Use of lectin histochemistry method in the characterization of biopolymer glycosylation processes in duodenal gland cells

Potentialities of lectin histochemistry method for analysis of glycosylation processes within secretory epitheliocytes have been investigated using duodenal glands of cow and sheep as a model system. It has been ascertained, that con A- and LCA-binding sites (mannosglycans) localize exclusively in basal cytoplasm of duodenal gland epitheliocytes, which corresponds to rough endoplasmic reticulum. Manifestation of D-galactose, L-fucose, N-acetyl-D-galactosamine residues within oligosaccharide changes (PNA-, RCA-, WGA- and LAA-reactivity) was detected in the supranuclear region and apical cytoplasm of glandulocytes, that corresponds to Golgi complex, SBA-reactive glycoconjugates were determined only in poorly differentiated cells, forming initial parts of glandular ducts, but were absent in more differentiated cells of superficial and profound acini. The results obtained demonstrate fine prospects that are opened by the use of lectin-peroxidase kits in precise analysis of glycopolymers processing within functionally active and differentiating glandular cells.     

Changes in the cell surface coat during the development ofXenopus laevis embryos,detected by lectins

Summary The composition of the surface coat in embryonic cells ofXenopus laevis was examined by agglutination and fluorescent staining with lectins.Cells of early and mid gastrula stages were agglutinated by lectins specific for D-mannose, D-galactose, L-fucose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. No differences in agglutinability among ectoderm, mesoderm and endoderm cells were observed with lectins specific for D-mannose, D-galactose and N-acetyl-D-galactosamine, though agglutination of gastrula cells with fluorescent lectins revealed considerable differences in the intensity of lectin binding among cells within an aggregate. These differences in amount of lectin bound were not related to cell size or morphology. Patches of fluorescent material formed on the cells, suggesting that lectin receptors are mobile in the plane of the plasma membrane.In the early cleavage stages intensive lectin binding occurs only at the boundary between preexisting and nascent plasma membranes. The external surface of the embryo has few lectin receptors up to the late gastrula stage. The unpigmented nascent plasma membranes, when exposed to fluorescent lectins, do not assume any fluorescence distinguishable from the background autofluorescence of yolk, in stages up to the mid-blastula. From this stage onwards lectin binding was observed on the membranes of the reverse side of surface layer cells and on the membranes of deep layer cells. During gastrulation there is an accumulation of lectin-binding material on surfaces involved in intercellular contacts.The significance of lectin binding material for morphogenesis is discussed.     

Glyconjugates in epidermal, branchial and digestive mucous cells and gastric glands of gilthead sea bream, Sparus aurata, Senegal sole, Solea senegalensis and Siberian sturgeon, Acipenser baeri development.

Epidermal, branchial and digestive mucous cells, and the gastric glands of larvae/postlarvae (from hatching until 45 days posthatching) of three fish species (two teleostean and a chondrostean) were investigated using conventional histochemical methods (periodic acid schiff -PAS-, diastase-PAS; alcian blue pH 0.5, 1 and 2.5) in order to distinguish neutral and acidic (carboxylated and sulphated) glycoconjugates, as well as bromophenol blue reaction for identification of proteins. Additionally, the presence and distribution of sugar residues in the oligosaccharide side chains of glycoconjugates were investigated using horseradish peroxidase (HPR)-conjugated lectins (Con A, DBA, WGA and UEA-I). Most mucous cells (digestive, epidermal and branchial) of Siberian sturgeon, Acipenser baeri, sea bream, Sparus aurata and Senegal sole, Solea senegalensis larvae were PAS- and alcian blue- (pH 2.5 and 0.5) positive, with small variations between organs/tissues and species. Bromophenol blue reaction (general proteins) was positive in a minority of the mucous cells, usually in those cells which were PAS-negative. Proteins rich in sulphydryl (-SH) and/or disulphide (-S-S-) groups related with the glycoprotein nature of the glycoconjugates present in mucous cells were also observed. Epidermal, branchial and digestive mucous cells of all studied larvae did not contain glycogen or lipids. Con A lectin staining was negative in all mucous cells types of sea bream and sole, but oesophageal mucous cell of sturgeon were reactive to different lectin reactions, suggesting the presence of mannose -Man- and/or glucose -Glc-, L-fucose -Fuc- ; N-acetyl-D-galactosamine -GalNAc-, as well as N-acetyl-D-glucosamine- GlcNAc - and/or sialic acid -NANA- residues. Digestive mucous cells of all studied larvae were positive to WGA and DBA lectins. Epidermal and branchial mucous cells of sea bream and sole were Con A, DBA and UEA-I unreactive. However, mucous cells of sturgeon larvae were stained with UEA-I lectin. Gastric glands appear very early in sturgeon stomach larvae development (between 5-6 days posthatching) but rather late (around 40 days) during the ontogeny of sole and sea bream larvae. These glands contain neutral glycoproteins with Man and/or Glc, Fuc, GlcNAc- and/or sialic acid and rich in GalNAc- sugar residues, as well as proteins moderately rich in arginine, and others particularly rich in tyrosine and tryptophan.     

