Distribution Of Lectin Binding Sites Research Articles

Characterization of two blood–brain barrier mimicking cell lines: Distribution of lectin-binding sites and perspectives for drug delivery

In the present study plant lectins with distinct sugar specificities were applied to two blood–brain barrier (BBB) mimicking cell lines, namely human ECV304 and porcine brain microvascular endothelial cells PBMEC/C1–2 in order to elucidate their glycosylation pattern and to evaluate the lectin-cell interaction for lectin-mediated targeting. The bioadhesive properties of fluorescein-labeled lectins were investigated with monolayers as well as single cells using fluorimetry and flow cytometry, followed by confirmation of the specificity of binding. For PBMEC/C1–2 layers highest binding capacity was found for wheat germ agglutinin (WGA), followed by Dolichus biflorus agglutinin (DBA) whereas single cell experiments revealed a predominance of DBA only. Analyzing ECV304 monolayers and single cells, WGA yielded the strongest interaction without any changes during cultivation. The binding capacities of the other lectins increased significantly during differentiation. As similar results to primary cells and brain sections were observed, both cell lines seem to be suitable as models for lectin-interaction studies. Thus, an additional focus was set on the mechanisms involved in uptake and intracellular fate of selected lectins. Cytoinvasion studies were performed with WGA for human ECV304 cells and WGA as well as DBA for PBMEC/C1–2 cells. For both lectins, the association rate to the cells was dependent on temperature which indicated cellular uptake.

In vitro and in situ localization of concanavalin A and wheat germ agglutinin binding sites on the surface of female cells in Torenia fournieri L.

The interaction between lectins and their specific binding sites is believed to play a critical role in fertilization in animals and some lower plants. However, for higher plants there is no information on lectins or their binding sites related to female gametes and fertilization. The present work was designed as a first attempt to reveal the general pattern of lectin binding site distribution on the surface of female cells, namely egg cells, central cells, and synergids of Torenia fournieri and, especially, to investigate the possible effects of cell isolation procedure on the distribution of lectin binding sites. Therefore, concanavalin A (Con A) and wheat germ agglutinin (WAG) binding sites on the surface of both in vitro and in situ living female cells were localized by using fluorescein isothiocyanate (FITC) conjugated Con A and WGA as probes. We demonstrated that enzymatic treatment and isolation procedures did not notably modify the surface character of the female cells and the distribution of Con ...

Distribution of lectin-binding glycosidic residues in the hamster follicular oocytes and their modifications in the zona pellucida after ovulation.

In the present study, we have employed a battery of colloidal gold-tagged lectins as probes in conjunction with quantitative analysis to demonstrate the distribution and changes of carbohydrate residues in the hamster zona pellucida (ZP) during ovarian follicular development and during transit of the oocyte through the oviduct after ovulation. High-resolution lectin-gold cytochemistry performed on thin sections of LR White-embedded ovaries revealed a moderate to strong reactivity to WGA, PNA, DSA, AAA, and MAA over the entire thickness of the ZP of ovarian oocytes at different stages of follicular development. Labeling intensity over the ZP progressively increased as follicles matured in the ovary. In parallel, there was an association of labeling by gold particles with cortical granules, stacks of Golgi saccules, and complex structures called vesicular aggregates in the oocyte proper especially during the late stages of follicular growth. In contrary, labeling with each of HPA, DBA, and BSAIB(4) was absent in the ovary but was found to be localized over Golgi complexes and secretory granules in the non-ciliated secretory cells of the oviduct. When ovulated oocytes were labeled with each of HPA, WGA, RCA-I, PNA, DSA, BSAIB(4), AAA, MAA, and DBA, the ZP and several organelles in the oocyte proper presented a differential distribution of lectin-binding sites. Quantitative analysis was also performed on labeling by lectin-gold complexes that bind specifically to the ZP of mature follicular and ovulated oocytes. Quantitative evaluation revealed heterogeneous labeling between the inner and the outer zone of the ZP. A significant increase in the labeling densities in both inner and outer ZP was noted when tissue sections of ovulated oocytes were labeled with RCA-I or AAA. Tissue sections of ovaries labeled with WGA demonstrated a significant increase in the density of labeling in the outer layer of the ZP. Labeling by PNA, DSA, and MAA, however, showed a significant decrease in both the inner and outer portions of the ZP. Together, these results suggest that in the hamster, glycoproteins carrying specific sugar residues are added to the ZP of ovarian follicles during the early stages of folliculogenesis and are processed through a common secretory machinery, and that there is a significant change in both the sugar moieties and distribution of glycoproteins in the ZP following ovulation. Our results also showed that the hamster oviduct plays an important role in contributing certain glycoproteins to the ZP suggesting that the sugar moieties of these oviductal glycoproteins may have functional significance in fertilization.

