Evaluation of structural and secretory glycoconjugates in normal human jejunum by means of lectin histochemistry

Summary The labelling pattern of eight lectins was studied in jejunal samples from ten normal subjects, in order to define the normal distribution of structural and secretory glycoconjugates in the small bowel.The following lectins were studied by means of a peroxidase technique on formalin-fixed samples: Arachis hypogaea, Ricinus communis, Canavalia ensiformis, Lens culinaris, Phaseolus vulgaris, Triticum vulgaris, Ulex europaeus, Dolichos biflorus. Phaseolus vulgaris reacted with goblet cell mucus throughout the villus-crypt axis.Conversely Ulex europaeus, Dolichos biflorus and Triticum vulgaris lectin labelling of globet cells appeared to be confined to the upper part of the villi. This finding suggests that during cell migration from crypt to villus tip, the continuing maturation of goblet cells is associated with the differentiation of secretory carbohydrates, which probably parallels the cell maturation cycle. Lectin histochemistry appears to be a reliable tool for the study of structural and secretory glycoconjugates in the jejunal mucosa, and might be of value in the study of diseases in which the cell-maturation cycle in the small bowel is altered.     

Lectins as Markers of Human Epidermal Cell Differentiation

The expression of sugar residues on human epidermal cells was investigated by means of lectin binding, as a way of determining membrane structural changes occurring during the differentiation of the epidermis. Fourteen lectins of different sugar specificity were conjugated with fluorescein isothiocyanate (FITC-lectins) and tested in fluorescence microscopy on frozen sections of normal human epidermis. In parallel, FITC-lectins were tested on psoriatic-involved epidermis to visualize differences in the expression of sugar residues that might occur during abnormal epidermal differentiation. No labelling could be obtained with lectins from Bandeira simplicifolia I, Dolichos biflorus, Limulus polyhemus, Tetragonolobus purpureas, Ulex europeus I , and Triticum vulgaris (group 1 lectins). A "pemphigus-like" intercellular labelling of the whole epidermis, except the stratum corneum, was obtained with lectins from Canavalia ensiformis, Maclura pomifera, Phaseolus vulgaris , and Ricinus communis I (group 2 lectins). A selective intercellular labelling of the stratum spinosum and the stratum granulosum was seen in normal epidermis with lectins from Arachis hypogaea, Glycine max, Helix pomatia , and Sophora japonica (group 3 lectins). In psoriatic epidermis, not only the basal cell layer, but also cells from the adjacent lower stratum spinosum were found to be negative, using FITC-lectins of group 3. These data indicate that the expression of lectin binding sites in normal epidermis differs according to the maturation of the cell from the basal cell to the more mature keratinocyte in the stratum granulosum. They suggest that lectins may be used as markers of epidermal cells in various stages of normal and abnormal differentiation.     

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

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

Cell surface carbohydrate of Leishmania mexicana amazonensis: differences between infective and non-infective forms

The cell surface carbohydrates of Leishmania mexicana amazonensis (amastigotes and promastigotes, both infective and non-infective forms) were comparatively analyzed by agglutination assay employing 28 highly purified lectins, and by binding assay using 125I-labeled lectins. Among the D-GalNAc binding lectins, Bandeiraea simplicifolia-I, Dolichos biflorus, Phaseolus vulgaris and Glycine max were highly specific for the amastigotes, while that from Maclura aurantiaca selectively agglutinated promastigotes. The lectins from Wistaria floribunda, Phaseolus lunatus (D-GalNAc), Arachis hypogaea (D-Gal) and Triticum vulgaris (D-GlcNAc) were selective for the infective forms (both amastigotes and promastigotes), not reacting with the non-infective ones. Conversely, no parasite agglutination occurred with the L-fucose binding lectins Lotus tetragonolobus and Ulex europaeus-I. Binding studies with 125I-labeled lectins from Wistaria floribunda, Triticum vulgaris and Arachis hypogaea were performed to find whether unagglutinated non-infective promastigotes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the selectivity, demonstrated in the agglutination assays of these lectins for the infective promastigotes.     