盲蝽科昆虫的食性

盲蝽科是半翅目最大的科 ,该科昆虫既有植食性和肉食性类群 ,又有杂食性类群 ,有很大的经济价值。作者综述了盲蝽科昆虫各食性类群中重要种类的寄主范围 ,指出在杂食性盲蝽中作为害虫的天敌作用远远大于对植物的危害 ,同时探讨了盲蝽科昆虫与植物的关系类型及食性的进化。     

Molecular anatomy of an endodermal gland: Investigations on mucus glycoproteins and cell turnover in Brunner's glands of didelphis verginiana using lectins and PCNA immunoreactivity

Brunner's glandsl are located in the submucosa of the proximal duodenum and are unique to mammalian species. The North American opossum (Didelphis virginiana) is generally regarded as a prototype marsupial that closely resembles fossil didelphids which can be placed at the beginning of mammalian evolution. The current investigation provided an opportunity for the analysis of secretory products from these glands in a species thought to be more closely related to earlier evolutionary forms. Extracts of Brunner's glands were subjected to SDS-PAGE and Western Blotting. The results indicate the presence of two high molecular weight PAS-positive glycoprotein bands. In addition to these two PAS-positive bands, several other glycoprotein bands were detected in the high molecular weight range that bind several lectins which typically recognize O-linked carbohydrates indicative of mucus type glycoproteins. The same lectins bind to glandular structures in tissue sections. Comparison of lectin binding sites with the pyloric glands of the stomach and duodenal goblet cells indicates that brunner's glands carbohydrate residues resemble those of the pyloric glands more closely than those of the duodenal goblet cells. The low cell turnover rate in brunner's glands is in contrast to the rapid turnover rate of goble cell precursors in the duodenal crypts. The mucus composition and the cell turnover rate correlate well with embryological data and suggest that Brunner's glands of Didelphis evolved from an epithelium more closely associated closely associated with the stomach than that of the duodenum as the topography of the gland would suggest. © Wiley-Liss, Inc.     

Production and characterization of the B chains of mistletoe toxic lectins

Mistletoe toxic lectins consist of two polypeptide chains: an enzymatically active A chain, which is a toxic component, and a disulfide-bonded B chain, which confers the lectin properties on the total molecule. Mistletoe leaves contain three toxic lectins encoded by three genes. The B chains of these lectins were overproduced in Escherichia coli in a soluble form. The recombinant proteins bound with asialofetuin, but had substantially lower affinity for simple sugars D-galactose and N-acetyl-D-galactosamine as compared with the natural proteins. The functional properties of the B chains strongly depended on the storage conditions (salt concentration and the presence of galactose); the dependence was explained by structural instability of nonglycosylated recombinant proteins. The lectin activity of one of the recombinant B chains was close to that of the native protein, which was attributed to the lack of N-glycosylation sites in the latter.     

