Lectin binding reveals divergent carbohydrate expression in human and mouse Peyer's patches

The nature of cell-associated carbohydrates in the human intestine that may mediate transepithelial transport of bacterial and dietary lectins and their processing by the lymphoid cells of Peyer's patches is not known. Because the cell surface carbohydrate receptors for lectins may vary in different species, the glycoconjugates of human and mouse follicle-associated epithelium and gut-associated lymphoid tissue were compared. A panel of 27, mainly recently isolated, lectins were used to identify glycoconjugate expression in M-cells, enterocytes, goblet cells, lymphocytes and macrophages in mouse and human intestine. Mouse M-cells were exclusively labelled by fucose-specific lectins but in human follicle-associated epithelium no distinct M-cell staining pattern was observed. In the human Peyer's patches,Bryonia dioica lectin bound selectively to paracortical T-lymphocytes andChelidonium majus lectin to germinal centre B-cells. Certain mannose-specific lectins (Galanthus nivalis, Hippeastrum hybrid) stained the tingible body macrophages in the germinal centre of human Peyer's patches but labelled the macrophages in the paracortical T-cell region of the mouse. The results indicate distinct differences in glycosylation between mouse and human Peyer's patches and their associated lymphoid cells. When considering cell surface glycoconjugates as target molecules for the gut immune system, care has to be taken to choose the appropriate lectin for each species.     

Localization of binding sites of Ulex europaeus I,Helix pomatia and Griffonia simplicifolia I-B4 lectins and analysis of their backbone structures by several glycosidases and poly-N-acetyllactosamine-specific lectins in human breast carcinomas

Several studies have shown the deletion of blood group A or B antigens and the accumulation of H antigens in human breast carcinomas. Other studies have independently demonstrated that the binding sites of lectins such asHelix pomatia agglutinin (HPA) andGriffonia simplicifolia agglutinin I-B₄ (GSAI-B₄) are highly expressed in these cells. In order to clarify the molecular mechanisms of malignant transformation and metastasis of carcinoma cells, it is important to understand the relationship between such phenotypically distinct events. For this purpose, we examined whether the binding sites of these lectins andUlex europaeus agglutinin I (UEA-I) are expressed concomitantly in the same carcinoma cells and analyzed their backbone structures. The expression of the binding sites of these lectins was observed independently of the blood group (ABO) of the patients and was not affected by the histological type of the carcinomas. Observation of serial sections stained with these lectins revealed that the distribution of HPA binding sites was almost identical to that of GSAI-B₄ in most cases. Furthermore, in some cases, UEA-I binding patterns were similar to those of HPA and GSAI-B₄ but in other cases, mosaic staining patterns with these lectins were also observed, i.e., some cell clusters were stained with both HPA and GSAI-B₄ but not with UEA-I and adjacent cell clusters were stained only with UEA-I. Digestion with endo-β-galactosidase orN-glycosidase F markedly reduced the staining intensity of these lectins. Together with the reduction of staining by these lectins, reactivity withGriffonia simplicifolia agglutinin II appeared in carcinoma cells following endo-β-galactosidase digestion. Among the lectins specific to poly-N-acetyllactosamine,Lycopersicon esculentum agglutinin (LEA) most vividly and consistently stained the cancer cells. Next to LEA, pokeweed mitogen agglutinin was also effective in staining these cells. Carcinoma cells reactive with these lectins corresponded well to those stained with both HPA and GSAI-B₄, and in some cases, with UEA-I. These results demonstrate that the binding sites of UEA-I, HPA, and GSAI-B₄ are expressed concomitantly in the same carcinoma cells and all carry linear and branched poly-N-acetyllactosamine onN-glycans, suggesting that the synthesis of this complex carbohydrate is one of the most important and basic processes leading to the malignant transformation of cells, invasion, and metastasis of carcinoma cells.     

