Post-embedding staining of rat gastric mucous cells with lectins

Summary The mucous cells of the rat stomach were stained with lectins by two post-embedding staining methods for electron microscopy. The mucous granules of surface mucous cells and foveolar mucous cells were stained weakly by Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of mucous neck cells were stained by concanavalin A-ferritin, Ricinus communis agglutinin-ferritin and wheat germ agglutinin-ferritin. The mucous granules of pyloric gland cells showed an affinity for wheat germ agglutinin-ferritin and concanavalin A-ferritin, while Ricinuscommunis agglutinin-ferritin only slightly stained the granules. The granules of mucous neck cells and pyloric gland cells were also stained by the concanavalin A-horseradish peroxidase-colloidal gold method, but the granules of surface and foveolar mucous cells were not stained by this method. Periodic acid oxidation of the sections before the standard concanavalin A-ferritin procedure enhanced the staining of the granules of mucous neck cells and pyloric gland cells slightly. Reduction of the sections after the periodic acid oxidation weakened the staining. Similar results were obtained using the concanavalin A-horseradish peroxidase-colloidal gold method. Though the staining with Ricinus communis agglutinin-ferritin was inhibited by periodic acid oxidation of the sections before staining, the staining with wheat germ agglutinin-ferritin was not inhibited by the oxidation. It is suggested that the paradoxical staining is closely related to the position of the concanavalin A-binding sugar residues in the carbohydrate chains.This work was supported in part by a grant-in-aid (No. 457008) from the Ministry of Education, Science and Culture, Japan and a grant-in-aid for cancer research (55-21) from the Ministry of Health and Welfare, Japan     

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.     

Localization of the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes.

Freeze-etch electron microscopy has been utilized to localize the binding sites for the Ricinus communis, Agaricus bisporus and wheat germ lectins on human erythrocyte membranes and to determine the relation of these different glycoprotein receptors to the intramembranous particles. A. bisporus lectin, which could be visualized directly on the surface of erythrocyte membranes, and ferritin conjugates of wheat germ agglutinin showed a distribution that correlates exactly with the intramembranous particles at all lectin concentrations tested. The binding sites for both of these lectins are located on the major sialoglycoprotein of the membrane. The R. communis agglutinin-ferritin conjugate which binds to receptors on membrane glycoproteins that are distinct from the major sialoglycoprotein showed a close correlation with the intramembranous particles at low lectin concentrations and a poor correlation at high lectin concentrations. High concentrations resulted in virtually complete coating of the surface of trypsinized ghosts which displayed marked aggregation of the intramembranous particles. We conclude that the intramembranous particles of erythrocyte membranes contain at least two glycoproteins and that some membrane lectin receptors are not associated with the intramembranous particles.     

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.     

Lectin-Reactive Components in White Matter Membranes from Normal and Multiple Sclerosis Brains

Abstract: Polypeptides derived from human white matter membranes reacted with the radioiodinated lectins concanavalin A, Lens culinaris phytohemagglutinin, Ricinus communis agglutinin and wheat germ agglutinin after electrophoresis in polyacrylamide pore gradient gels. The molecular weights of these lectin-reactive bands were estimated by comparison with radioiodinated protein standards by using the linear relationship between log of the molecular weight and log of the gel concentration reached by the protein after electrophoresis in a polyacrylamide gradient gel. The molecular weight estimates for components reactive with concanavalin A were 176,800, 141,200, 72,800, 52,800, 44,700, 40,000, 24,800 and 23,900. The molecular weights of the bands reactive with both wheat germ agglutinin and Lens culinaris phytohemagglutinin were 138,000, 113,500, 92,100, 52,800, 44,700, 24,800 and 23,900. Wheat germ agglutinin was bound also to a band with a molecular weight of 72,800. Ricinus communis agglutinin bound to bands with estimated molecular weights of 138,000, 72,800, 52,800, 44,700, 24,800 and 23,900. The electrophoretic pattern of lectin-reactive polypeptides derived from normal-appearing white matter of multiple sclerosis brains was not qualitatively different from the lectin-binding pattern of control brain membrane polypeptides.     

Lectin affinity chromatography of cell surface proteins of novikoff tumor cells

Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/¹²⁵I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.     