Localization of carbohydrate components in rat colon with fluoresceinated lectins

Cryostat sections of rat descending colon were studied by fluorescence microscopy after exposure to conjugates of fluorescein isothicoyanate with lectins from Glycine max (soybean), Triticum vulgaris (wheat germ), Ricinus communis (castor bean), Ulex europaeus, (gorse), Dolichos biflorus (horse gram) and Canavalia ensiformis (concanavalin A) (Jack bean). No two lectins showed identical patterns of fluorescence. FITC-conjugates of soybean and D. biflorus lectins reacted strongly with the mucus present in the crypt lumens and with the surface (as well as cytoplasm) of the epithelial cells suggesting that these sites are rich in terminal, non-reducing, N-acetylgalactosamine residues. Wheat germ, R. communis, U. europaeus and concanavalin A-FITC conjugates did not stain mucus but showed fluorescence in the cytoplasm of absorptive cells as well as in the lamina propria and submucosa. The FITC-R. communis conjugate also reacted with structures in the apical portion of epithelial cells that may correspond to the Golgi apparatus.     

Glycoconjugates in normal human kidney. A histochemical study using 13 biotinylated lectins

The avidin-biotin-peroxidase complex technique was used with 13 lectins to study the glycoconjugates of normal human renal tissue. The evaluated lectins included Triticum vulgaris (WGA), Concanavalin ensiformis (ConA), Phaseolus vulgaris leukoagglutinin and erythroagglutinin (PHA-L and PHA-E), Lens culinaris (LCA), Pisum sativum (PSA), Dolichos biflorus (DBA), Glycine max (SBA), Arachis hypogaea (PNA), Sophora japonica (SJA), Bandeiraea simplicifolia I (BSL-I), Ulex europaeus I (UEA-I) and Ricinus communis I (RCA-I). Characteristic and reproducible staining patterns were observed. WGA and ConA stained all tubules; PHA-L, PHA-E, LCA, PSA stained predominantly proximal tubules; DBA, SBA, PNA, SJA and BSL-I stained predominantly distal portions of nephrons. In glomeruli, WGA and PHA-L stained predominantly visceral epithelial cells; ConA stained predominantly basement membranes and UEA-I stained exclusively endothelial cells. UEA-I also stained endothelial cells of other blood vessels and medullary collecting ducts. Sialidase treatment before staining caused marked changes of the binding patterns of several lectins including a focal loss of glomerular and tubular staining by WGA; an acquired staining of endothelium by PNA and SBA; and of glomeruli by PNA, SBA, PHA-E, LCA, PSA and RCA-I. The known saccharide specificities and binding patterns of the lectins employed in this study allowed some conclusions about the nature and the distribution of the sugar residues in the oligosaccharide chains of renal glycoconjugates. The technique used in this report may be applicable to other studies such as evaluation of normal renal maturation, classification of renal cysts and pathogenesis of nephrotic syndrome. The observations herein reported may serve as a reference for these studies.     

Human sperm surface mapping with lectins.

Ten fluorescein isothiocyanate (FITC)-linked lectins [Bauhimia purpurea, Concanavalin A, Dolichos biflorus (DBA), Griffonia simplicifolia I, Griffonia simplicifolia II, Maclura pomifera, Arachis hypogea (PNA), Glycine max, Ulex europaeus (UEA) and Triticum vulgaris agglutinin] have been used to study their binding features on the human ejaculate spermatozoa. Qualitative changes in the labeling pattern have been observed in unfixed and acetone-treated spermatozoa. Furthermore, ultrastructural localization of some of the colloidal gold-linked lectins, namely PNA, UEA and DBA, has been attempted to delineate the binding domains of the specific sugars on the sperm surface. It needs to be emphasized that flow-cytometric methods employed in our study, which provide quantitative slant to qualitative data, should be utilized to evaluate the functional status of the spermatozoa.     