The wound-healing responses of Antithamnion nipponicum and Griffithsia pacifica (Ceramiales, Rhodophyta) monitored by lectins

The binding of FITC labeled lectins to repair cells of Antithamnion nipponicum Yamada et Inagaki and Griffithsia pacifica Kylin, and their physiological effects on somatic cell fusion have been studied. Results indicate that repair cells strongly bind the lectins ConA and LCA, whereas other lectins did not bind to the cell, The binding of these lectins to the dead cell wall shows ConA and LCA specific substances are secreted from the tip of the repair cells. When fluorescently labeled ConA or LCA was added at various time intervals after wounding, it firstly bound (3 h post-wounding) as a thin layer at the tips of the adjacent cells. Later (4–5 h post-wounding) labeling also appeared at the tips of the repair ceils. Intense labeling at these sites continued throughout the wound-healing process until repair cell fusion, at which time the lectin labeling was reduced to a narrow ring around the area of fusion, When added to plants prior to wounding and with continued monitoring, these same lectins were found to act as inhibitors to the wound-healing response. Other control lectins showed no inhibitory effects. These results suggest that a signal glycoprotein with α-D-mannosyl residues is involved in the wound-healing process of Antithamnion nipponicum. Lectins conjugated with visible tags can be used as a very fast and useful tool to monitor these signal substances.     

Characterization of sinusoidal endothelial cells of the liver and bone marrow using an intravital lectin injection method

The vascular endothelia express a variety of structural and biological phenotypes. We used an intravital injection method of plant derived lectins (Lycopersicon esculentum lectin (LEL), Ricinus communis Agglutinin-I (RCA-I), Ulex europaeus Agglutinin-I (UEA-I) and Concanavalin A (ConA)) to elucidate the morphology and function of the sinusoidal endothelium of the liver and bone marrow. All four lectins stained the sinusoidal endothelia of the liver and bone marrow in a patchy granular pattern which differed from the uniform and smooth staining pattern of non-sinusoidal vessels in other organs. By transmission electron microscopy, the granular pattern was identified as internalization of these lectins and subsequent accumulation within the endothelial cells, suggesting their active endocytosis. The endocytosis of these lectins emphasizes the fact that sinusoidal endothelial cells of the liver and bone marrow belong to the reticuloendothelial system (RES), a cell system characterized by internalization of foreign material. We introduce this intravital lectin injection as a useful technique to discriminate sinusoidal endothelial of the liver and bone marrow from other vascular endothelia.     

Effect of lectins on endocytosis and secretion of lysosomal enzymes by cultured fibroblasts.

1. Pretreatment of cultured human skin fibroblasts with convanavalin A and wheat germ agglutinin inhibited endocytosis of alpha-N-acetylglucosaminidase and increased extracellular accumulation of beta-N-acetylglucosaminidase. 2. These effects were dose-dependent, reversible and could be prevented by haptenic carbohydrates, such as methyl alpha-D-mannoside or N-acetylglucosamine. 3. Pretreatment of fibroblasts with di- and monovalent succinylated concanavalin A inhibited alpha-N-acetylglucosaminidase endocytosis, but had no effect on extracellular beta-N-acetylglucosaminidase accumulation. 4. Concanavalin A-alpha-N-acetylglucosaminidase complexes become internalized via the recognition of the lectin. Complex formation prevents recognition of the phosphorylated carbohydrate on lysosomal enzymes that interacts with cell surface receptors specific for lysosomal enzymes. The inhibitory effect of all lectins tested on lysosomal enzyme endocytosis suggests that the cell surface receptors for lysosomal enzymes interact either directly with lectins or are closely linked to lectin receptors. The effect of polyvalent lectins on extracellular lysosomal enzyme accumulation is ascribed to their alteration of membrane fluidity.     