Transport of membrane-bound macromolecules by M cells in follicle-associated epithelium of rabbit Peyer's patch

Summary M cells in Peyer's patch epithelium conduct transepithelial transport of luminal antigens to cells of the mucosal immune system. To determine the distribution of specific lectin-binding sites on luminal membranes of living M cells and to follow the transport route of membranebound molecules, lectin-ferritin conjugates and cationized ferritin were applied to rabbit Peyer's patch mucosa in vivo and in vitro. The degree to which binding enhances transport was estimated by comparing quantitatively the transport of an adherent probe, wheat germ agglutinin-ferritin, to that of a nonadherent BSA-colloidal gold probe. When applied to fixed tissue, the lectins tested bound equally well to M cells and columnar absorptive cells. On living mucosa, however, ferritin conjugates of wheat germ agglutinin and Ricinus communis agglutinins I and II bound more avidly to M cells. Absorptive cells conducted little uptake and no detectable transepithelial transport. Lectins on M cell membranes were endocytosed from coated pits, rapidly transported in a complex system of tubulocisternae and vesicles, and remained adherent to M cell basolateral membranes. Cationized ferritin adhered to anionic sites and was similarly transported, but was released as free clusters at M cell basolateral surfaces. When applied simultaneously to Peyer's patch mucosa, wheat germ agglutinin-ferritin was transported about 50 times more efficiently than was bovine serum albumin-colloidal gold.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell.

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

Lectin histochemistry of mammalian endothelium

Lectin-histochemical studies were performed on formalin-fixed, paraffin-embedded tissues from ten mammalian species to demonstrate the pattern of carbohydrate residues in vascular endothelium. Ten different biotinylated lectins were used as probes and avidin-biotin-peroxidase complex (ABC) was used as visualant. Ricinus communis agglutinin-I (RCA-I) and wheat germ agglutinin (WGA) stained vascular endothelium in all species. Peanut agglutinin (PNA) stained vascular endothelium in all species only after preincubation with neuraminidase. Bandeirea simplicifolia agglutinin-I (BS-I) stained vascular endothelium in all species but human, while Ulex europeus agglutinin-I (UEA-I) stained only human endothelium. Individual differences in staining of human vascular endothelium were noted with BS-I and succinylated-WGA (SWGA). Similarly, individual differences in staining of animal vascular endothelium were noted with soybean agglutinin (SBA) after preincubation with neuraminidase. Finally, Concanavalia ensiformis agglutinin (Con A), Dolichos biflorus agglutinin (DBA) and Lens culinaris agglutinin (LCA) did not stain vascular endothelium in any of the species studied.     

Ultrastructural visualization of carbohydrates in oxytalan fibers in monkey periodontal ligaments

Fullmer's oxytalan fibers appear to be special connective tissue fibers belonging to elastic system fibers. We have ultrastructurally examined carbohydrates in oxytalan fibers in monkey periodontal ligaments after glutaraldehyde fixation and ethylenediaminetetraacetic acid (EDTA) decalcification using: Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for thin-section staining of vicinal glycol-containing complex carbohydrates, and the concanavalin A-ferritin (Con A-ferritin) and Con A-horseradish peroxidase (Con-A-HRP) en bloc staining methods specific for alpha-D-mannosyl and alpha-D-glucosyl groups. PA-TCH-SP stained collagen fibrils weakly to moderately and stained oxytalan fibers moderately. Con A-ferritin and Con A-HRP stained collagen fibrils weakly or moderately and stained oxytalan fibers intensely within the superficial region of specimen blocks. The penetration of staining reagents was improved by prior saponin treatment and/or chondroitinase ABC digestion. Thus, these studies demonstrate that PA-TCH-SP and Con A staining of carbohydrates is very useful in identifying oxytalan fibers at the ultrastructural level and that more carbohydrate components are present in oxytalan fibers than in collagen fibrils.     

Synergistic effects of lectins in the interaction of thrombin with human platelets

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.     

Changes in lectin-binding pattern in the digestive tract of Xenopus laevis during metamorphosis. II. Small intestine.

The binding of seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin, UEA-I; and wheat germ agglutinin, WGA) to the small intestine in metamorphosing Xenopus laevis was studied by the avidin-biotin-peroxidase (ABC) method. The staining pattern of the epithelium with all lectins except for UEA-I and Con A changed gradually during metamorphic climax; the main component of the epithelium, absorptive cells, gradually became positive for DBA, PNA, and SBA and the scattered goblet cells for RCA-I and WGA. On the other hand, the change of the staining pattern in the connective tissue occurred only for Con A, RCA-I, and WGA, and this change took place rapidly at the beginning of climax (stage 60). Increased staining for Con A and WGA at stage 60 was observed only in a group of connective tissue cells close to the epithelium and in the basement membrane. As metamorphosis progressed, this localization of the staining intensity became less clear. At the completion of metamorphosis (stage 66), the absorptive cells were stained with all lectins except for UEA-I, whereas the goblet cells stained only with RCA-I and WGA. These results indicate that lectin histochemistry can distinguish between larval and adult cells of both two epithelial types (absorptive and goblet cells). The technique may also identify a group of connective tissue cells, close to the epithelium, that possibly induce the metamorphic epithelial changes.     

