Fluorescent-labeled Lectin Research Articles

Diverse Localization Patterns of an R-Type Lectin in Marine Annelids.

Lectins facilitate cell–cell contact and are critical in many cellular processes. Studying lectins may help us understand the mechanisms underlying tissue regeneration. We investigated the localization of an R-type lectin in a marine annelid (Perinereis sp.) with remarkable tissue regeneration abilities. Perinereis nuntia lectin (PnL), a galactose-binding lectin with repeating Gln-X-Trp motifs, is derived from the ricin B-chain. An antiserum was raised against PnL to specifically detect a 32-kDa lectin in the crude extracts from homogenized lugworms. The antiserum detected PnL in the epidermis, setae, oblique muscle, acicula, nerve cord, and nephridium of the annelid. Some of these tissues and organs also produced Galactose (Gal) or N-acetylgalactosamine (GalNAc), which was detected by fluorescent-labeled plant lectin. These results indicated that the PnL was produced in the tissues originating from the endoderm, mesoderm, and ectoderm. Besides, the localizing pattern of PnL partially merged with the binding pattern of a fluorescent-labeled mushroom lectin that binds to Gal and GalNAc. It suggested that PnL co-localized with galactose-containing glycans in Annelid tissue; this might be the reason PnL needed to be extracted with haptenic sugar, such as d-galactose, in the buffer. Furthermore, we found that a fluorescein isothiocyanate-labeled Gal/GalNAc-binding mushroom lectin binding pattern in the annelid tissue overlapped with the localizing pattern of PnL. These findings suggest that lectin functions by interacting with Gal-containing glycoconjugates in the tissues.

The investigation of the changes in the surface glycoconjugates using two different spheroid models of breast cancer cells and availability assessment of these spheroid models for rapid diagnosis

The importance of early cancer diagnosis has led to development of many different diagnostic methods. In this context, the studies investigating the presence and amount of sugar residues to use as indicators in the identification of cancer cell type have become prominent. In the present study, sialic acids found on the membrane surfaces of ER (+) MCF-7 and ER (-) MDA-MB-231 breast cancer cell lines were labeled using three-dimensional (3D) cell culture (Spheroid) model as the closest method to the patient sample, thus its natural environment, among the cell culture methods. These sugar units that play a role in regulation of important immune characteristics such as recognition, binding and metastasis were made visualizable by applying fluorescent-labeled lectins such as FITC-(Wheat Germ Agglutinin) specifically binding to sialic acid units (GlcNAc, Neu5Ac) including particularly ß-GlcNAc and FITC-(Maackia Amurensis-Lectin-1) specifically binding to Galß4GlcNAc type sialic acids. These glycan units were specifically labeled with FITC-(Maackia Amurensis-Lectin-1) and FITC- (Wheat Germ Agglutinin) and radiation intensities were analyzed relatively. The two different breast cancer cell cultures were compared with respect to change in the amounts of sialic acid residues containing α-2,3- and α-2,6 bonds using fluorescent-labeled lectins. In the present study, we have performed a precise, accurate and rapid determination of the sugar content in the different breast cancer cell surface lines by means of fluorescent-labeled lectins and carried out a relative comparison between the micrographs.

Looking inside the heart: a see-through view of the vascular tree.

The ability to acquire 3D images of the heart and its vasculature at cellular resolution facilitates a more detailed study of many heart diseases. Here, we describe a novel technique to image in 3D the heart vasculature by combining the CUBIC clearing protocol combined with in vivo administration of fluorescent-labeled lectin. The use of these techniques in combination with Selective Plane Illumination Microscopy (SPIM) made it possible to obtain high resolution 3D images of the cardiac vascular tree. This methodological approach may enhance the visualization of 3D images of the cardiac vasculature remodeling associated with coronary disease.

Glycocalyx and its involvement in clinical pathophysiologies.