Binding Patterns of Lectins with GalNAc specificity in the mouse dorsal root ganglia and spinal cord

To localize membrane glycoconjugates in neurons of the mouse spinal cord and dorsal root ganglia (DRG), cryostat sections of newborn (P0), 7 day-old (P7), P14, P21 and P31 animals were stained with ten FITC-conjugated plant lectins, the majority of them recognizing N-acetyl-D-galactosamine (GalNAc) terminal sugar residues. In the dorsal root ganglia of P0 animals, the different lectins showed distinct patterns of labeling in either cells of the nervous system, including neurons, or other structures such as nerves or blood vessels. Moreover, some of these lectins showed important changes in their pattern of labeling during postnatal development. This was especially relevant for lectins that label a subpopulation of small-sized cells that have been previously identified as the nociceptive cells of the DRG. Enzymatic digestion of sections with neuraminidase removes sialic acid from the carbohydrate chains of glycoconjugates thus exposing novel sugar residues. When this treatment was applied to DRG sections from postnatal animals the pattern of lectin staining was either changed or eliminated and heterogeneous subsets of glycoconjugates normally masked by this sugar were exposed. In the spinal cord of PO animals, none of the lectins labeled cells in the central gray matter. However, after the enzymatic digestion of sections with neuraminidase, spinal cord motoneurons and some other cells were labeled by two of the lectins suggesting that GalNAc residues present in these cells are normally masked by terminal sialic acid. Altogether, these results show important changes in the temporal and spatial expression of glycoconjugates that may be relevant for the postnatal development of the CNS and PNS of mice.     

Evaluation of glycoconjugates on the K562 cell surface by means of lectin binding — comparison with human erythrocytes

The binding of five radiolabelled lectins (Vicia graminea, peanut,Phaseolus vulgaris isolectins E-PHA and L-PHA,Evonymus europaeus) to untreated and desialylated K562 cells and human erythrocytes was compared. The number of glycophorin A receptors recognized on the K562 cells by anti-blood group NV. graminea lectin was comparable to that found on the MN or NN erythrocyte surface. However, K562 cells had a severalfold higher number of oligosaccharide chains (presumablyO-glycosidic) which after desialylation became high-affinity receptors for peanut agglutinin, and of complex typeN-glycosidic chains available for the reaction with E-PHA and also with L-PHA (the latter lectin was not bound to erythrocytes). Moreover, K562 cells not treated with neuraminidase had a significant amount of extremely low affinity receptors for peanut agglutinin, whereas binding of this lectin to untreated erythrocytes was undetectable. On the other hand, the untreated K562 cells did not bind anti-blood group B and HE. europaeus lectin, but a small amount of binding by the desialylated cells was observed. Some other differences observed in the mode of lectin binding to K562 cells and erythrocytes are discussed.     

Experimental substantiation of differences in the extent of development of the duodenal glands in mammals

The glandulo-duodenal index was lower in carnivorous animals than in herbivores. The character of the distribution of the duodenal glands in the intestinal wall of animals with different types of nutrition differed. The given indices also varied within the range for each group of animals; this could be associated with fine differences in nutritional specialization. A diet rich in cellulose led to an increase in the glandulo-duodenal index and to changes in gland topography in the rat duodenum.     