Cytochemical localization of blood group substances in human salivary glands using lectin-gold complexes

We investigated localization of blood group antigens and their related substances in human labial salivary and submandibular glands by application of a post-embedding cytochemical staining procedure using lectin- or glycoprotein-gold complexes. Surgical tissue was obtained from 10 patients. Blood group-specific lectins, such as Dolichos biflorus agglutinin or Helix pomatia agglutinin (group A-specific), Griffonia simplicifolia agglutinin-I B4 (group B-specific), and Ulex europaeus agglutinin I (group H-specific) could recognize A, B, and H antigens, respectively, only in mature secretory granules (mature SG), which were found preferentially in cells in the late phase of the maturation cycle. In immature secretory granules (immature SG), which were found in cells in the early or middle phase of the maturation cycle, no binding with these lectins was observed. The Golgi complexes and endoplasmic reticula also were not labeled with these lectins. In blood group O and B secretors, blood group antigens were uniformly distributed throughout all the mature SG examined. However, in blood group A secretors, the distribution was heterogeneous, i.e., in some granules only H antigen was demonstrated, whereas in others both A antigens and a small amount of H antigens were detected. Among the blood group-nonspecific lectins, wheat germ agglutinin (WGA) was found to bind more preferentially to immature SG than to mature SG. This was demonstrated irrespective of the blood group and secretor status of the tissue donor, except that in blood group A secretors WGA bound strongly to some mature SG which possessed A antigen. We discuss the significance of cellular and subcellular mosaic distribution of blood group antigens in connection with morphological differences of secretory granules and the maturation cycle of mucous cells.     

Lectin-induced abnormalities of mouse blastocyst hatching and outgrowth in vitro

We have examined the role of cell surface glycoconjugates during mouse blastocyst maturation, hatching, attachment, and outgrowth by monitoring the influence of six lectins on blastocyst development in vitro. Two lectins, concanavalin A and wheat germ agglutinin were toxic to blastocysts at the concentrations used. Bandierea simplicifolia lectin 1 (BSL-1) induced abnormal growth, developmental arrest at the hatching stage, and some disruption of cell contacts. Culture with Lotus tetragonolobus lectin-1 (LTA-1) also disrupted cell contacts and caused developmental arrest. The remaining lectins, Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin (UEA), retarded blastocyst hatching and outgrowth but did not induce any major defects, although differentiation of the inner cell mass was limited by both. This study demonstrates that very low concentrations of lectins can disrupt blastocyst development, suggesting that exposed surface saccharide moieties may be involved in interactions between blastomeres and their environment.     

The carbohydrate components of corn prolamins]

This work was aimed to study the patterns of zein glycosylation. Zein proteins included 1-3% of sugars. The affinity of different lectins, such as concanavalin A (Con A), Lens culinaris lectin (LCL) and lectins of Arachis hypogaea (PNA), of Triticum vulgaris (WGA), of Dolichos biflorus (DBA), of Glycin max (SBA), of Lotus tetragonolobus (LTA), of Laburnum anagiroides (LAL), of Ricinus communis (RCA), of Phaseolus vulgaris (PHA) was used to analyze the glycosylation sites. All selected lectins interacted with zein proteins. It may serve a basis for determination of mannose, galactose, fucose and aminosugars. Some lectins were bound only by prolamines of some inbred lines, while others were connected with all lines.     

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line

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

Glycoproteins histochemistry of the gills of Odontesthes bonariensis (Teleostei, Atherinopsidae)

The histochemistry of glycoproteins (GP) in the mucous cells of the gills of the silverside Odontesthes bonariensis was identified with: (1) oxidizable vicinal diols; (2) sialic acid and some of their chain variants, carbon 7 ((7) C), carbon 8 ((8) C) or carbon 9 ((9) C); (3) sialic acid residues without O-acyl substitution and with O-acyl substitution at (7) C, (8) C or (9) C; (4) carboxyl groups and (5) sulphate groups. A battery of seven biotinylated lectins allowed GPs sugar residues to be distinguished. Mucous cells showed the presence of neutral, sulphated and sialylated GPs. Dolichos biflorus agglutinin (DBA) and Glycine max agglutinin (SBA) showed strong positive staining; Arachis hypogaea agglutinin (PNA), Ricinus communis agglutinin-I (RCA-I) and Triticum vulgaris agglutinin (WGA) showed moderate staining, while Ulex europaeus agglutinin-I (UEA-I) was completely negative.     