Electron microscopic demonstration of lectin binding sites in the taste buds of the European catfish Silurus glanis (Teleostei)

Summary Taste buds in the European catfish Silurus glanis were examined with electron microscopic lectin histochemistry. For detection of carbohydrate residues in sensory cells and adjacent epithelial cells, gold-, ferritin-and biotin-labeled lectins were used. A post-embedding procedure carried out on tissue sections embedded in LR-White was applied to differentiate between the sensory cells: The lectins from Helix pomatia (HPA) and Triticum vulgare (WGA) bound to N- acetyl-galactosamine and to N-acetylglucosamine residues occurring especially in vesicles of dark sensory cells. This indicates a secretory function of these cells. Most light sensory cells — with some exceptions, probably immature cells —, are HPA-negative. The mucus of the receptor field and at the top of the adjacent epithelial cells was strongly HPA-positive. Pre-embedding studies were performed in order to obtain information about the reaction of the mucus with lectins under supravital conditions. The mucus of the taste bud receptor field exhibited intensive binding to WGA, but not to the other lectins tested. Most lectins bound predominantly to the surface mucus of the nonsensory epithelium and to the marginal cells close to the receptor field. The strong lectin binding to mucins and the relatively weak lectin binding to cell surface membranes in pre-embedding studies suggest that the mucus possibly serves as a barrier which is passed selectively only by a small amount of lectins or lectincarbohydrate complexes. Lectin-carbohydrate interactions may play a role in recognition phenomena on the plasmalemmata of the taste bud sensory cells. Recognition processes directed to bacteria or viruses should be considered as well.Parts of this investigation were presented at the XI. Annual Meeting of the Association for Chemoreception Sciences (AChemS XI), held at Sarasota, Fl, April 12–14, 1989 (Witt and Reutter 1989)     

Difference in the ability of blood group-specific lectins and monoclonal antibodies to recognize the ABH antigens in human tissues

Summary Twelve different kinds of blood group-specific lectins have been used along with monoclonal anti-A,-B and-H antibodies for detecting the corresponding antigens in selected human tissues. Although most of the lectins recognized the antigens in the tissue sections examined, they displayed marked differences in their recognition patterns in certain tissues.Helix asparsa agglutinin (HAA),Helix pomatia agglutinin (HPA) and monoclonal anti-A antibody recognized A antigens in the mucous cells of salivary glands from blood group A or AB nonsecretor as well as secretor individuals, whereasDolichos biflorus agglutinin (DBA).Griffonia simplicifolia agglutinin-I (GSA-I),Sophora japonica agglutinin (SJA) andVicia villosa agglutinin (VVA) did not bind to them from nonsecretors. A antigens in endothelial cells, lateral membrane of pancreatic acinar cells and small mucous-like cells of submandibular glands from some individuals were likewise recognized by HAA and HPA but not by other blood group A-specific lections. In contrast, both HAA and HPA did not recognize the A antigens in mucous cells of Brunner's glands while other A-specific lectins and monoclonal anti-A antibody reacted specifically with the antigens. Such a difference was not observed with lectins specific for blood group B. However, the B antigens in Brunner's glands were recognized by these lectins but not with monoclonal anti-B antibody. The difference in labelling ability was also noted among the blood group H-specific lectins and monoclonal anti-H antibody in endothelial cells of blood vessels.Ulex europaeus agglutinin-I reacted with these cells irrespective of ABO and the secretor status of the individuals, whileAnguilla anguilla agglutinin and monoclonal anti-H antibody reacted only with those cells from blood group O individuals. No reaction was observed withLotus tetragonolobus agglutinin in these tissue sites. These results suggest a great diversity of blood group antigens in different human tissues.     

Biochemical, histochemical and cell biological investigations on the actions of mistletoe lectins I, II and III with human breast cancer cell lines

Cytotoxicity of the mistletoe lectins I, II and III towards six human breast cancer cell lines was assessed using the Mossman assay. In addition, binding of the three mistletoe lectins to the separated membrane glycoproteins of these cell lines, the binding and uptake of these lectins into the cells in tissue culture and the binding of the lectins to histological preparations of these cell lines were analysed. The results indicate that there are quantitative differences concerning the toxicity of these three lectins towards the different cell lines. Furthermore, the lectin binding pattern in the cell lines differed. In Western blots, several membrane glycoproteins were labelled by the lectins. Our results indicate subtle differences between the three lectins with regard to the parameters mentioned above; however, the toxicity of all three lectins from mistletoe is so similar that they all seem suitable for the construction of immunotoxins.Dedication: This work is dedicated to one of the discoverers (amongst many other important contributions) ofHelix pomatia agglutinin, which plays an important role in metastasis research, Professor Dr G. Uhlenbruck on the occasion of his 65th birthday.     

