities of the other lectins were reduced dramatically. Low concentrations of sodium dodecyl sulfate (0.05%) inhibited only the activity of immobilized SBA, but at higher concentration (0.1%) and prolonged periods of incubation (16 h, 23 degrees C) most of the lectins were inactivated. These data are compared with previous reports on the use of detergents in lectin affinity chromatography, and the conditions for the optimal use of detergents are detailed.     

GalNAc moieties in O-linked oligosaccharides of the primordial germ cells of Xenopus embryos

Glycoconjugates could play a role in cell adhesion and migration mechanisms, including the locomotive movements of the primordial germ cells (PGCs) during the development of the embryo. In the present work, we have studied by lectin histochemistry the presence of N-acetylgalactosamine (GalNAc) in the glycans of the Xenopus PGCs, as a first approach to identifying their glycoconjugates which could be involved in the migration mechanism. The PGCs were negative for three of the GalNAc-binding lectins employed (from soybean, SBA; from lima bean, LBA; and from snail, HPA). However, when sialic acid (NeuAc) was previously removed by acid hydrolysis, SBA and HPA, but not LBA, labeled the PGCs, except if the staining was combined with the beta-elimination procedure. This suggests the presence of GalNAc alpha(1,3)-linked to galactose (Gal) in O-linked oligosaccharides, in a subterminal position to NeuAc. As the PGCs were always negative for LBA, the absence of fucose alpha(1,2)-linked to subterminal Gal is suggested. With the lectin from horse gram (DBA), the PGCs were stained, although beta-elimination turned the cells negative and acid hydrolysis increased the labeling, suggesting that GalNAc(alpha)(1,3)GalNAc was in O-linked glycans in terminal and subterminal to NeuAc position.