SOLUBLE AND MEMBRANE LECTIN-BINDING GLYCOPROTEINS OF THE CHROMAFFIN GRANULE

Abstract— Fluorescein isothiocyanate-labelled lectins were used to identify lectin-binding glycoproteins of the chromaffin granule after electrophoresis of the membrane and soluble granule proteins on sodium dodecyl sulphate polyacrylamide slab gels. The glycoprotein nature of all lectin-binding bands was confirmed by staining the gels for carbohydrates, and the specificity of the lectin-binding was demonstrated by hapten sugar inhibition of binding. In samples of granule membrane proteins reduced with dithiothreitol 10 concanavalin A (Con A), 5 wheat germ agglutinin, 8 Ricinus communis agglutinin-60, and 7 Ricinus communis agglutinin-120 (RCA-120) binding glycoproteins were identified. Molecular weights of these glycoproteins varied from 20,000 to 200,000 daltons. All but two of the Con A-binding bands and one of the RCA-120 binding bands appeared to react with more than one lectin, suggesting possible carbohydrate heterogeneity in these membrane glycoproteins. The band identified as dopamine β-hydroxylase reacted most intensely with all four lectin tested, and in the soluble core material this enzyme was the sole significant lectin binding glycoprotein.     

Lectin histochemistry of feline sphingomyelinosis.

The brain from a Siamese cat with sphingomyelinosis was examined with lectin histochemistry. Swollen neurons were stained with Canavalia ensiformis agglutinin (Con A). Some of them were also stained with Ricinus communis agglutinin-I (RCA-I) and Ulex europaeus agglutinin-I (UEA-I). A small number of axonal spheroids and glia cells were positive for Con A, RCA-I, UEA-I and wheat germ agglutinin. Control tissues were weakly stained with Con A, but not with any of the other lectins. These results indicate that affected neurons contain mannose and glucose residues in addition to sphingomyelin. This study points to the possibility that the characteristics of lectin histochemical study might be helpful for the diagnosis of sphingomyelinosis.     

A statistical evaluation of the binding of ferritin-conjugated lectins to the surface of rat cells. Topographical variations as a function of lectin concentration and cell type.

Statistical procedures were utilized to evaluate the concentration dependence of labeling by ferritin-conjugated lectins on four different rat cells: hepatocytes, normal thymocytes, Friend virus-induced rat tumor cells and feline sarcoma virus-induced rat sarcoma cells. Labeling by ferritin conjugates of concanavalin A, wheat germ agglutinin and Ricinus communis agglutinins I and II was quantitated by counting the number of ferritin granules on 600 Angstrom membrane segments. Relationships between the arithmetic means and variances for sample populations from each cell and ferritin-lectin combination were used to define four types of topographical distributions: uniform/ordered, uniform/random, random and clustered. It was found that the distribution and/or density of surface-bound lectin was concentration-dependent for all four ferritin-lectins. The nature of this dependency was complex and varied with both lectin and cell type.     

Changes in lectin-binding pattern in the digestive tract of Xenopus laevis during metamorphosis. I. Gastric region.

The distribution of structural and secretory glycoconjugates in the gastric region of metamorphosing Xenopus laevis was studied by the avidin-biotin-peroxidase (ABC) histochemical staining method using seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin I, UEA-I; and wheat germ agglutinin, WGA). Throughout the larval period to stage 60, the epithelium consisting of surface cells and gland cells was stained in various patterns with all lectins examined, whereas the thin layer of connective tissue was positive only for RCA-I. At the beginning of metamorphic climax, the connective tissue became stained with Con A, SBA, and WGA, and its staining pattern varied with different lectins. The region just beneath the surface cells was strongly stained only with RCA-I. With the progression of development, both the epithelium and the connective tissue gradually changed their staining patterns. The surface cells, the gland cells, and the connective tissue conspicuously changed their staining patterns, respectively, for Con A and WGA; for Con A, PNA, RCA-I, SBA, and WGA; and for Con A, RCA-I, and WGA. At the completion of metamorphosis (stage 66), mucous neck cells became clearly identifiable in the epithelium, and their cytoplasm was strongly stained with DBA, PNA, RCA-I, and SBA. These results indicate that lectin histochemistry can provide good criteria for distinguishing among three epithelial cell types, namely, surface cells, gland cells, and mucous neck cells, and between adult and larval cells of each type.     