Concanavalin A promotes adherence of Salmonella typhimurium to small intestinal mucosa of rats

A number of dietary lectins have been shown to resist proteolytic digestion. These lectins interact with the small intestinal mucosa causing structural and functional changes. Concomitant to these changes, bacterial overgrowth was reported and a possible interaction between lectins and bacteria in the small intestine was postulated. The aim of this study was to investigate the effect of various lectins on adherence of Salmonella typhimurium to both isolated small intestinal enterocytes and ligated intestinal loops. Isolated intestinal cells or ligated intestinal loops were incubated with [3H] adenine-S. typhimurium in the presence or absence of concanavalin A, phytohemagglutinin, peanut agglutinin, and wheat germ agglutinin. Only concanavalin A promoted the adherence of various strains of nonfimbriated S. typhimurium to isolated viable intestinal cells. Other lectins showed no effect on the adherence. In situ studies showed that bacterial binding was increased in concanavalin A-treated intestinal loops, supporting the significance of the experiments in vitro. These data suggest that lectins may act by promoting bacterial adherence to the small intestine, thereby facilitating colonization and infection, and leading to bacterial overgrowth.     

Characterization and biological implications of membrane lectins in tumor, lymphoid and myeloid cells

Complex carbohydrates and sugar receptors at the surface of eukaryotic cells are involved in recognition phenomena. Membrane lectins have been characterized, using biochemical, biological and cytological methods. Their biological activities have been assessed using labeled glycoproteins or neoglycoproteins. Specific glycoproteins or neoglycoproteins have been used to inhibit their binding capacity in both in vitro and in vivo experiments. In adults, lymphoid and myeloid cells as well as tumor cells grow in a given organ and eventually migrate and home in another organ; these phenomena are known as the homing process or metastasis, respectively. In specific cases, membrane lectins of endothelial cells recognize cell surface glycoconjugates of lymphocytes or tumor cells, while membrane lectins of lymphocytes and of tumor cells recognize glycoconjugates of extracellular matrices or of non-migrating cells. Therefore, membrane lectins are involved in cell-cell recognition phenomena. Membrane lectins are also involved in endocytosis and intracellular traffic of glycoconjugates. This property has been demonstrated not only in hepatocytes, fibroblasts, macrophages and histiocytes but also in tumor cells, monocytes, thyrocytes, etc. Upon endocytosis, membrane lectins are present in endosomes, whose luminal pH rapidly decreases. In cells such as tumor cells or macrophages, endosomes fuse with lysosomes; it is therefore possible to target cytotoxic drugs or activators, by binding them to specific glycoconjugates or neoglycoproteins through a linkage specifically hydrolyzed by lysosomal enzymes. In cells such as monocytes, the delivery of glycoconjugates to lysosomes is not active; in this case, it would be preferable to use an acid-labile linkage. Cell surface membrane lectins are developmentally regulated; they are present at given stages of differentiation and of malignant transformation. Cell surface membrane lectins usually bind glycoconjugates at neutral pH but not in acidic medium: their ligand is released in acidic specialized organelles; the internalized ligand may be then delivered into lysosomes, while the membrane lectin is recycled. Some membrane lectins, however, do bind their ligand in relatively acidic medium as in the case of thyrocytes. The presence of cell surface membrane lectins which recognize specific sugar moieties opens the way to interesting applications: for instance, isolation of cell subpopulations such as human suppressor T cells, targeting of anti-tumor or anti-viral drugs, targeting of immunomodulators or biological response modifiers.     