The apical membrane glycocalyx of MDCK cells.

The microenvironment near the apical membrane of MDCK cells was studied by quantitation of the fluorescence of wheat germ agglutinin attached to fluorescein (WGA). WGA was shown to bind to sialic acid residues attached to galactose at the alpha-2,3 position in the glycocalyx on the apical membrane. Young MDCK cells (5-8 days after splitting) showed a patchy distribution of WGA at stable sites that returned to the same locations after removal of sialic acid residues by neuraminidase treatment. Other lectins also showed stable binding to patches on the apical membrane of young cells. The ratio of WGA fluorescence emission at two excitation wavelengths was used to measure near-membrane pH. The near-membrane pH was markedly acidic to the pH 7.4 bathing solution in both young and older cells (13-21 days after splitting). Patches on the apical membrane of young cells exhibited a range of near-membrane pH values with a mean +/- SEM of 6.86 +/- 0.04 (n = 121) while the near-membrane pH of older cells was 6.61 +/- 0.04 (n = 120) with a uniform WGA distribution. We conclude that the distribution of lectin binding sites in young cells reflects the underlying nonrandom location of membrane proteins in the apical membrane and that nonuniformities in the pH of patches may indicate regional differences in membrane acid-base transport as well as in the location of charged sugars in the glycocalyx.

Distribution of Lectin Binding Sites in Xenopus laevis Egg Jelly

Eggs from the anuran Xenopus laevis are surrounded by a thick jelly coat that is required during fertilization. The jelly coat contains three morphologically distinct layers, designated J1, J2, and J3. We examined the lectin binding properties of the individual jelly coat layers as a step in identifying jelly glycoproteins that may be essential in fertilization. The reactivity of 31 lectins with isolated jelly coat layers was examined with enzyme-linked lectin-assays (ELLAs). Using ELLA we found that most of the lectins tested showed some reactivity to all three jelly layers; however, two lectins showed jelly layer selectivity. The lectin Maackia amurensis (MAA) reacted only with J1 and J2, while the lectin Trichosanthes kirilowii (TKA) reacted only with J2 and J3. Some lectins were localized in the jelly coat using confocal microscopy, which revealed substantial heterogeneity in lectin binding site distribution among and within jelly coat layers. Wheat germ agglutinin (WGA) bound only to the outermost region of J3 and produced a thin, but very intense, band of fluorescence at the J1/J2 interface while the remainder of J2 stained lightly. The lectin MAA produced an intense fluorescence-staining pattern only at the J1/J2 interface. Several lectins were also tested for the ability to inhibit fertilization. WGA, MAA, and concanavalin A significantly inhibited fertilization and WGA was found to block fertilization by preventing sperm from penetrating the jelly. Using Western blotting, we identified high-molecular-weight components in J1 and J2 that may be important in fertilization.

Detection of glycoconjugates by lectin gold labelling, silver enhancement, and scanning electron microscopy.

A method for detecting glycoconjugates on cell surfaces in scanning electron microscopy is described. Terminal saccharides were specifically recognized by a lectin conjugated to biotin, and, after incubation with an anti-biotin antibody conjugated to colloidal gold, silver enhancement was used to produce deposits large enough to be detected in standard scanning electron microscopes. Secondary electron images revealed the ultrastructure of the tissue investigated, while backscattered electron images showed the distribution of lectin binding sites. Using digital recording and processing, the two channels were combined in colour-encoded images. The new method brings together lectin histochemistry and scanning electron microscopy and thus allows the three-dimensional distribution of glycoconjugates to be analysed at an ultrastructural level.