Selective staining of human platelet glycoproteins using nitrocellulose transfer of electrophoresed proteins and peroxidase-conjugated lectins

Platelet proteins (0.5-5 micrograms) were electrophoresed in a one-dimensional or an unreduced-reduced, two-dimensional sodium dodecyl sulfate gel system. The separated proteins were then transferred electrophoretically to nitrocellulose and reacted with peroxidase-conjugated lectins. Visualization of specific glycoproteins which bound the lectins was made by the chromogenic reaction catalyzed by peroxidase utilizing 3,3'-diaminobenzidine as the substrate. Wheat germ agglutinin specifically reacted with and allowed the visualization of glycoprotein Ib. Peanut agglutinin also specifically stained glycoprotein Ib after treatment of the nitrocellulose transferred proteins with neuraminidase. Ricinus communis agglutinin I stained thrombospondin, a 260 kDa protein, and factor VIII. Concanavalin A stained mainly glycoproteins IIb, III, IV, and V. Glycoproteins Ia, Ic, IIa, and other minor glycoproteins could be separated by unreduced-reduced, two-dimensional gel electrophoresis and were stained weakly with wheat germ agglutinin conjugates. These techniques were found to be reproducible as well as easily applied to the analysis and identification of platelet glycoproteins, particularly when dealing with a limited amount of platelets.     

Lectin histochemistry of human skeletal muscle

Biotinyl derivatives of seven plant lectins-concanavalin A (Con A), peanut agglutinin (PNA), Ricinus communis agglutinin I (RCA I), Ulex europeus agglutinin I (UEA I), soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), and wheat germ agglutinin (WGA)-were bound to cryostat sections of biopsied normal human muscle and visualized with avidin-horseradish peroxidase conjugates. A distinct staining pattern was observed with each lectin. The most general staining was observed with Con A, RCA I, and WGA, which permitted strong visualization of the plasmalemma-basement membrane unit, tubular profiles in the interior of muscle fibers, blood vessels, and connective tissue. PNA gave virtually no intracellular staining, while SBA and UEA I selectively stained blood vessels. DBA was unique in providing good visualization of myonuclei. In each case, lectin staining could be blocked by appropriate sugar inhibitors. Neuraminidase pretreatment of the cryostat sections altered the pattern of staining by all lectins except UEA I and Con A; staining with RCA I became stronger and that with WGA became less intense, while staining with PNA, SBA and DBA became stronger and more generalized, resembling that of RCA I. These effects of neuraminidase pretreatment are in conformity with the known structure of the oligosaccharide chains of membrane glycoproteins and specificities of the lectins involved.     

Lectin receptor sites on rat liver cell nuclear membranes.

The presence and localization of lectin receptor sites on rat liver cell nuclear and other endomembranes was studied by light and electron microscopy using fluorescein and ferritin-coupled lectin conjugates. Isolated nuclei labelled with fluorescein-conjugated Concanavalin A (Con A) or wheat germ agglutinin (WGA) often showed membrane staining, which sometimes was especially bright on small stretches of the nuclear surface. Unlabelled nuclei and nuclei with a complete ring fluorescence were also seen. The nuclear fluorescence corresponded in intensity to that seen on the surface of isolated rat liver cells. Con A-ferritin particles were seldom detected on the cytoplasmic surface of the intact nuclear envelope. However, at places where the 2 leaflets of the envelope were widely separated or where the outer nuclear membrane was partly torn away, heavy labelling was seen on the cisternal surface of both the inner and outer nuclear membranes. Labelling with Con A-ferritin was also found on the cisternal side of rough endoplasmic reticulum present in the specimens. No labelling was seen on the cytoplasmic surface of mitochondrial outer membrane. The results demonstrate the presence of binding sites for Con A and WGA in nuclei and an asymmetric localization of these sites on the cisternal side of ribosome-carrying endomembranes in rat liver cells.     

A histochemical comparison of human corneal stromal glycoconjugates with eight other species. Distinct species-dictated differences in binding sites of Griffonia simplicifolia I

Frozen sections of human, calf, rabbit, rat, cat, dog, goat, lamb, and hog corneas were stained with various lectins using an avidin-biotin-peroxidase complex to study glycoconjugates of stromal matrix. Staining of the stromal matrix and keratocytes with an alpha-galactose-specific lectin, Griffonia simplicifolia I (GSA-I) was species-dependent. The stromal matrices of cat, dog, and hog corneas invariably reacted intensely with this lectin, whereas those of the human, calf, rabbit, rat, and lamb did not react. A positive reaction with GSA-I could be abolished in each instance by preincubation of the sections with alpha-galactosidase. The stromal matrices and keratocytes of all nine species reacted positively with wheat germ agglutinin, concanavalin A, and Ricinus communis agglutinin but did not react with soybean agglutinin. Results of this study may help select an appropriate animal model for further investigate human corneal stromal glycoconjugates.