A new mitogenic D-galactosephilic lectin isolated from seeds of the coral-tree Erythrina corallodendron. Comparison with Glycine max (soybean) and Pseudomonas aeruginosa lectins

The lectin of Erythrina corallodendron (Caesalpiniaceae) seeds was purified by heating, ammonium sulfate fractionation, and affinity chromatography on acid-treated Sepharose. The purified lectin is similar to the soybean lectin in being a glycoprotein of molecular weight around 110 000 - 120 000 and having D-galactosephilic activity. This lectin, like the soybean and Pseudomonas aeruginosa lectins, binds to D-galactosamine, N-acetyl-D-galactosamine, alpha- and beta-galactosides as well as to D-galactose. Like these lectins it absorbs onto either untreated or enzyme (papain or neuraminidase) treated human red blood cells, but exhibits a considerable mitogenic activity towards human lymphocytes (predominantly T cells) only after their treatment with neuraminidase. This mitogenic stimulation of lymphocytes is inhibited by D-galactose and its derivatives. Despite the great similarity between them, the E. corallodendron, soybean, and Pseudomonas lectins differ in regard to the intensity of their agglutinating activity towards erythrocytes obtained from different animals and human donors of diverse ABO blood groups. This phenomenon may be attributed to the difference in the affinities of the three lectins to the various D-galactose derivatives and to their molecular properties.     

Structural differences between two lectins from Cytisus scoparius, both specific for D-galactose and N-acetyl-D-galactosamine.

Three lectin fractions were obtained from seeds of the leguminous plant Cytisus scoparius (Scotch broom) by means of affinity chromatography on a N-acetyl-D-galactosamine medium. The first fraction, termed CSIa, was equally well inhibited in haemagglutination experiments by D-galactose and by N-acetyl-D-galactosamine and consisted of a group of isolectins formed from closely related polypeptide chains of approx. Mr 30000. The second fraction, CSIb, was closely related to CSIa in specificity, c.d. and other properties. The third fraction contained a homogeneous lectin, CSII, formed from subunits again of approx. Mr 30000. CSII was 100 times more readily inhibited by N-acetyl-D-galactosamine than by D-galactose. Despite the similarity in specificity, comparative studies of their amino acid composition, c.d. and N-terminal amino acid sequence showed that the CSIa and CSII lectins diverged considerably in structure. The lectin from Cytisus sessilifolius, specific for chitobiose, was also examined and resembled CSIa in composition and c.d. properties.     

Ultrastructural localization of glycoconjugates in human bronchial glands: the subcellular organization of N- and O-linked oligosaccharide chains.

We investigated the glycoconjugates of the human bronchial glands at light and electron microscopic level by means of lectin histochemistry in combination with neuraminidase digestion and beta-elimination reaction. Both direct and indirect techniques using lectin-peroxidase, lectin-gold, and glycoprotein-gold complexes were applied. The binding pattern of the six lectins (ConA, HPA, DSA, WGA, LEA, and PNA) used in the present study suggests that mucous and serous cells of human bronchial glands contain both N- and O-glycosylated proteins in the secretory granules. Asparagine-linked oligosaccharides containing Gal(beta-1,4) GlcNAc and Man residues were abundant in serous cells. The demonstration of both the terminal Neu 5Ac (alpha-2,3, or 6) Gal (beta-1,4) GlcNAc sequence in the N-linked oligosaccharides of mucous cells and the terminal disaccharide Gal (beta-1,4) GlcNAc in the N-linked oligosaccharide chains of serous cells suggests the existence of complex type sugar chains N-glycosidically linked to the peptide region of the glycoproteins. The binding pattern of the DSA and the neuraminidase-DSA sequence provides evidence for the existence of sialyltransferase activity in the forming mucous granules of mucous bronchial cells.     

Histochemical study of rabbit duodenal mucosa carried out using lectins

The Authors report histochemical findings about rabbit's duodenal mucosa. The present study has been carried out using five different lectins (Peanut Agglutinin (PNA), Dolichos Biflorus Agglutinin (DBA), Wheat Germ Agglutinin (WGA), Soybean Agglutinin (SBA), Ulex Europaeus Agglutinin I (UEA-I). These lectins have been labelled with Horseradish Peroxidase and binding sites have been stained with 3-3' Diaminobenzidine, according to Farragiana et al. The PNA reacted with the glandular cells, while the reaction was negative in the superficial cells. The DBA reacted exclusively with the glandular cells. The superficial and the glandular cells showed strong positive binding sites to the WGA and slight positive binding sites to the SBA. The UEA-I did not react with the epithelial cells. The presence of binding sites for the lectins we have used in the present study, shows a different glycoprotein composition of the cellular secretion, in comparison with the other animals we have already studied. In addition, these lectins can not be used as cellular differentiation markers in the epithelial cells of the rabbit's duodenal mucosa.     