The Role of Cell-Surface Lectins in the Aggregation of Azospirilla

The mutant strain Azospirillum brasilenseSp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A. brasilenseSp7(S) both in the exponential and stationary growth phases. The pretreatment of bacterial cells with the specific haptens (L-fucose and D-galactose) of a lectin located at the cell surface of the mutant strain was found to inhibit the aggregation of azospirilla. The specific binding of the A. brasilenseSp7(S) lectin to the extracellular polysaccharide-containing complexes of this strain was revealed by dot immunoblotting on nitrocellulose membrane filters. The interaction of the lectins of A. brasilense75, A. brasilenseSp7, and A. lipoferum59b with the polysaccharide-containing complexes that were isolated from these strains was not specific. No interstrain cross-interaction between the exopolysaccharides and lectins of azospirilla was found. A coflocculation of A. brasilenseSp7 cells with Bacillus polymyxa1460 cells was shown. The involvement of autogenous lectins in the aggregation of bacterial cells is discussed.     

Properties of cell surface carbohydrates in sexual reproduction of the Closterium peracerosum–strigosum–littorale complex (Zygnematophyceae,Charophyta)

The Closterium peracerosum–strigosum–littorale complex is a best characterized zygnematophycean green alga with respect to the process of sexual reproduction. Intercellular communication mediated by two sex pheromones has been well documented in this organism, but information concerning direct cell–cell recognition and fusion of cells involved in conjugation processes has not yet been clarified. In this study, we examined the properties of cell surface carbohydrates in vegetative and reproductive cells using a variety of fluorescein isothiocyanate labeled lectins as probes. Among 20 lectins tested, 10 bound to the Closterium cell surface, and eight of these were specific for the cells involved in sexual reproduction. In addition, some of the lectins inhibited the progress of zygote formation. In particular, Lycopersicon esculentum lectin (LEL) and ConcanavalinA (ConA) considerably inhibited zygote formation (23.6% and 0% of zygotes formed, respectively, compared with the control). LEL mainly accumulated on conjugation papillae and on the surface and lumens of empty cell walls remaining after zygote formation. ConA bound to both vegetative and sexually reproductive cells and strongly accumulated on the conjugation papillae of the latter, indicating ConA binding material(s) are non‐specifically present in Closterium cells but some of the material(s) would be essential for zygote formation. These results suggest that different carbohydrates specifically recognized by these lectins are involved in cell recognition and/or fusion during conjugation processes in the C. psl. complex.     

Localization of mannose- and galactose-binding lectins in an effective peanut nodule

Summary Mannose/glucose- and galactose-binding lectins (ML and GL respectively, were located by immunogold labelling in tissues of a peanut (Arachis hypogaea) nodule induced by an effectiveBradyrhizobium sp. strain. Light and electron microscopic examination of silver-enhanced semithin and ultrathin sections, respectively, revealed that both lectins were widely distributed throughout the cortex and bacteroidal zones although ML was more abundant. The lectins were predominantly in the vacuoles of cortical cells but GL was absent from, or at low concentration in, a two-cell-thick layer of cortical cells surrounding the bacteroidal region. Only ML was detected in cells of the vascular bundle endodermis and in central vascular bundle cells; neither lectin was found in pericycle cells. Bacteroidal cells contained abundant ML in the nuclei and cytoplasm surrounding bacteroids while GL was mainly located in the central vacuoles of these cells. Neither lectin was associated with bacteroid surfaces, peribacteroid membranes, plant cell walls or cell organelles and membranes. The above observations indicate that the nodule lectins are not symbiotic cell recognition determinants and suggest that they have protein storage functions.Abbreviations BSA bovine serum albumin - GL galactose-binding lectin - ML mannose-binding lectin - PBS phosphate-buffered saline - PBST phosphate-buffered saline plus Tween     

Cloning and characterization of the lectin cDNA clones from onion,shallot and leek

Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells.cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level.Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5–13 kDa) after post-translational modifications.Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.     