Vascular hyperpermeability is a frequent intractable feature involved in a wide range of diseases in the intensive care unit. The glycocalyx (GCX) seemingly plays a key role to control vascular permeability. The GCX has attracted the attention of clinicians working on vascular permeability involving angiopathies, and several clinical approaches to examine the involvement of the GCX have been attempted. The GCX is a major constituent of the endothelial surface layer (ESL), which covers most of the surface of the endothelial cells and reduces the access of cellular and macromolecular components of the blood to the surface of the endothelium. It has become evident that this structure is not just a barrier for vascular permeability but contributes to various functions including signal sensing and transmission to the endothelium. Because GCX is a highly fragile and unstable layer, the image had been only obtained by conventional transmission electron microscopy. Recently, advanced microscopy techniques have enabled direct visualization of the GCX in vivo, most of which use fluorescent-labeled lectins that bind to specific disaccharide moieties of glycosaminoglycan (GAG) chains. Fluorescent-labeled solutes also enabled to demonstrate vascular leakage under the in vivo microscope. Thus, functional analysis of GCX is advancing. A biomarker of GCX degradation has been clinically applied as a marker of vascular damage caused by surgery. Fragments of the GCX, such as syndecan-1 and/or hyaluronan (HA), have been examined, and their validity is now being examined. It is expected that GCX fragments can be a reliable diagnostic or prognostic indicator in various pathological conditions. Since GCX degradation is strongly correlated with disease progression, pharmacological intervention to prevent GCX degradation has been widely considered. HA and other GAGs are candidates to repair GCX; further studies are needed to establish pharmacological intervention. Recent advancement of GCX research has demonstrated that vascular permeability is not regulated by simple Starling’s law. Biological regulation of vascular permeability by GCX opens the way to develop medical intervention to control vascular permeability in critical care patients.

Cardiomyocyte A Disintegrin And Metalloproteinase 17 (ADAM17) Is Essential in Post-Myocardial Infarction Repair by Regulating Angiogenesis.

A disintegrin and metalloproteinase 17 (ADAM17) is a membrane-bound enzyme that mediates shedding of many membrane-bound molecules, thereby regulating multiple cellular responses. We investigated the role of cardiomyocyte ADAM17 in myocardial infarction (MI). Cardiomyocyte-specific ADAM17 knockdown mice (ADAM17(flox/flox)/α-MHC-Cre; f/f/Cre) and parallel controls (ADAM17(flox/flox); f/f) were subjected to MI by ligation of the left anterior descending artery. Post MI, f/f/Cre mice showed compromised survival, higher rates of cardiac rupture, more severe left ventricular dilation, and suppressed ejection fraction compared with parallel f/f-MI mice. Ex vivo ischemic injury (isolated hearts) resulted in comparable recovery in both genotypes. Myocardial vascular density (fluorescent-labeled lectin perfusion and CD31 immunofluorescence staining) was significantly lower in the infarct areas of f/f/Cre-MI compared with f/f-MI mice. Activation of vascular endothelial growth factor receptor 2 (VEGFR2), its mRNA, and total protein levels were reduced in infarcted myocardium in ADAM17 knockdown mice. Transcriptional regulation of VEGFR2 by ADAM17 was confirmed in cocultured cardiomyocyte-fibroblast as ischemia-induced VEGFR2 expression was blocked by ADAM17-siRNA. Meanwhile, ADAM17-siRNA did not alter VEGFA bioavailability in the conditioned media. ADAM17 knockdown mice (f/f/Cre-MI) exhibited reduced nuclear factor-κB activation (DNA binding) in the infarcted myocardium, which could underlie the suppressed VEGFR2 expression in these hearts. Post MI, inflammatory response was not altered by ADAM17 downregulation. This study highlights the key role of cardiomyocyte ADAM17 in post-MI recovery by regulating VEGFR2 transcription and angiogenesis, thereby limiting left ventricular dilation and dysfunction. Therefore, ADAM17 upregulation, within the physiological range, could provide protective effects in ischemic cardiomyopathy.