An anti-A1 lectin in the seeds of Amphicarpaea bracteata

An anti-A1 lectin has been isolated from the extract of Amphicarpaea bracteata seeds by affinity chromatography on Epoxy-activated Sepharose 6B coupled to N-acetyl-D-galactosamine. The yield of the purified lectin was 86 microgram/g of seeds. The purified lectin shows one main band on electrophoresis in sodium dodecyl sulfate-polyacrylamide. The amino acid and neutral sugar composition indicate that this lectin is an acidic glycoprotein with a neutral sugar content of approx. 2%. The composition of the lectin is different from that of the Dolichos biflorus lectin but the two lectins have some common characteristics. The most powerful inhibitors of the agglutination of A1 red blood cells by the A. bracteata lectin is N-acetyl-D-galactosamine. Much weaker inhibitors of the agglutination are alpha-lactose, D-fucose, and five other sugars.     

Pregnancy-related changes in the human endometrium revealed by lectin histochemistry

The binding of 22 fluorescein isothiocyanate (FITC) conjugated lectins to human proliferative phase and pregnant endometrium was studied histochemically. Only the lectin from Bauhinia purpurea (BPA) reacted exclusively with the epithelial cells. All the others reacted to a certain extent with glandular and/or stromal cells. Lectins from soybean (SBA), and Vicia villosa seeds (VVA) reacted with endometrial glands of pregnancy but not with the glands of the proliferative endometrium. In the proliferative endometrium SBA reacted only with cells of the surface endometrium. Lectin from peanuts (PNA) reacted only with some glands in the proliferative endometrium but was unreactive with others. In pregnant endometrium PNA reacted with all glands. Lectins from lentils (LCA) and red kidney beans (PHA-E and PHA-L) reacted with endometrial glands of the proliferative phase but not with the glands from pregnant endometrium. We thus show that FITC labeled lectins define specific carbohydrate moieties selectively expressed on either proliferative phase or pregnant endometrial glands.     

A method of selective histochemical analysis of sialoglycoproteins using lectins from elder (Sambucus nigra L.)]

Good potentialities in application of elderberry (Sambucus nigra L.) bark lectin for selective histochemical identification of sialylated glycoconjugates has been demonstrated using lectin-peroxidase technique. In order to omit this lectin binding to D-galactose and N-acetyl-D-galactosamine residues, preincubation of tissue sections with non-marked PNA and SBA (or other lectins with similar carbohydrate specificity) is proposed. By means of neuraminidase digestion it has been ascertained, that oligosaccharide chains of secretory glycopolymers, synthesised in ovine submandibular gland mucocytes, contain DGal and DGalNAc residues penultimate to terminal sialic acids.     

Lectin histochemistry of normal human gastric mucosa

Information about the saccharides expressed in gastric mucosa is mostly limited to the glycan content of gastric mucins and there are only a few studies of the glycoprofiling of the constituent cells and their components. Knowledge of the glycan expression of normal gastric mucosa is necessary for the interpretation of the significance of changes of expression in disease. A lectin histochemical study of normal human gastric (body) mucosa was performed using 27 lectins chosen to probe for a wide range of oligosaccharide sequences within several categories of glycoprotein glycans. There were marked differences in staining reactions in the various microanatomical structures of the mucosa, particularly between pits and glands with the former more closely resembling the surface epithelium. A notable feature was the degree of difference in the staining between a substantial sub-population of cells within the neck region and the epithelium of both the pits and glands. These neck cells resembled the pit cells with some lectins, glandular cells with some others and neither with some other lectins. Overall, the differences between the pit, gland and neck epithelia were diverse and numerous, and could not be explained by altered activity of a small set of glycosyltransferases. Widespread alterations of glycans must have occurred (affecting terminal and internal parts of their structures) and the very different glycotypes of the pit, neck and gland epithelia are, therefore, suggestive of the existence of three cell lineages within normal gastric epithelium. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Vitelline coat of Unio elongatulus: III. Glycan chain analysis of the 220- and 180-kD components by means of lectins

