Lectin binding sites inParamecium tetraurelia cells

Summary Though all three lectins tested (ConA, RCA II, WGA) bound to the entire cell membrane, none bound selectively to the docking site of secretory organelles (trichocysts); the same results were achieved with FITC-conjugates, or, on the EM level, with peroxidase- or gold-labeling. Only WGA triggered the release of trichocysts and none of the lectins tested inhibited AED-induced synchronous exocytosis.When exocytosis was triggered synchronously in the presence of any of these three lectins (FITC-conjugates), the resulting ghosts trapped the FITC-lectins and the cell surface was immediately afterwards studded with regularly spaced dots (corresponding to the ghosts located on the regularly spaced exocytosis sites). These disappeared within about 10 min from the cell surface (thus reflecting ghost internalization with a half life of 3 min) and fluorescent label was then found in 6–10 vacuoles, which are several m in diameter, stain for acid phosphatase and, on the EM level, contain numerous membrane fragments (other-wise not found in this form in digesting vacuoles). We conclude that synchronous massive exocytosis involves lysosomal breakdown rather than reutilization of internalized trichocyst membranes and that these contain lectin binding sites (given the fact free fluorescent probes did not efficiently stain ghosts).Trichocyst contents were analyzed for their lectin binding capacity in situ and on polyacrylamide gels. RCA II yielded intense staining (particularly of tips), while ConA (fluorescence concentrated over bodies) and WGA yielded less staining of trichocyst contents on the light and electron microscopic level. Only ConA- and WGA-staining was inhibitable by an excess of specific sugars, while RCA II binding was not. ConA binding was also confirmed on polyacrylamide gels which also allowed us to assess the rather low degree of glycosylation (1% by comparison with known glycoprotein standards) of the main trichocyst proteins contained in their expandable matrix.Since RCA II binding could be due to its own glycosylation residues we looked for an endogenous lectin. The conjecture was substantiated by the binding of FITC-lactose-albumin (inhibitable by a mixture of glucose-galactose). This preliminary new finding may be important for the elucidation of trichocyst function.Abbreviations AED aminoethyldextran - BSE backscatter electrons - ConA Concanavalin A - DAB 3,3-diaminobenzidine - EM electron microscope - FITC fluorescein-isothiocyanate - kD kiloDalton - ME mercaptoethanol - MIP membrane-intercalate particle - M_r apparent molecular weight - PAGE polyacrylamide-gel-electrophoresis - PAS periodic acid Schiff - pI isoelectric point - POX peroxidase - RCA II Ricinus communis agglutinin II - SDS sodium dodecylsulphate - SEM scanning electron microscope - WGA wheat germ agglutinin     

Lectin binding sites during Drosophila embryogenesis

The spectrum of lectin binding sites as it emerges during embryonic development of Drosophila was analysed by means of fluorescein-labelled lectins. As development and morphogenesis proceed, the reaction pattern becomes more and more complex. Mannose/glucose-, mannose-, N-acetylglucosamine- and poly-N-ace-tylglucosamine-specific lectins bind ubiquitously. Nuclear envelopes only have binding sites for wheat germ agglutinin. N-acetylgalactosamine-binding lectins are specific for ectodermal derivatives. Ga-3-N-acetylgalac-tosamine-binding lectins are highly selective markers for neural structures, haemocytes and Garland cells. It is also shown that Drosophila laminin is differentially glycosylated. The possible implications of differential and germ layer-specific glycosylation are discussed.Dedicated to the memory of Jan Callaerts     

Lectin binding in the diabetic rat kidney

In this study metal-conjugated concanavalin A (Con A) and Bandieraea simplicifolia isolectin II (BSA II) have been applied to sections from kidneys of control rats and rats which had untreated diabetes for 70 days or for 200 days. Lectin binding was measured by atomic absorption spectrophotometric analysis of ferritin-iron or hemocyanin-copper. Con A binding increased significantly with diabetes; was totally blocked by alpha-D-mannoside; was not inhibited by fructose lysine; and was enhanced by NaHB4 preincubation. BSA II binding also increased significantly with diabetes.     