Incorporation of Fluorescent Nonnatural Amino Acid into Sialic Acid-Binding Lectin for Fluorescence Detection of Ligand-Binding

Abstract Lectins are important not only as biological functional molecules but as useful probes for glycan analysis. Here, we developed fluorescent-labeled lectins showing fluorescence changes upon ligand-binding. BODIPYFL-aminophenylalanine was incorporated at eight respective positions near the ligand-binding site of a sialic acid-binding lectin in response to UAG codon in a cell-free translation system. Fluorescence imaging of SDS-PAGE gel indicated the successful expression of the fluorescent-labeled lectins containing BODIPYFL-linked amino acid. Fluorescence spectral measurements revealed that the fluorescent-labeled lectins showed significant increases in the fluorescence intensity upon the binding of sialyllactose for all the incorporation positions. Particularly, the fluorescence intensity increased 3.7-fold for the labeled lectin at Thr38 position. The increase in the fluorescence intensity could occur because the fluorescence of BODIPYFL is quenched by amino acid residues located near the ligand-binding site in the absence of ligand, but the ligand-binding reduces the fluorescence quenching by removing the BODIPYFL moiety from the ligand-binding site. The W31F substitution decreased the fluorescence change of the labeled lectin, suggesting that the Trp31 residue may partially contribute to the ligand-dependent quenching. The present strategy will be utilized for producing various fluorescent-labeled lectin probes showing ligand-dependent fluorescence change.

Inhibition of lectin adherence to tissue culture cells by prebiotic carbohydrates

Prebiotic carbohydrates, in addition to their ability to influence the colonic microbiota, are also able to inhibit attachment of pathogenic bacteria to epithelial cells. This effect is mediated via their structural similarity to the carbohydrate ligands, located on the mucosal cell surface to which bacterial lectins attach. However, the mechanism for this inhibition is not well understood. The goal of this research was to measure the effect of two prebiotic carbohydrates, galactooli-gosaccharide and polydextrose, on the binding kinetics of plant lectins, having known ligand specificity, to tissue culture cells. To measure adherence, competetion experiments were conducted with HEp-2 cells exposed to nine fluorescent-labeled lectins and either the cognate ligand or a prebiotic. Fluorescence microscopy and image analysis were used to quantify adherence. Lectins that were able to bind to target cells were significantly inhibited in the presence of the cognate ligands. When prebiotics were added, inhibittion of lectin binding was observed, depending on the structural similarity between the prebiotic and the cognate ligands. In general, PDX did not inhibit lectin attachment, whereas GOS significantly inhibited most lectins. This research suggests that receptor sites located on the surface of epithelial HEp-2 cells are structurally similar to GOS.

Differences in human and porcine platelet oligosaccharides may influence phagocytosis by liver sinusoidal cells in vitro

Acute thrombocytopenia was revealed as a limiting factor to porcine liver xenotransplantation from in vitro and in vivo studies using porcine liver in human and baboon transplant models. The asialoglycoprotein receptor 1 (ASGR1) on liver sinusoidal endothelial cells (LSEC) and macrophage antigen complex-1 (Mac-1) on Kupffer cells (KC) mediate platelet phagocytosis and have carbohydrate-binding sites that recognize galactose and N-acetyl glucosamine in the beta conformation. Analysis of these receptor carbohydrate-binding domains and surface carbohydrates on human and porcine platelets may shed light on the mechanism of xenotransplantation-induced thrombocytopenia. An amino acid sequence comparison of human and porcine ASGR1 lectin-binding domains was performed. Using fluorescent labeled-lectins, human platelets, domestic and α1,3 galactosyltransferase knockout/human decay accelerating factor, porcine platelets were characterized by flow cytometry and lectin blot analyses. After desialylation, human and porcine platelets were examined by flow cytometry to determine whether sialic acid capping of galactose and N-acetyl glucosamine oligosaccharides in the beta conformation was a factor. Further, desialylated human platelets were studied on primary porcine liver sinusoidal cells with regard to binding and phagocytosis. Human platelets have four times more exposed galactose β1-4 N-acetyl glucosamine (Galβ) and N-acetyl glucosamine β1-4 N-acetyl glucosamine (βGlcNAc) than fresh porcine platelets. Galβ and βGlcNAc moieties on porcine platelets were not masked by sialic acid. Removal of sialic acid from human platelets increased binding and phagocytosis by LSEC and KC. Differences between human and porcine ASGR1 and Mac-1, in combination with a significantly higher number of galactose and N-acetyl glucosamine-containing oligosaccharides on human platelets contribute, in part, to platelet loss seen in porcine liver xenotransplantation.

