Effect on fertilization and localized binding of lectins in the ascidian Phallusia mammillata

The effect of concanavalin A (conA), fucose-binding protein (FBP), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA) on fertilization of the ascidian Phallusia mammillata was investigated. ConA, FBP, and RCA had no influence on fertilization and did not bind to the chorion or sperm, as determined with FITC-conjugated conA and by electron microscopy with gold-labelled FBP. WGA (100 μg/ml) prevented fertilization of eggs by sperms in concentrations which gave 100% fertilization in controls (2 × 10⁷ sperm/ml). N-Acetyl-glucosamine (50 mM) abolished the effect of WGA, whereas an excess (100 mM) of this competitive sugar alone did not affect fertilization. FITC-conjugated and gold-labelled WGA revealed binding sites on the chorion, but not on follicle cells nor sperms. Electron microscopy showed that WGA gold-markers are bound to the fibrillar network forming the outer layer of the chorion and indicate that WGA inhibits fertilization by interfering with sperm binding to the chorion. Binding of WGA to the chorion may either mask sperm binding receptors or cause chorion resistance to sperm enzymes.     

Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens.

The cell surface glycoproteins of goat epididymal maturing spermatozoa have been investigated using lectins as surface probes that interact with specific sugars with high affinity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epididymal sperm agglutination, whereas RCA2, kidney beans lectin and peanut agglutinin caused much lower or little agglutination of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm for binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-labelled lectins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC-WGA binds to the acrosomal cap region of the head. The FITC-RCA2 mainly labelled the posterior head of mature cauda sperm. However, no WGA-specific glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather than glycoproteins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-Sepharose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunoblot of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunoresponsive.     

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.     

137 Gamete Recognition and Sexual Isolation in a Red Alga,Aglaothamnion Byssoides (Rhdophyta)

The binding of fluorescein isothiocyanate (FITC) conjugated lectins to gametes of Aglaothamnion byssoides Itono during the fertilization was studied by the use of confocal microscope. The physiological effects of lectins and carbohydrates on gamete binding were also examined. Three lectins, concanavalin A (ConA), Soybean agglutinin (SBA) and wheat germ agglutinin (WGA) bound to the surface of spermatia, but each lectin labeled different region of the spermatium. SBA bound only to the spermatial appendages but ConA bound to the whole spermatial surface except spermatial appendages. WGA labeled narrow region which connects spermatial body and appendages. During fertilization, ConA and WGA specific substances on the spermatial surface moved towards the area contacting with trichogyne and accumulated on the surface of fertilization canal. Spermatial binding to trichogynes was inhibited by pre‐incubation of spermatia with SBA, while trichogyne receptors were blocked by the complementary carbohydrate, N‐acetyl‐D‐galactosamine. WGA and its complementary carbohydrate had little effect on gamete binding. For searching the step of sexual isolation, crossing experiment was performed between Aglaothamnion byssoides and twelve other red algal species. Results showed that the gamete recognition was genus‐specific: the gametes bound freely with their partners of the same genus. When two species from same genus were crossed, sexual isolation occurs gradually during the fertilization process. Therefore, sexual isolation in red algae appears to be determined by multi‐step process and gamete binding is the initial step.     

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)     

138 Molecular Evolution of Glutamine Synthetase In Protists

The binding of fluorescein isothiocyanate (FITC) conjugated lectins to gametes of Aglaothamnion byssoides Itono during the fertilization was studied by the use of confocal microscope. The physiological effects of lectins and carbohydrates on gamete binding were also examined. Three lectins, concanavalin A (ConA), Soybean agglutinin (SBA) and wheat germ agglutinin (WGA) bound to the surface of spermatia, but each lectin labeled different region of the spermatium. SBA bound only to the spermatial appendages but ConA bound to the whole spermatial surface except spermatial appendages. WGA labeled narrow region which connects spermatial body and appendages. During fertilization, ConA and WGA specific substances on the spermatial surface moved towards the area contacting with trichogyne and accumulated on the surface of fertilization canal. Spermatial binding to trichogynes was inhibited by pre-incubation of spermatia with SBA, while trichogyne receptors were blocked by the complementary carbohydrate, N-acetyl-D-galactosamine. WGA and its complementary carbohydrate had little effect on gamete binding. For searching the step of sexual isolation, crossing experiment was performed between Aglaothamnion byssoides and twelve other red algal species. Results showed that the gamete recognition was genus-specific: the gametes bound freely with their partners of the same genus. When two species from same genus were crossed, sexual isolation occurs gradually during the fertilization process. Therefore, sexual isolation in red algae appears to be determined by multi-step process and gamete binding is the initial step.     