Lectin binding in the human foetal testis

In the present study we have investigated the oligosaccharidic content of the glycoconjugates within the human foetal testis starting from its earliest differentiation phase (8, 10 and 12 weeks of gestation). To this purpose we have used a battery of six horseradish peroxidase-labelled lectins (SBA, PNA, WGA, UEAI, LTA and ConA). We have obtained a complete distributional map of the sugar residues of the glycoconjugates in the coelomic mesothelium, tunica albuginea, pre-Sertoli cells, pre-gonocytes, Leydig cells, basement membrane of the sex cords, interstitial tissue, mastocytes and endothelial cells of the capillary vessels. Since the beginning of the testis differentiation phase the cells of the coelomic mesothelium showed a large amount of sugar residues. In the pre-Sertoli cells and in the pre-gonocytes a role played as structural molecules by some oligosaccharides could be hypothesized. D-galactose-(beta1-->3)-N-acetyl-D-galactosamine, sialic acid, N-acetyl-D-glucosamine and alpha-D-mannose could be involved in inducing and maintaining the cellular activity of the Leydig cells.     

Lectin binding in the diabetic rat kidney

Summary In this study metal-conjugated concanavalin A (Con A) andBandieraea simplicifolia isolectin II (BSA II) have been applied to sections from kidneys of controls rats and rats which had untreated diabetes for 70 days or for 200 days. Lectin binding was measured by atomic absorption spectrophotometric analysis of ferritin-iron or hemocyanin-copper.Con A binding increased significantly with diabetes; was totally blocked by alpha-D-mannoside; was not inhibited by fructose lysine; and was enhanced by NaHB₄ preincubation. BSA II binding also increased significantly with diabetes.     

Lectin binding by microvillous membranes and coated-pit regions of human syncytial trophoblast

Summary The distribution of saccharides on the microvillous membrane of the human syncytial trophoblast was investigated using ferritin conjugates of four lectins: concanavalin A (specific for -d-manno- and -d-glucopyranosyl residues), wheatgerm agglutinin (specific forN-acetylglucosamine),Limulus polyphemus lectin (specific forN-acetylneuraminic acid), andLotus tetragonolobus lectin (specific for -l-fucose). Concanavalin A and wheatgerm agglutinin (WGA) reacted strongly with the surface membrane and ferritin deposits were also observed in coated pit regions of the membrane. Lectins fromL. polyphemus andL. tetragonolobus, however, reacted only weakly with the microvillous border and neither reacted with coated pits.Enhanced agglutinability of trophoblast cells in comparison with other foetal cells from the same conceptus was seen with WGA. This agglutination was inhibited by addition of acetylglucosamine or by a solubilized membrane fraction which was bound by a column of WGA-Sepharose. The membrane fraction which did not bind to the column did not inhibit agglutination. Electrophoresis of the WGA-bound membrane proteins revealed six subunits, the major band having an apparent mol. wt. of 55 000. A protein of this mol. wt was also seen in coated vesicles isolated from equivalent human placentae.     

Lectin binding sites of glycoproteins as revealed by lectin-gold-silver and lectin-peroxidase-diaminobenzidine methods

Summary For the precise histochemical detection of lectin binding sites of glycoproteins, the results obtained by lectin-gold-silver (LT-G-S) staining methods have been systematicaly compared with those revealed by alternative techniques of lectin-peroxidase-diaminobenzidine (LT-PO-DAB) reactions in a series of organs from different mammalian species.Ricinus communis agglutinin-I and concanavalin A were the lectins used in the present study. In the tissues subjected to the LT-G-S procedures, reactive tissue structures exhibited positive reactions of varying intensities of black. The results of control staining for the LT-G-S methods substantiated the view that the reaction products demonstrated the precise lectin binding sites of glycoproteins. The staining images obtained by the LT-PO-DAB techniques were not necessarily correlated precisely with those revealed by the LT-G-S procedures, and unavoidable background staining in pale brownish shades was noted in the majority of LT-G-S negative tissue structures. In view of these results, the LT-G-S staining methods employed in the present study are believed to be a reliable technique for the precise localization of saccharide residues of glycoproteins in light microscopy.     

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.     