In situ Characterization of Biofilm Exopolymers Involved in the Accumulation of Chlorinated Organics

The chemical nature and spatial arrangements of exopolymers in a degradative biofilm community were studied using a panel of fluorescein isothiocyanate– and tetramethyl rhodamine isothiocyanate–conjugated probes. Image analysis and dual channel imaging, in conjunction with scanning confocal laser microscopy, allowed detection and quantification of lectin binding to a variety of glycoconjugates. Relative abundance of these components varied between 0 and 67% of biofilm area at any depth. Lectin binding sites were distributed nonuniformly, both horizontally and vertically, within the >30-μm thick biofilms when the herbicide diclofop methyl was provided as the sole carbon source. A more uniform distribution of lectin binding sites was formed by the same biofilm community, when grown on a labile medium. Diclofop and its metabolites accumulated in extracellular polymers when biofilms were grown with diclofop as the sole source of carbon and energy, but not in the presence of the labile carbon source. There was a nearly 1:1 correspondence between the distribution of regions that accumulated diclofop (and other chlorinated ring compounds) and regions with binding sites for the α-l-fucose-specific Ulex europaeus Type I lectin. These regions also bound polyanionic and cationic fluor-conjugated dextrans, and a hydrophobic-specific dye, demonstrating the nonuniform distribution of charged and hydrophobic regions in the biofilm matrix. Hydrolytic enzymes, some of them selected for their specificity against residues identified by the lectin assay, had no effect on either structural integrity or diclofop binding. The distribution of diclofop binding, lectin binding, and charged regions observed in these biofilms indicated a degree of spatial organization and differentiation within the biofilm community. In addition, based on cell morphology and fluorescent gram reaction, these regions were primarily associated with one community member, a Bacillus coagulans strain.

A histochemical study of the distribution of lectin binding sites in the developing oocytes of the lancelet Branchiostoma belcheri

The distribution of carbohydrate moieties in lancelet (Branchiostoma belcheri) oocytes has been studied at different stages of development, using a peroxidase-labeled lectin incubation technique, the PAS-reaction and Alcian Blue staining. Binding sites of 5 lectins, indicating the presence of different sugar moieties (Wheat germ agglutinin (WGA) for N-acetylglucosamine, Concanavalin A (Con A) for glucose/mannose, Helix pomatia agglutinin (HPA) for N-acetyl-D-galactosamine, Ricinus communis agglutinin (RCA-I) for galactose and Ulex europaeus agglutinin (UEA-I) for fucose), were identified and were shown to undergo considerable variation during oocyte development. In the previtellogenic stage, HPA, RCA-I and UEA-I were not identified on the oocyte surface, but WGA and Con A gave strongly positive reactions at this site. In the cytoplasm, 4 lectins (Con A, HPA, RCA-I and UEA-I) gave a weak or moderate reaction, and Con A was also observed in the perinuclear region. In vitellogenic oocytes, these 4 lectins were found to also bind to the nuclear envelope, karyoplasm and nucleolus, and, with the exception of Con A, could also be found in the nuclei of more mature stages. The cytoplasmic yolk granules and Golgi vesicles of the vitellogenic oocyte, were moderately positive for Con A, HPA, RCA-I and UEA-I, but HPA, RCA-I and UEA-I were only weakly bound at the oocyte surface. In mature oocytes, all 5 lectins bound moderately or strongly to yolk granules and cell surface. HPA, RCA-I and UEA-I bound moderately or strongly to various nuclear compartments.(ABSTRACT TRUNCATED AT 250 WORDS)

A histochemical study of the distribution of lectin binding sites in the developing oocytes of the lancelet Branchiostoma belcheri

A histochemical study of the distribution of lectin binding sites in the developing oocytes of the lancelet Branchiostoma belcheri

Distribution of lectin binding sites in the oviducts of cycling and hormone-treated pigs.