Characterization of Laurencia arbuscula spore mucilage and cell walls with stains and FITC-labelled lectins

In most red algae, spores are liberated without a cell wall, within a sheath of mucilage that is responsible for its primary attachment. Utilizing fluorescent-labelled lectins, we identified carbohydrate residues and their location in the mucilage and cell walls of spores of Laurencia arbuscula. Cell wall formation and mucilage composition were studied with Calcofluor, Toluidine Blue (AT-O), Alcian Blue (AB) and periodic acid-Schiff (PAS). In the mucilage, we identified α- d-mannose, α- d-glucose, N-acetyl-glucosamine, N-acetyl-galactosamine and β- d-galactose. All sugar residues were found in the cell wall, in the spore body rather than in the rhizoid, which suggests that the residues may be related to initial substrate adhesion. A cell wall is produced soon after the spore's attachment, beginning with a deposition of cellulose around the spore, as indicated by Calcofluor. A polarization of the cell wall triggers the process of germination. The cell-wall matrix was positive to AB and metachromatic to AT-O, indicating acidic polysaccharides, while neutral polysaccharides were positive to PAS.

Spore Adhesion and Cell Wall Formation in Gelidium Floridanum (Rhodophyta, Gelidiales)

The attachment of spores to a substratum is essential for their germination and, therefore, to the completion of the life cycle of the red algae. In most red algae, spores are liberated without a cell wall, within a sheath of mucilage which is responsible for their primary attachment. Utilizing fluorescent-labeled lectins, we identified carbohydrate residues and their locations in the mucilage and cell walls of spores of Gelidium floridanum. Cell wall formation and mucilage composition were studied with calcofluor, toluidine blue — O (AT-O), alcian blue (AB) and periodic acid-Schiff (PAS). In the mucilage we identified α-D mannose, α-D glucose, β-D-galactose, N-acetyl-glucosamine and N-acetyl-galactosamine. The first two sugar residues were not found in the cell wall of the germ tube but they were present on the rhizoid’s cell wall indicating their importance to substrate adhesion. A cell wall is produced soon after the spore’s attachment, beginning with a polar deposition of cellulose and its gradual spread around the spore as indicated by calcofluor. The cell wall matrix was positive to AB and metachromatic to AT-O, indicating acidic polysaccharides, while cellulose microfibrills were positive to PAS. A polar disorganization of the cell wall triggers the process of germination. As spores are the natural form of propagation of Gelidium, the understanding of the mechanisms of spore attachment may contribute to the cultivation of this valuable seaweed.

The effect of fungal species on the fluorescent lectin test

Fungal (mold) contamination is an important indicator of low-quality raw product used in food processing operations. Fluorescent-labeled lectins, specific for chitin, have been shown to be valuable for quantitative detection of mold in raw tomatoes. In this research, the response of individual fungal species to a rapid fluorescent lectin assay was investigated. Ten of the most common mold species were grown on two types of artificial broth media, and added to blended field tomatoes. The assay was conducted on each species, and linear regressions were developed, comparing the fluorescent lectin assay score with the fungal dry weight. The assay was able to detect all molds at sensitivities required for the tomato industry, and had high linearity ( r 2 ranging from 0.72 to 0.99) and low variability (standard error of calibration ranging from 20 to 116 μg of fungal biomass/ml of tomato juice) for individual species grown on V-8 juice broth.