A comparison of lectin binding in rat and human peripheral nerve

Eleven different fluorescein- or peroxidase-conjugated lectins with different sugar-binding affinities were employed to analyze and compare glycoconjugates of rat and human peripheral nerves at the light microscopic level. A majority of lectins showed a distinct binding pattern in different structures of the nerve. Lectin binding was similar but not identical in rat and human nerves. Limulus polyhemus agglutinin did not stain any structures in rat or human nerves. In both species, all other lectins bound to the perineurium. Perineurial staining was intense with Canavalia ensiformis (Con A), Triticum vulgaris (WGA), Maclura pomifera (MPA); moderate with Glycine max (SBA), Griffonia simplicifolia-I (GS-I) and GS-II; weak with Ulex europaeus (UEA), Dolichos biflorus (DBA), and Ricinus communis (RCA). In the endoneurium of both species, ConA staining was intense, MPA and WGA moderate, SBA, GS-II, PNA, and RCA weak, and UEA and DBA absent. Interestingly, GS-I stained rat but not human endoneurium. Most lectins bound to blood vessels. GS-I bound to rat but not human, whereas UEA bound to human but not rat vessels. The results show that lectins can be used to reveal heterogeneity in sugar residues of glycoconjugates within neural and vascular components of nerves. They may therefore be potentially useful in detecting changes in glycoconjugates during nerve degeneration and subsequent regeneration after trauma or in pathological states.     

Heterogeneity of the blood group ABH antigens and variation in the expression of these antigens of secretory granules in human cervical glands. An electron microscopic observation using lectins and monoclonal antibodies

Cytochemical localization of blood group ABH antigens was examined in secretory cells of human cervical glands by application of a post-embedding lectin-gold as well as immuno-gold labeling procedure using monoclonal antibodies. Blood group specific lectins such as Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) and Ulex europaeus agglutinin-I (UEA-I) reacted with secretory granules but not with other cytoplasmic organellae such as nucleus and cell membrane. The reactivity of secretory granules with these lectins showed strict dependence on the blood group and secretor status of tissue donors. The binding patterns with these lectins were not homogeneous, but exhibited marked cellular and subcellular heterogeneity. Thus, for example, in blood group A individuals, some granules were stained strongly with DBA and others were weakly or not at all with the lectin. Such a heterogenous labeling with the lectin was observed even in the same cells. Similar results were obtained with UEA-I and GSAI-B4 staining in blood group O and B secretor individuals, respectively. Monoclonal antibodies likewise reacted specifically with the granules but they occasionally bound to some nucleus. The labeling pattern of the antibodies with the granules was essentially the same as those of lectins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental changes in the distribution of cecal lectin-binding sites of Balb-c mice.

The existence of lectin-binding sites was investigated in the cecum of Balb-c mice at seven developmental stages ranging from 18 days post conception (p.c.) to 8 weeks after birth. Nine horseradish-peroxidase-conjugated lectins (concanavalin A, Triticum vulgaris, Dolichus biflorus, Helix pomatia, Arachis hypogaea, Glycine maximus, Lotus tetragonolobus, Ulex europaeus, Limulus polyphemus) were applied to 5- to 7-microns thin paraffin sections of Bouin-fixed tissue. After DAB staining the sections were evaluated by light microscopy. It was shown that each lectin exhibits a unique developmental pattern. The adult binding patterns were established at the age of 3-4 weeks with only minor changes occurring thereafter. Considerable differences in binding patterns occurred not only between lectins of different groups but also between lectins with the same nominal monosaccharide specificity.     

Distinguishing benign from malignant pleural effusions by lectin immunocytochemistry

Since distinguishing malignant from benign cells in pleural effusions can be difficult, with reactive mesothelial cells simulating adenocarcinoma cells, the binding patterns of a battery of lectins on cells in eight benign and eight malignant effusions were studied using the avidin-biotin peroxidase complex method. The following lectins were used: concanavalin A, Dolichos biflorus agglutinin, peanut agglutinin, Phaseolus vulgaris agglutinin, Ricinus communis germ agglutinin, soybean agglutinin, Ulex europeaus agglutinin (UEA) and wheat germ agglutinin. Several patterns of staining were seen with the lectins, but only UEA was helpful in distinguishing between benign and malignant effusions. Sixty percent of the adenocarcinomas stained with UEA, whereas none of the cells in the benign effusions did. These results imply that UEA positivity is indicative of carcinoma and can be useful in separating reactive or atypical mesothelial cells from adenocarcinoma cells.     