Molecular demarcation of surface domains as established by label-fracture cytochemistry of boar spermatozoa

We used "label-fracture" (J Cell Biol 99:1156, 1984) to establish high-resolution maps of wheat germ agglutinin (WGA) and concanavalin A (ConA) receptor sites on the cell surface of boar spermatozoa and to investigate the possible association of these receptors to integral membrane components. Label-fracture reveals intense WGA labeling over the region of the plasma membrane that overlies the acrosome, including the equatorial segment. The density of WGA receptors decreases from the post-acrosomal area to the posterior ring. The WGA receptor domain changes abruptly into a microdomain with an unusually high density of WGA receptors over a sharply delimited, particle-free zone at the base of the head. Over the tail, the density of WGA receptors in the tail is high and uniform over the midpiece, annulus, and principal piece, but narrow patches of rectilinear arrays of pits close to the annulus are not labeled. Labeling of the entire sperm head by ConA is both intense and uniform, except for the particle-free zone at the base of the head, which is barren of receptors. Over the tail, ConA labeling is strong over the midpiece, absent over the annulus, and sparse over the principal piece and the end piece. In contrast to WGA, ConA receptors co-distribute with the intramembrane particles. Our results confirm the potential of label-fracture for high-resolution mapping of the distribution of cell surface receptors. They show that in this specialized cell membrane domains can be sharply defined, i.e., apparent without free lateral diffusion of components.     

The Cell Surface of Trypanosoma musculi Bloodstream Forms. II. Lectin and Immunologic Studies*

SYNOPSIS. Living Trypanosoma musculi bloodstream trypomastigotes were agglutinated specifically with concanavalin A (ConA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose-binding protein (FBP). The agglutination with these lectins of living cells from which the coat was removed by trypsinization was the same as with intact trypanosomes. Glutaraldehyde or formalin fixation did not affect the results with regard to agglutination with WGA, SBA, and FBP, but lower agglutination with ConA was observed upon fixation. By using a dense iron-dextran marker many fewer ConA marker particles were localized at the fine structural level in the intact than in trypsin-treated trypanosomes. On the basis of the results obtained by agglutination and electron microscopy, it is likely that fixation cross-links intact surface-coat components associated with the ConA binding sites. It is evident from the studies in which lectins were employed that ligands containing α-D-mannose, N-acetylglucosamine, N-acetylgalactosamine, and α-L-fucose are randomly distributed in the outer surface of the pellicular and flagellar membranes of T. musculi trypomastigotes. Results obtained with α-amylase- and dextranase-treated trypanosomes suggested that lectin-binding sugar ligands in the cell surface were not directly associated with α-1,4 or repetitive α-1,6 glucan-bonded polysaccharide moieties. Similar conclusions can be drawn on the basis of neuraminidase treatment with regard to N-acetylated neuraminic acids. After thorough washing, intact, but not trypsin-treated trypomastigotes were agglutinated specifically with antisera against whole mouse serum and against mouse IgG. Evidently, adsorbed constituents of mouse serum are regular components of the T. musculi surface coat. After incubation in dilute whole mouse serum or in mouse IgG solutions, also the trypsinized cells were agglutinated by the 2 antisera. No such results were obtained with trypsinized cells incubated in serum-free buffers. It was concluded that mouse serum proteins were readily readsorbed on, and firmly bound to the trypsinized cells' surfaces. Specific agglutinations were obtained with trypsinized cells after incubation in dilute rat, rabbit, bovine, and human sera and in solutions of rat and rabbit IgG in reactions with the corresponding antisera. It seems, therefore, that the host serum proteins are adsorbed nonspecifically to the cell surface of trypsinized T. musculi bloodstream forms. When examined by electron microscopy, the intact trypomastigotes were covered by an ununiform, slightly granular, fibrillar extracellular coat, applied to the entire outer lamina of the pellicular and flagellar membranes. No indication of such a coat was noted in the trypsinized organisms. Flocculent surface coat-like matrix could, however, be discerned in cells which, after trypsinization, were incubated in various sera.     