Mycophenolic acid does not inhibit protein glycosylation in T lymphocytes

Background: Mycophenolic acid inhibits guanosine nucleotide synthesis and has been shown to be a potent inhibitor of lymphocyte proliferation as well as being effective at decreasing the incidence of graft rejection. Guanosine nucleosides are essential for protein glycosylation and many cell surface proteins including adhesion molecules, which are important for graft infiltration and rejection, are glycoproteins. There have been conflicting reports concerning the ability of MPA to interfere with glycosylation in lymphoid cells. Therefore, the purpose of this study was to investigate the effects of MPA on cell surface protein glycosylation in lymphoid cells. Methods: Cells were cultured in the presence of increasing concentrations of MPA for different lengths of time and stained with fluorescent-labelled lectins specific for either mannose or fucose residues on glycoproteins. Analysis was then performed by flow cytometry. Results: MPA treatment had no effect on the binding of either fucose or mannose-specific lectins to Con A stimulated human PBLs and rat lymph node lymphocytes or to a CEMC7a T cell line. Conclusion: The results show that, contrary to previous reports, MPA does not affect cell surface glycosylation in T cells using T cells from different sources of both human and non-human origin.

Major carbohydrate epitopes in tissues of domestic and African wild animals of potential interest for xenotransplantation research.

We investigated the main glycotopes expressed on the tissues of 44 animal species, including primates, nonprimate mammals, marsupials, birds, and a reptile. Paraffin-embedded tissue sections of kidney, heart, liver, pancreas, lung, brain and intestine of 24 domestic animal species were stained with seven fluorescent-labeled lectins. Testis sections of 20 African wild animal species were tested with the same lectins. Overall, three main immunofluorescence patterns were found in the vascular compartment. First, humans and Old World monkeys express genetically polymorphic ABH antigens and do not express alphaGal. Second, New World monkeys, other mammals, and marsupials do not express ABH antigens, but have large amounts of a genetically monomorphic alphaGal. Third, birds and reptiles do not express either ABH or alphaGal, but have monomorphic betaGal, probably different from the lactosamine precursor of ABH and alphaGal. Epithelial cells producing exocrine secretions also expressed carbohydrate epitopes. The fluorescence patterns of the cells of the exocrine compartment are similar, but not identical, to those expressed in the vascular compartment. All the animals tested have some ABH and betaGal in exocrine tissues, but New World monkeys and lower mammals are the only ones expressing alphaGal in exocrine tissues.

Lectin binding to human colonocytes is predictive of colonic neoplasia

OBJECTIVE: The aim of this study was to determine whether lectin binding to exfoliated human colonocytes could be used as a noninvasive test for colorectal polyps or cancer. METHODS: Colonocytes were harvested from 31 patients (10 controls, 10 with adenomatous polyps, and 11 with cancer), incubated with a panel of fluorescent-labeled lectins, and assayed by flow cytometry. RESULTS: The lectins jacalin (JAC) and wheat germ agglutinin (WGA) were useful in predicting the presence of a colorectal neoplasm ( p = 0.0018 for JAC and p = 0.0099 for WGA). For JAC, sensitivity reached 81% with a specificity of 80%, and for WGA the sensitivity and specificity were both 75%. CONCLUSIONS: Lectin binding to human colonocytes can predict the presence of malignant and premalignant lesions of the colon, and has potential as a noninvasive screening tool for colorectal neoplasms.

Lectin binding to human colonocytes is predictive of colonic neoplasia.

The aim of this study was to determine whether lectin binding to exfoliated human colonocytes could be used as a noninvasive test for colorectal polyps or cancer. Colonocytes were harvested from 31 patients (10 controls, 10 with adenomatous polyps, and 11 with cancer), incubated with a panel of fluorescent-labeled lectins, and assayed by flow cytometry. The lectins jacalin (JAC) and wheat germ agglutinin (WGA) were useful in predicting the presence of a colorectal neoplasm (p = 0.0018 for JAC and p = 0.0099 for WGA). For JAC, sensitivity reached 81% with a specificity of 80%, and for WGA the sensitivity and specificity were both 75%. Lectin binding to human colonocytes can predict the presence of malignant and premalignant lesions of the colon, and has potential as a noninvasive screening tool for colorectal neoplasms.

Alpha-Galactosidase A deficient mice: a model of Fabry disease.