Gamete transport, fertilization, and early embryonic development take place in different regions of the oviduct and under different hormonal conditions. The objective of this study was to use lectins to detect variation in the distribution of glycosylated molecules on the surface of the epithelia that influence these events. Oviducts were collected from gilts on day 1 (estrus) and day 16 (diestrus) of the estrous cycle, and from gilts that were bilaterally ovariectomized on day 4 of the estrous cycle, and were subsequently treated with estradiol valerate (100 micrograms/day), progesterone (200 mg/day), or corn oil vehicle for 11 days. Six biotinylated lectins, Triticum vulgaris (WGA), Arachis hypogaea (PNA), Ulex europeus (UEA-I), Dolichos biflorus (DBA), Ricinus communis (RCA-I), and Canavalia ensiformis (Con A), were used to probe tissue from the isthmus and ampulla using peroxidase-conjugated avidin as the marker. In cyclic gilts, WGA and DBA stained more strongly in the isthmus than the ampulla on both cycle days. Staining with PNA was patchy, but greater on the apical surface of isthmic epithelium on day 16 than day 1. With UEA-I, staining was more intense in the ampulla on day 16 than day 1. Staining by PNA and UEA-I was suppressed in both the ampulla and isthmus with estradiol treatment. DBA staining was suppressed in the isthmus with both estradiol and progesterone treatments. Cilia were labelled by all lectins except WGA and PNA. Thus glycosylation patterns vary in the porcine oviduct with region and hormonal state, reflecting the variety of events that may be influenced.

Ultrastructural distribution of lectin-binding sites on gastric superficial mucus-secreting epithelial cells

Normal human gastric epithelial cells were examined by electron microscopy using each of five biotinylated lectins [Ulex europaeus agglutinin I (UEA-I), peanut agglutinin (PNA), wheat germ agglutinin (WGA), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA)] as a probe. We employed 35 gastric surgical specimens removed from complicated peptic disease. The lectin-binding sites were revealed with streptavidin-colloidal gold complex. All specimens were embedded in Spurr and LR White resins. In superficial foveolar epithelial cells, the lectins used were generally positive in all cell types (mainly UEA-1 and PNA) on the Golgi region and mucus cytoplasmic vacuoles, with many variations among cells in the same case. On the other hand, extracellular mucus was negative for WGA. Labelling with PNA revealed a biphasic pattern (peripheral positivity) on mucous droplets in surface and foveolar cells. The cis side of the Golgi apparatus was labelled with SBA and PNA and rough endoplasmic reticulum with SBA (only five cases). Lectin-binding variability could be related to heterogeneous composition of gastric mucus. Our results with SBA suggest initiation of O-glycosylation at the Golgi apparatus; however a role of the rough endoplasmic reticulum cannot be excluded (N-glycosylation). We propose the following sequence of sugar addition to the carbohydrate side-chains of gastric glycoproteins: (1) GaNAc (Golgi apparatus cis-side), (2) GlcNAc (Golgi apparatus intermediate face), (3) GalNac or Gal, alpha-L-fucose (Golgi apparatus trans-side).

Detection of lectin binding sites in the trophoblast of cattle during early pregnancy

In the present study we report on the histotopographical distribution of lectin binding sites in the trophoblasts of day 18 to day 40 bovine embryos, using the FITC-labeled lectins BPA, Con A, DBA, GS I, GS II, MPA, PNA, SBA, UEA I and WGA. Lectin binding sites localized in giant binucleate cells differ from those localized in uninucleate cells, indicating changes in the biochemical structure of cell surfaces taking place during differentiation. In the trophoblast of the day 40 embryo, a distinct staining of uninucleate cells was seen after incubation with GS I, Con A and MPA, demonstrating N-acetylgalactosamine (GS I), Mannose (Con A) and Galactose (MPA) moieties, whereas giant binucleate cells showed intense reactions after incubation with DBA and WGA, indicating presence of N-acetylgalactosamine (DBA) and N-acetylglucosamine (WGA). GS II (specific for N-acetylglucosamine), SBA (specific for N-acetylgalactosamine) and UEA I (specific for L-Fucose) showed no affinity toward any of the examined tissues. We assume, that carbohydrate moieties in trophoblast cells play an important role in fetomaternal cell-cell adhesion and cell migration during implantation and placentation period.

The lectin binding characteristics of spontaneous and phenobarbitone induced hepatic lesions in C3H/He mice.

The surface membrane glycoprotein patterns of spontaneous hepatic nodules, phenobarbitone induced nodules and hepatocellular carcinoma were studied in the C3H mouse using lectin histochemistry. The lectin binding patterns of hepatocellular carcinoma cells were markedly different to those of non-tumour cells and similar to the pattern in chemically induced hepatocellular carcinomas. This supports the hypothesis that changes in surface glycoprotein are a consistent feature associated with malignancy. Similar changes in the distribution of lectin binding sites were also seen in the phenobarbitone induced eosinophilic nodules and in a proportion of spontaneous basophilic nodules. Two populations of early basophilic nodules were identified on the basis of their lectin binding patterns, and this may indicate a link between one nodular type and carcinoma.