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas

Summary Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): l-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis-to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.     

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas

Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): L-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis- to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.     

Lectin-binding pattern on the surface epidermis of Ambystoma tigrinum larvae

Summary The surface epidermis of Ambystoma tigrinum larvae was examined at the light- and electron-microscope levels using five different lectin conjugates as probes for the detection of sugar residues on the cell membranes. Concanavalin A (Con-A), wheat-germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Dolichos biflorus agglutinin and soybean agglutinin (SBA) conjugates clearly labelled the surface cells, especially their apical surfaces. At electron microscopy, the labelling on plasma membranes was found to exhibit regional differences. Among the lectins tested WGA displayed a particularly characteristic binding pattern. WGA also bound to basolateral cell surfaces, including the tight-junction zone wich was also stained by the RCA-I conjugate. The different labelling intensity and staining patterns obtained with the conjugates indicated the polarity of the cell surfaces. It is also assumed that the WGA staining of the basolateral membranes and intercellular spaces reflected transcellular transport, which is facilitated by acidic glycoconjugates. Other functional aspects of the polarized distribution of the lectin conjugates were also correlated with the receptor sites of certain sugar residues.     

Fluorophore-conjugated lectin labeling of the cell surface of isolated male and female gametes,central cells and synergids before and after fertilization in maize

Three fluorescein isothiocyanate (FITC)-conjugated lectins, Canavalia ensiformis agglutinin (Con A), Triticum vulgaris agglutinin (WGA) and Phaseolus vulgaris erythroagglutinin (PHA-E), were used as probes to localize sugar moieties of glycoconjugates on the cell surface of isolated maize sperm, egg, central, antipodal cells, synergids, and in vitro- and in vivo-fertilized zygotes. Fluorescence signals on the surface of the cells were due to specific binding. Calcium was necessary for WGA and PHA-E binding and enhanced Con A labeling. Differences in glycoconjugate composition of the membranes of gametes and other embryo sac component cells were found. FITC-Con A strongly labeled egg and central cells, but labeled sperm only weakly. FITC-WGA binding sites were detected on egg, but not sperm cells. Con A and WGA binding sites were equally distributed around egg and central cell protoplasts. FITC-PHA-E binding sites were not found on sperm and egg cells before fertilization. Binding sites of these lectins were located on synergids, especially on their filiform apparatus. Interestingly, WGA binding to egg cells was enhanced after fertilization, whereas PHA-E binding to egg cell membranes could only be detected after fertilization. These results suggest the occurrence of fertilization-induced changes in glycoconjugate composition of the maize egg cell membrane. An increase in the number of WGA and PHA-E binding sites was also observed on newly formed cell walls of cultured two-celled embryos derived from in vitro-produced zygotes.     

Lectin binding to injured corneal endothelium mimics patterns observed during development

Summary Fluorochrome conjugated lectins were used to observe cell surface changes in the corneal endothelium during wound repair in the adult rat and during normal fetal development. Fluorescence microscopy of non-injured adult corneal endothelia incubated in wheat-germ agglutinin (WGA), Concanavalin A (Con A), and Ricinus communis agglutinin I (RCA), revealed that these lectins bound to cell surfaces. Conversely, binding was not observed for either Griffonia simplicifolia I (GS-I), soybean agglutinin (SBA) or Ulex europaeus agglutinin (UEA). Twenty-four hours after a circular freeze injury, endothelial cells surrounding the wound demonstrated decreased binding for WGA and Con A, whereas, RCA binding appeared reduced but centrally clustered on the apical cell surface. Furthermore, SBA now bound to endothelial cells adjacent to the wound area, but not to cells near the tissue periphery. Neither GS-I nor UEA exhibited any binding to injured tissue. By 48 h post-injury, the wound area repopulates and endothelial cells begin reestablishing the monolayer. These cells now exhibit increased binding for WGA, especially along regions of cell-to-cell contact, whereas, Con A, RCA and SBA binding patterns remain unchanged. Seventy-two hours after injury, the monolayer is well organized with WGA, Con A and RCA binding patterns becoming similar to those observed for non-injured tissue. However, at this time, SBA binding decreases dramatically. By 1 week post-injury, binding patterns for WGA, ConA and RCA closely resemble their non-injured counterparts while SBA continues to demonstrate low levels of binding. In early stages of its development, the endothelium actively proliferates and morphologically resembles adult tissue during wound repair. The 16-day fetal tissue is mitotically active, does not exhibit a well defined monolayer, and demonstrates weak fluorescence binding for WGA, Con A and RCA. Conversely, SBA binding is readily detected on many cell surfaces. By 19 days in utero, the endothelial monolayers becomes organized and cell proliferation greatly diminishes. WGA, Con A and RCA now exhibit binding similar to that seen in the adult tissue. SBA binding is not detected at this time. Thus, changes in lectin binding during wound repair of the adult rat corneal endothelium mimic changes in lectin binding seen during the development of the tissue.Supported by grant EY-06435 from The National Institutes of Health     