The preputial gland of the Japanese serow Capricornis crispus: ultrastructure and lectin histochemistry

Preputial glands of the Japanese serow were found to consist of the modified sebaceous gland and apocrine gland in both sexes. In the modified sebaceous gland, cells transformed gradually toward the lumen from plentiful cytoplasm to a sponge-like network. Lipid droplets were observed in mitochondria of differentiating cells. By lectin histochemistry, differentiating cells were stained with Arachis hypogaea (PNA), Triticum vulgaris (WGA) and Canavalia ensiformis (Con A) lectins, while secretory cells of the apocrine gland revealed various stainings with PNA, Glycine max, Dolichos biflorus, WGA and Con A lectins. These findings suggest that glycoconjugates may have an active function in the preputial gland.     

Exposed and hidden lectin-binding epitopes at the surface of<Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

The presence of surface- and subsurface-located lectin-binding epitopes of Borrelia burgdorferi was examined by electron microscopy using a variety of gold-labeled lectins. Concanavalin A reacted predominantly with extracellular material adjacent to the spirochetes. Wheat germ agglutinin bound weakly to the surface of borreliae; however, alterations of the outer membrane by preincubation in 100 ppm Triton X-100 or boiling uncovered numerous periplasmic sites recognized by the lectin. The periplasmic flagella liberated by some cells after detergent treatment were labeled with concanavalin A, wheat germ agglutinin and Ulex europaeus agglutinin UEA-I. No surface-exposed or periplasmic epitopes for the lectins from Glycine max, Dolichos biflorus or Helix pomatia were detected.     

A versatile immunoadsorbent capable of binding lectins of various specificities and its use for the separation of cell populations

A procedure for cell fractionation using lectin-affinity chromatography is described. It consists of a single affinity adsorbent, hog gastric mucin blood group A+H substance covalently coupled to Sephadex or Sepharose, to which lectins of various specificities can bind. The complex formed, lectin in equilibrium hog A+H substance-Sephadex, then serves as an affinity probe for isolating and fractionating cells. The lectins from Ulex europaeus, Lotus tetragonolobus, Helix pomatia, Dolichos biflorus, and Phaseolus lunatus were used with the same blood group substance as adsorbent. The affinity columns retained erythrocytes with blood group specificity for the adsorbed lectin and thus fractionate cells in mixtures. Cells as well as lectins are eluted by specific sugar inhibitors. Mixtures of two kinds of cells can be separated when the proportion of the adsorbed cells is not too low.     

Opisthorchis viverrini: ultrastructure and cytochemistry of the glycocalyx of the tegument

The ultrastructure and cytochemistry of the glycocalyx of the tegument of Opisthorchis viverrini during maturation from newly excysted juvenile to adult stages were investigated using colloidal iron, ruthenium red and lectin stainings. The results showed that the glycocalyx was intensely stained by the first two dyes, thus indicating the presence of relatively high amounts of negative charges. However, the thickness and intensity of the staining decreased during the fluke's maturation. Binding studies using lectin probes on the surface of adult parasites showed that binding sites for Canavalia ensiformis (Con A), Triticum vulgaris (WGA) and Ricinus communis I (RCA I) were present in relative large amounts on the glycocalyx of the adult tegument, whereas those for Dolichos biflorus (DBA) were relatively fewer in number, and those for Ulex europaeus I (UEA I) were absent. The binding patterns of Con A, WGA, RCA I and DBA were generally similar, and the reaction product was uniformly distributed over the dorsal and ventral surfaces of the parasite's body. These bindings, therefore, indicate the presence of D-mannose/D-glucose, N-acetyl-D-glucosamine/sialic acid, D-galactose and N-acetyl-D-galactosamine residues on the glycocalyx of the adult tegument.