Distribution of lectin-binding sites in oxyntic and chief cells of isolated rabbit gastric glands

The distribution of lectin-binding sites in oxyntic and chief cells of isolated rabbit gastric glands was determined with seven fluoresceinated lectins, to ascertain which lectins might best be used in the biochemical characterization of cell membranes and glycoproteins of these two cell types. Oxyntic cell canaliculi were labeled by wheat germ, Helix pomatia, and peanut lectins, suggesting a predominance of N-acetylhexosamines. Tubulovesicles were heavily stained by wheat germ, Helix pomatia, and Ricinus communis I lectins, indicative of N-acetylhexosamine- and galactose-containing glycoconjugates. Diffuse oxyntic cell cytoplasmic staining was observed with the mannose-binding lectin concanavalin A. This lectin, along with wheat germ, soybean, Helix pomatia, and Ricinus communis I lectins, bound to oxyntic cell basolateral membranes, indicating mannose, N-acetylhexosamine, and galactose residues. Chief cell apical membranes were labeled with peanut, Ricinus communis I, Helix pomatia, and Ulex europaeus lectins, suggesting a predominance of N-acetylhexosamine, galactose, and fucose residues. None of the lectins demonstrated any significant affinity for chief cell cytoplasm or basolateral membrane. Ulex europaeus agglutinin-binding sites were additionally concentrated in lateral intercellular spaces. The results of this study indicate that the range of utility of isolated rabbit gastric glands can be expanded to include histochemical work. In addition, the data suggest the applicability of lectin affinity chromatography in the isolation and characterization of oxyntic and chief cell membranes and glycoproteins.

Backscattered electron imaging of lectin binding sites in tissues following freeze‐fracture cytochemistry

Recently, cytochemical techniques have been applied for localizing membrane components; however, transmission electron microscopy only provides two-dimensional information about their distribution. Scanning electron microscopy, on the other hand, offers the possibility of examining the three-dimensional architecture of biological samples. The fracture-label cytochemical technique was combined with the backscattered electron imaging (BEI) mode of the scanning electron microscope to visualize the in vivo distribution of lectin binding sites on freeze-fractured biological membranes in tissues and cells. Pancreatic and testicular tissues, fixed with glutaraldehyde, were freeze-fractured and labeled with Helix pomatia lectin-gold or Ricinus communis I-gold complexes. The labeled specimens were then critical-point dried and replicated with platinum-carbon for routine transmission electron microscopy or with carbon alone for BEI. Lectin-gold labeling of fractured plasma and intracellular membranes observed with BEI showed a labeling pattern similar to that seen by the replica method. However, BEI-fracture-label provided additional information about the distribution of the labeling with respect to three-dimensional organization of tissues and cells. Large sample areas could be examined, making this technique particularly useful as a survey method for specimens that are either differentially labeled or composed of heterogenous cell populations.

Distribution of lectin binding sites in the colonic mucosa in ulcerative colitis

The distribution of binding sites for three lectins labeled with rhodamine was studied by using immunofluorescence microscopy in the colonic mucosa in ulcerative colitis.Binding sites for Dolichos biflorus agglutinin (DBA), a lectin specific to terminal α-N-acetylgalactosamine residue, and soy bean agglutinin (SBA), specific to teminal α or β-N-acetylgalactosamine residue of surface glycoprotein in the endoscopically uninvolved mucosa in ulcerative colitis were almost equal in distribution to those of normal human colon. These two lectins were markedly decreased in distribution in the endoscopically involved mucosa, whereas they were also decreased in the boundary zone which was considered as locating between the involved and the uninvolved portion. On the other hand, there was no definite difference in binding sites for concanavalin A (Con A), a lectin specific to terminal α-mannopyranoside, α-glucopyranoside, between normal colon and ulcerative colitis.These results suggest that a quantitative and/or qualitative alteration in the colonic mucin has an important role in the colonic mucosa in ulcerative colitis. And lectins may be developed into a powerful probe for the detection of biochemical change in the colonic mucosa in ulcerative colitis.