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.     

Polarized distribution of binding sites for concanavalin A and wheat-germ agglutinin in the zona pellucida of goodeid oocytes (teleostei)

Summary Zonae pellucidae of the viviparous goodeid teleosts Girardinichthys viviparus, Xenoophorus captivus, and Xenotoca eiseni were investigated ultrastructurally, and binding sites for ConA and WGA were localized on cross-sections using a colloidal gold technique. In late stages of development, the oocytes are surrounded by a three-zonated acellular matrix multiply perforated by pore canals allowing long microvilli of the oocyte to penetrate interstices of the follicle epithelium. Together, the surface of the microvilli and zona pellucida is coated by a thin layer of homogeneous slightly electron-dense material. In early oogenesis, the thin acellular layer is entirely packed with binding sites for WGA, whereas those for ConA occur only sparsely. Three-zonated zonae pellucidae amply contain both WGA and ConA receptors. The asymmetric labelling pattern obtained with both lectin protein gold preparations indicates a polarized organization of the different glycoconjugates. WGA receptors are concentrated within the outer region of the zona pellucida. Labelling with ConA-HRP-Au complexes produced heavy deposits of marker beads within the inner two thirds of the zona pellucida and weak labelling of the superficial coat. After prolonged digestion with neuraminidase, WGA binding sites were no longer detectable.Supported by the Deutsche Forschungsgemeinschaft (Schi 268/1-1)     

Cell type-specific binding of Ricinus lectin to murine cerebellar cell surfaces in vitro

Summary The binding of several plant lectins, Concanavalin A (ConA), Lens culinarisA (LCA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin 120 (RCA120) to cell surfaces of developing mouse cerebellar cells was assayed by the use of fluorescein isothiocyanate (FITC)-conjugated compounds. Freshly dissociated, live single-cell suspensions from 6-day-old mouse cerebellum contain 93% ConA, 99% LCA, 98% WGA, and 59% RCA 120-positive cells with ring fluorescence. Of the RCA 120-positive cells, 4% express a high and 55% a lower or very low number of lectin receptors. Flow cytometric analysis of fluorescent lectin binding yields results qualitatively similar to those obtained by scoring positive and negative cells in the fluorescence microscope.In monolayer cultures of 6-day-old mouse cerebellum practically all cells express receptors for ConA, LCA, and WGA, whereas RCA 120 binding sites are absent from neurons with small cell bodies (granule, basket and stellate cells) and present in large number on neurons with large cell bodies (Purkinje and possibly Golgi Type-II cells) and fibroblasts. RCA 120 receptors are weakly expressed on astro-and oligodendroglia. Cell type-specific expression of RCA 120 receptors is constant throughout all ages studied (embryonic day 13 to postnatal day 9). At early embryonic ages the proportion of highly fluorescent neurons with large cell bodies is significantly increased.     

Lectin-binding patterns on the plasma membranes of dissociated rat liver cells

Summary Carbohydrate moieties on the surface of dissociated rat liver cells were examined electron microscopically using ferritin-or horseradish peroxidase (HRP)-conjugated lectins as probes. Rat liver was fixed by perfusion with 0.7% glutaraldehyde via the portal vein and dissociated into single cells with gentle homogenization. Concanavalin A (ConA), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA) bound almost evenly to the entire cell surface of hepatocytes as well as of endothelial cells. Ulex europaeus agglutinin I (UEA-I) and peanut agglutinin (PNA) revealed no binding to any region. Dolichos biflorus agglutinin (DBA) was found to bind exclusively to the sinusoidal surface of hepatocytes and to endothelial cell surfaces. Soybean agglutinin (SBA)-binding was restricted to the endothelial cell surfaces and part of the sinusoidal microvilli of hepatocytes. Regional differences in lectin-binding pattern were visualized between the sinusoidal and the lateral or bile-canalicular surfaces of the hepatocytes. A polarity may exist on the hepatocyte cell surfaces in terms of the distribution pattern of the carbohydrate moieties, especiàlly those of N-acetylgalactosamine.     