Opposite effects on human colon cancer cell proliferation of two dietary Thomsen-Friedenreich antigen-binding lectins

Increased cell surface expression of the Thomsen-Friedenreich antigen (TF antigen, Galbeta1-3GalNAcalpha-) is a common feature in malignant and pre-malignant epithelia. Our previous studies have shown that dietary TF-binding lectins from peanut (Arachis hypogea) and edible mushroom (Agaricus bisporus) produce marked but different effects on human intestinal epithelial cell proliferation. This study investigates the proliferative effects of the other two known dietary TF-binding lectins: jacalin (Artocarpus integrifolia, JAC) and amaranth lectin (Amaranthus caudatus, ACA). JAC produced dose-dependent and non-cytotoxic inhibition of proliferation in HT29 human colon cancer cells with maximal effects of 46 +/- 4% at 20 microg/ml, whereas ACA produced dose-dependent stimulation of proliferation with maximal effects of 22 +/- 3% at 20 microg/ml when assessed both by incorporation of [3H]thymidine into DNA and by cell counting. The lectin-mediated effects were inhibitable by the presence of appropriate Galbeta1-3GalNAc-expressing glycoproteins but differences existed between JAC and ACA in their patterns of inhibition by such substances. Ligand binding equilibrium studies using iodinated lectins revealed different Kd of the two lectins for HT29 cell surface glycoproteins. Lectin blots of cell membrane extracts showed different binding patterns in all the four TF-binding lectins. These results provide further evidence that dietary TF-binding lectins can have marked effects on the proliferation of human malignant gastro-intestinal epithelial cells and hence may play a role in intestinal cancer development, and also show that the biological effects of dietary lectins cannot be predicted solely from their carbohydrate binding properties.     

Lectin histochemical studies of mouse granulated metrial gland cells

A lectin histochemical study has been carried out on mouse granulated metrial gland cells, the major leucocyte population that differentiates in the uterine wall in pregnancy. The binding characteristics of 26 lectins were examined using light microscopical methods. Fourteen of the lectins, with affinities ranging through N-acetylgalactosamine, galactose, N-acetylglucosamine, mannose and sialic acid residues, bound to the cytoplasmic granules of granulated metrial gland cells, and each appeared to bind to the limiting membrane of the granules. The binding characteristics of three of these lectins (Wheat germ agglutinin, Concanavalin A and Helix pomatia agglutinin) were examined using electron microscopical methods. These showed a different binding pattern to the cytoplasmic granules of granulated metrial gland cells compared with that found using light microscopical methods, as they appeared to bind evenly across the granule's matrix. This binding pattern corresponds to the reactivity of the granule matrix in the periodic acid--Schiff technique. Six lectins bound to the cell membranes of granulated metrial gland cells. These included the E and L isoforms of Phaseolus vulgaris agglutinin, with affinities for complex carbohydrates, whose binding differences were related to the stage of differentiation of the granulated metrial gland cells. The lectin binding described presents additional markers of granulated metrial gland cells and tools for investigating carbohydrate moieties in the functional activities of granulated metrial gland cells This revised version was published online in November 2006 with corrections to the Cover Date.     

Snowdrop and wheatgerm lectins and avidin as antimetabolites for the control of sugarcane whitegrubs

Snowdrop and wheatgerm lectins were found to be insecticidal and growth inhibiting dietary proteins for larvae of the sugarcane whitegrub Antitrogus parvulus. At concentrations as low as 0.5 mg of snowdrop lectin per gram of semi-artificial diet, growth was inhibited by 21 days of feeding and significant mortality was apparent by 28 days. Wheatgerm lectin was active at similar concentrations, although expression of the effects was slower. Avidin was found to be a growth inhibiting dietary protein for larvae of Antitrogus consanguineus. At levels as low as 0.01 mg g^-1 of diet, growth was inhibited by 28 days of feeding. Avidin caused no significant mortality after 35 days of feeding. Snowdrop and wheatgerm lectins and avidin are insect growth-inhibiting proteins whose genes potentially could be manipulated into sugarcane and improve host-plant resistance to whitegrubs.