Changes in the glycosylation pattern during embryonic development of mouse kidney as revealed with lectin conjugates.

Distribution of lectin-binding sites in adult and developing mouse kidney was studied with fluorochrome- and peroxidase-coupled lectins. Effects of fixation methods on lectin-binding patterns were also compared. Un-induced mesenchymal cells and ureter bud of the early metanephros reacted with Concanavalin A, Lens culinaris, Ricinus communis I, and wheat germ agglutinins, whereas binding sites for both soybean and peanut (PNA) agglutinins were seen only in ureter bud tissue. On induction, PNA positivity rapidly appeared in the induced, condensed areas of the metanephrogenic mesenchyme. Early glomeruli expressed heterogeneously terminal galactosyl and N-acetylgalactosaminyl moieties in the podocytes. Later, these sites disappeared and were apparently covered by sialic acids. Endothelia also displayed a comparable sialylation of terminal saccharide moieties during maturation. Binding sites for many of the above lectins were also found in the developing proximal and distal tubules. Terminal fucosyl residues, characteristic of mature proximal tubules, appeared during day 13 of development. Dolichos biflorus agglutinin reactivity, typically seen in the collecting ducts, appeared by day 13. Griffonia simplicifolia-I-B4 isolectin reactivity was exclusively localized to endothelial in adult kidney cortex, but in embryonic kidneys reactivity with collecting duct and podocytes was also seen. These results suggest that the compartmentalized expression of cell glycoconjugates in adult mouse kidney is acquired in a sequential manner during development. Such sequential appearance of the mature glycosylation pattern probably reflects functional maturation of the nephron.

Lectin-binding sites in Paget's disease.

The presence and distribution of lectin-binding sites on neoplastic cells of Paget's disease was studied using fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA), and FITC-conjugated wheatgerm agglutinin (WGA), and compared with such lectin-binding sites on keratinocytes, and cells of eccrine glands, apocrine glands, and mammary glands. Neoplastic cells of both mammary and extramammary Paget's disease showed cytoplasmic staining with both lectins. There were however fewer stained cells in mammary Paget's disease than in extramammary Paget's disease. The cytoplasmic staining of lectin-binding sites in cells of apocrine glands was in sharp contrast to the cell-surface staining seen on keratinocytes, or cells of eccrine glands or mammary glands. These results indicate that the lectin-binding sites of neoplastic cells of Paget's disease more closely resemble those of cells of apocrine glands than of keratinocytes, cells of eccrine glands or cells of mammary glands.

Distribution of lectin binding sites in rabbit oviduct.

The carbohydrate histochemistry of the rabbit oviduct has been examined by the use of four lectins conjugated with horseradish peroxidase as histochemical reagents. Each lectin gave a very distinct typical pattern of binding, but for each lectin there was no difference between the distribution of binding sites in ampulla and isthmus. Wheat germ lectin bound exclusively with the connective tissue of the oviduct folds; winged pea lectin was detected only in the ciliated cells; peanut lectin binding sites were visualized in the secretory cells; the binding reactivity of soybean lectin was limited to the basal part of the cilia. Although it is very difficult at present to correlate the distribution of lectin binding sites with the function of the positive cells, some hypotheses have been advanced.

Histochemical observations of lectin-binding sites and keratin distribution in calcifying epithelioma of Malherbe.

Histochemical distribution of lectin binding sites and keratin protein was studied for epithelial components, stromal elements and giant cells in calcifying epithelioma of Malherbe as well as for normal hair follicles. The HRP (horseradish peroxidase) -conjugated lectin technique was employed; the lectins used were Con A, PNA, RCA-1, DBA, SBA, WGA and UEA-1. Immunocytochemical detection of keratin protein was made using the indirect PAP technique. Basophilic cells and shadow cells in the tumor epithelial components revealed histochemical properties similar to those of matrix cells and cortex cells in growing hair follicles, respectively. Most prominent lectin binding was observed in Con A staining. In the tumor stroma, many fibrillar elements which were distinguished as immature or mature hair were found by lectin and keratin staining. Tumor giant cells showed a stronger staining for Con A and keratin, which were enhanced following treatment with amylase. The staining intensity of lectins was usually decreased in both epithelial cells and connective tissue stroma following treatment with neuraminidase.