Lectins of different binding specificity were used to analyze the oligosaccharide chains of the 220- and 180-kD proteins of the Unio elongatulus egg vitelline coat (vc). The lectins ConA and RCA₁ reacted with both glycoproteins, and four other lectins reacted with one or other vc components. The lectin from Galanthus nivalis, which recognizes terminal mannose residues of N-linked high mannose type oligosaccharide chains, bound specifically to the 180-kD protein. Binding sites for this lectin were found throughout the vc of the differentiating oocyte and the mature egg. Lectins specific for the O-linked oligosaccharide chains, such as AIA and PNA, reacted only with the 220-kD protein species. Binding sites for these lectins were found only in the crater region. The presence of fucosyl residues on the glycan chains was investigated with lectins from Lotus tetragonolobus and Aleuria aurantia. The latter was positive on both glycoproteins, whereas LTA was only positive to the 220-kD species. The binding sites of both these lectins were in the same areas as those of PNA and AIA. These results suggest that while the 180-kD protein is part of the entire vc structure, the 220-kD protein is prevalently accumulated in the crater region. Since this is where sperm recognition and interaction take place, it has been suggested the 220-kD protein acts as a ligand molecule in the sperm-egg interaction. © 1995 Wiley-Liss, Inc.     

Lectin binding of surface epithelia and concomitant glands of gallbladder and biliary ducts in the guinea pig

Summary Complex carbohydrate components of secretory granules and the glycocalix were analysed in surface epithelia, endoepithelial glands and exoepithelial tubuloalveolar glands of the biliary-ductular system (guinea pig). Brunner glands and pyloric glands were studied for comparison. The columnar epithelial cells of the gallbladder and biliary ducts displayed a well-developed PAS-positive apical glycocalix. These materials strongly bound Ricinus communis AI, Ulex europaeus I, Lotus tetragonolobus A and wheat-germ-A lectins. With the exception of Lotus A lectin which did not bind at all, the same lectins stained the basolateral cell surface. The secretory granules in the supranuclear regions of surface epithelia and in the exoepithelial glands strongly bound Ricinus A I, Ulex europaeus I, wheat-germ-A and Helix pomatia lectins. Concanavalin A was less intensively bound by the secretions of tubuloalveolar glands than by the secretory granules in surface epithelia. The luminal and basolateral cell surfaces of glandular cells in the exoepithelial glands were stained by the same spectrum of lectins as were the less distinct. In the guinea pig, the lectin-binding patterns of tubuloalveolar glands in the biliary ducts closely resembled those of Brunner glands and pyloric glands. The secretions of the tubuloalveolar glands were different from the secretion of surface epithelia, as they bound Concanavalin A less intensively.     

Lectin histochemistry of epidermal glandular cells in the earthworm Lumbricus terrestris (Annelida Oligochaeta)

Carbohydrate residues were localized in the glandular cells of the epidermis of Lumbricus terrestris by lectin histochemistry. The following biotinylated lectins were used: ConA, PNA, WGA, UEA-I. Each lectin has a specific binding pattern in the epidermal glandular cells. The ConA binding is evident in the orthochromatic mucous cells; PNA in the metachromatic mucous cells; WGA in the neuroendocrine-like cells; UEA-I in the cuticle. The epidermal glandular cells possess specific sites for the different lectins in relation to their functional characteristics. Therefore, these sugar residues indicate different behaviours of the cells in epidermal functions related to ion transport, receptor-secretory processes and defence.