Lectin binding pattern in normal human labial mucosa

Summary The pattern of lectin binding in normal human labial mucosa was examined by light and electron microscopy using eight different lectins (ConA, LCA, WGA, UEA-1, RCA-1, SBA, DBA and PNA) and compared with the patterns in normal human skin and oesophageal mucosa. As seen by light microscopy, ConA, LCA, and WGA stained cell membranes in all layers of the mucosae. RCA-1 stained the plasma membrane of cells in the basal and middle layers, whereas cells in the superficial layers showed little positive staining. UEA-1, SBA, and PNA stained the cells in the middle layers weakly in some cases. No positive staining for DBA was seen. By electron microscopy, reaction product indicating ConA-binding sites was observed in the plasma membrane, cisternae of the endoplasmic reticulum, nuclear envelope and the Golgi apparatus. Binding of LCA, WGA, and RCA-1 was observed in the plasma membrane. These results show that the binding pattern of PNA, SBA, and RCA-1 in labial mucosa is different from that in the normal skin or oesophageal mucosa, although the labial mucosal epithelium, epidermis, and oesophageal epithelium are all stratified squamous epithelia. These differences in the cell-surface sugar residues are likely to be related to the possible functional differences in these tissues.     

Lectin binding sites in duodeno-jejunal mucosae from coeliac children

The distribution pattern of some labelled lectins (WGA, SBA, PNA, DBA, ConA, LCA) has been investigated in small intestinal mucosa of coeliac children (119 cases) and normal (short stature) and pathological (postenteritis syndrome) controls. The oligosaccharide side chains of glycoproteins present in the cytoplasm and in the brush border of enterocytes, goblet cells and luminal crypt surface have been revealed. The most important differences in lectin binding reactivity between coeliac and controls mucosae are discussed.     

Lectin binding ability of B-chronic lymphocytic leukaemia cells.

The binding of five fluorescein-labelled lectins: peanut agglutinin (PNA), lentil agglutinin (LEN), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and asparagus pea agglutinin (ASP) to human B-cell chronic lymphocytic leukaemia (B-CLL) and B lymphocytes of normal donors was studied. The specificity of the fluorescence was demonstrated by inhibition with appropriate saccharides. The proportion of B cells was estimated using anti-B cell monoclonal antibody. Both leukaemic and normal B cells showed the binding ability of all except of one (ASP) studied lectins. We have found the differences in surface carbohydrate patterns between B-CLL and normal B lymphocytes. B-CLL cells showed the considerably lower ability to bind SBA and slightly higher expression of PNA and LEN receptors in comparison to normal B cells. The analysis of WGA binding allowed for recognizing two groups of CLL patients: one with high and the second one with low WGA receptor expression. The double marker studies revealed that B cells could simultaneously react with anti-B cell monoclonal antibody and fluorochrome labelled lectins.     

Lectin binding sites in duodeno-jejunal mucosae from coeliac children

Summary The distribution pattern of some labelled lectins (WGA, SBA, PNA, DBA, ConA, LCA) has been investigated in small intestinal mucosa of coeliac children (119 cases) and normal (short stature) and pathological (postenteritis syndrome) controls. The oligosaccharide side chains of glycoproteins present in the cytoplasm and in the brush border of enterocytes, goblet cells and luminal crypt surface have been revealed. The most important differences in lectin binding reactivity between coeliac and controls mucosae are discussed.     

Lectin binding to collagen strands in histologic tissue sections

Histologic sections from human skin and uterine ligaments were stained with the following FITC conjugated lectins: Con A, WGA, s-WGA, SBA, DBA, UEA I, PNA, RCA I, BPA, GSA I, GSA II, MPA and LPA. The staining of the connective tissue was similar in the dermis and the uterine ligaments and it was most intense in the extracellular matrix containing collagen strands whereas the fibrocytes remained unstained. The staining was clear with glucose or N-acetylglucosamine binding lectins like Con A, WGA, s-WGA and GSA II, which may be related to the presence of glucose residues in collagenous hydroxylysine. The staining with some of the galactose or N-acetylgalactosamine binding lectins like RCA I, DBA, and BPA was less intense. This may reflect the presence of terminal galactose sugars in the hydroxylysine of collagen. No staining was found with SBA, UEA I, PNA, GSAI, MPA or LPA. The results show that different particularly glucose specific lectins bind to the extracellular matrix and especially to collagenous strands in connective tissue. It is suggested that this might be used in histochemical studies of connective tissue and particularly concerning the changes that may occur in different disease states.     