Morphological evidence of sperm maturation in the ampulla ductus deferentis of the bull

The present lectin histochemical comparison of cauda epididymal and ampullary bovine sperm was conducted to investigate whether ampullary secretions are involved in altering the plasma-membrane glycoconjugates of sperm. A marked redistribution of glycoconjugates between sperm from these two regions was indeed revealed on the basis of changes in binding patterns of the following fluoroscein-isothiocyanate (FITC)-labelled lectins: wheat-germ agglutinin (WGA), Maclura pomifera agglutinin (MPA), Griffonia simplicifolia I agglutinin (GS I) and Bauhinea purpurea agglutinin (BPA). This was evidenced in the first three cases by a relative reversal of staining intensity between the acrosomal and post-acrosomal regions, and by a pronounced increase in the staining of the midpiece. Changes in the distribution of BPA binding sites were limited to the latter phenomenon. The results are compared with previous findings, discussed in the context of the hypermotility characteristic of ampullary sperm and related to previously reported differences in the lectin-binding patterns of the luminal and glandular epithelia.     

Lectin binding to injured corneal endothelium mimics patterns observed during development

Fluorochrome conjugated lectins were used to observe cell surface changes in the corneal endothelium during wound repair in the adult rat and during normal fetal development. Fluorescence microscopy of non-injured adult corneal endothelia incubated in wheat-germ agglutinin (WGA), Concanavalin A (Con A), and Ricinus communis agglutinin I (RCA), revealed that these lectins bound to cell surfaces. Conversely, binding was not observed for either Griffonia simplicifolia I (GS-I), soybean agglutinin (SBA) or Ulex europaeus agglutinin (UEA). Twenty-four hours after a circular freeze injury, endothelial cells surrounding the wound demonstrated decreased binding for WGA and Con A, whereas, RCA binding appeared reduced but centrally clustered on the apical cell surface. Furthermore, SBA now bound to endothelial cells adjacent to the wound area, but not to cells near the tissue periphery. Neither GS-I nor UEA exhibited any binding to injured tissue. By 48 h post-injury, the wound area repopulates and endothelial cells begin reestablishing the monolayer. These cells now exhibit increased binding for WGA, especially along regions of cell-to-cell contact, whereas, Con A, RCA and SBA binding patterns remain unchanged. Seventy-two hours after injury, the monolayer is well organized with WGA, Con A and RCA binding patterns becoming similar to those observed for non-injured tissue. However, at this time, SBA binding decreases dramatically. By 1 week post-injury, binding patterns for WGA, ConA and RCA closely resemble their non-injured counterparts while SBA continues to demonstrate low levels of binding. In early stages of its development, the endothelium actively proliferates and morphologically resembles adult tissue during wound repair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycoconjugates of human sperm surface. A study with fluorescent lectin conjugates and lens culinaris agglutinin affinity chromatography

Distribution of glycocompounds in human spermatozoa was studied by using fluorescent lectin-conjugates. Con A bound predominantly to acrosomal and posterior head regions whereas RCA I bound to the acrosomal region of intact spermatozoa, stained in suspension. Other lectins used (LCA, WGA, SBA, PNA) stained the the entire sperm surface. In airdried sperm smears binding of both Con A and RCA I were identical with the staining pattern obtained with living cells whereas LCA, WGA, SBA and PNA now bound heavily into acrosomal region. As a similar staining pattern was obtained with permeabilized sperm cells, this staining is apparently due to binding to intracellular structures. The efficiency of Lens culinaris agglutinin affinity chromatography in purification of human sperm glycoproteins was tested after their external radiolabelling with the neuraminidase/galactose oxidase/sodium borohydride method. 22% of applicated radioactivity could be eluted from the column with the specific inhibitory saccharide, and most of the radiolabelled surface glycoproteins of the whole sperm lysate, were also present in the LCA affinity column eluate. LCA affinity chromatography seems thus be an effective method to enrich membrane glycoproteins of human spermatozoa.