Lectin binding in Cryptomonas and Chroomonas (Cryptophyceae)

The cell envelopes of Cryptomonas and Chroomonas exhibited significant fluorescence using FITC-labelled concanavalin A and wheat germ agglutinin when the cells were fixed prior to lectin binding. The periplast became intensely labelled in Chroomonas whereas Cryptomonas showed fluorescing granula in its gullet/furrow region and on the cell surface. Lectin labelling followed by fixation showed only label of periplast remnants of lysed cells and of the flagella of Chroomonas. Isolated periplasts of Cryptomonas and Chroomonas were intensively labelled with both concanavalin A and wheat germ agglutinin. Glycostaining of gels, onto which total cell protein extracts were loaded, showed a glycoprotein of high molecular weight for Cryptomonas and Chroomonas and an additional glycoprotein for Cryptomonas species.     

Lectin binding to parietal cells of human gastric mucosa

A light microscopic and ultrastructural analysis of lectin receptors on parietal cells from human gastric mucosa was performed utilizing 12 biotinylated lectins in conjunction with an avidin-biotin-peroxidase complex. Peanut agglutinin conjugated directly to peroxidase was also used. Several fixatives and fixation regimens were evaluated for optimal preservation of parietal cell saccharide moieties. Formalin proved to be the most practical fixative for light microscopic studies. A periodate-lysine-paraformaldehyde (PLP) combination provided good preservation of lectin binding capacity but yielded relatively poor ultrastructure. Conversely, glutaraldehyde provided excellent preservation of ultrastructure but a somewhat diminished lectin binding activity, which was overcome by using long incubation times and high concentrations of reagents. Parietal cells reacted strongly with Bandieraea simplicifolia, Dolichos biflorus, peanut agglutinin, and soybean agglutinin (all specific for galactosyl/galactosaminyl groups) and weakly with Ulex europaeus (specific for fucose). At the light microscopic level a beaded, perinuclear staining pattern was observed which, ultrastructurally, corresponded to an intense staining of intracytoplasmic canaliculi. The membranes of the intracytoplasmic canaliculi were characterized by an abundance of galactosyl residues, a paucity of fucosyl groups, and a lack of mannosyl and glucosyl residues. The biochemical and physiological significance of these findings is 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.     

Lectin specificity and binding characteristics of human C-reactive protein.

C-reactive protein (CRP) is thought to play an important role in immunomodulation. The exact biologic function of this pentraxin protein is, however, still unclear. Here we report experiments designed to further characterize the binding properties of CRP. Using purified human CRP it could be shown that CRP immobilized onto polystyrene surfaces or onto latex beads binds distinct plasma glycoproteins including IgG, asialofetuin, asialo-beta 2-glycoprotein I and, likewise, synthetic glycoproteins as a lectin, exhibiting binding specificity for terminal galactosyl residues of the glycoprotein glycans. Binding of CRP to IgA, IgM, IgG, asialofetuin, asialo-beta 2-glycoprotein I and to synthetic glycoproteins requires immobilization onto surfaces of both CRP and the ligand. Fibronectin and fibrinogen are bound by surface-immobilized CRP also in soluble phase. Comparing various mono-, di-, and trisaccharides as competitive inhibitors of the lectin binding activity of CRP, only beta-D-Gal-(1-3)-D-GalNAc, beta-D-Gal-(1-4)-D-GalNAc, and beta-D-Gal-(1-4)-beta-D-Gal-(1-4)-D-GlcNAc had significant inhibitory power at a concentration of 8 mmol/liter. Binding activity of CRP was pH-dependent with an optimum at pH 5 to 6 and was reduced by 90% when pH was shifted from 6 to the physiologic pH value of 7.4. CRP exhibited lectin-like properties with binding specificity for galactosyl residues also when bound to K-562 erythroleukemia cells. It is therefore suggested that CRP immobilized onto surfaces exhibits lectin activity toward galactosyl groups preferentially in a mildly acidic environment as present at sites of inflammation.