Fabry disease is an X-linked inherited metabolic disorder that is caused by a deficiency of alpha-galactosidase A (alpha-Gal A). Progressive deposition of neutral glycosphingolipids that have terminal a-linked galactosyl moieties in vascular endothelial cells causes renal failure along with premature myocardial infarctions and strokes in patients with this condition. No specific treatment is available for patients with this disorder at this time. An animal model of this condition would be valuable for exploring therapeutic strategies for patients with Fabry disease. We report here the generation of alpha-Gal A deficient mice by gene targeting and an analysis of the resulting phenotype. The knockout mice display a complete lack of alpha-Gal A activity. The mice, however, appeared clinically normal at 10 weeks of age. Ultrastructural analysis revealed concentric lamellar inclusions in the kidneys, and confocal microscopy using a fluorescent-labeled lectin specific for alpha-D-galactosyl residues showed accumulation of substrate in the kidneys as well as in cultured fibroblasts. Lipid analysis revealed a marked accumulation of ceramidetrihexoside in the liver and the kidneys. These findings indicate the similarity of the pathophysiological process in the mutant mice and in patients with Fabry disease. The deficiency of alpha-Gal A activity and the accumulation of material containing terminal alpha-galactosyl residues in cultured embryonic fibroblasts derived from alpha-Gal A(-/0) mice were corrected by transducing these cells with bicistronic multidrug resistance retroviruses containing human alpha-Gal A cDNA.

The Examination of Seliberia stellata Exopolymers Using Lectin Assays

Holdfast exopolymers of the dimorphic oligotrophic bacterium Seliberia stellata were examined using fluorescent lectins under light microscopy and colloidal gold lectins using transmission electron microscopy. Examination using fluorescent-labeled lectins revealed that lectins specific for polysaccharides and monosaccharides such as glucose and/or mannose, galactose, N-acetylgalactosamine, and N-acetylglucosamine (and its dimer) adhered to holdfast structure. Colloidal gold-labeled lectin assays also suggested the presence of these sugars. Both the holdfast that mediates swarmer cell adhesion and the holdfast that facilitates rosette formation gave similar results, suggesting the structures may be the same. Another exopolymer produced later in the growth cycle was observed using transmission electron microscopy. It appeared as an amorphous glycocalyx-like material very different from holdfast exo-polymers. Retention of the gold lectin (Wheat Germ Agglutinin (WGA), suggested the presence of N-acetylglucosamine, but fluorescent analyses were unsuccessful. The data suggest that S. stellata produces at least two different exopolymers: (a) the exopolymer of the swarmer cell and rosette holdfast whose function is adhesion and whose composition is (but may not be limited to) polysaccharides and (b) a slime-like exopolymer whose composition and function remain unknown.

Tissue treatment for whole mount internal lectin staining in the nematodes Caenorhabditis elegans, Panagrolaimus superbus and Acrobeloides maximus.

Four different fixation schemes, using ten fluorescent-labelled lectins, were investigated for whole mount internal staining of three rhabditid nematodes: Caenorhabditis elegans, Panagrolaimus superbus and Acrobeloides maximus. Acetone-only fixation was found to give strong and reproducible staining, which could be prevented either by periodate treatment of the organisms or by specific inhibitory sugars of the lectins under investigation. Whereas the use of either phosphate or TRIS buffers had no effect on the staining pattern or the fluorescence intensity, the incubation time as well as the incubation temperature affected the staining reaction. The best results were obtained upon overnight incubation at 4 degrees C: the lectin staining could be inhibited in all cases, except for the intestinal brush border of C. elegans by the lectin of Lens culinaris.

Surface properties of developing stages of Trichuris muris.

Surface properties of developing stages of Trichuris muris were investigated by analysis of binding affinities for specific anti-parasite antibodies present in a range of infection sera; in vitro eosinophil adherence studies; and binding of the fluorescent-labelled lectins, Con A, WGA, PNA and RCA. In general, larvae of any one particular stage did not bind anti-parasite antibodies present in serum collected at an earlier stage of the infection, thus indicating a considerable degree of antigenic stage-specificity. Forty day and older parasites displayed similar binding properties and it is suggested that no major changes in surface antigenicity occur after the final moult at day 25-30 after infection. Surface properties were also examined by means of complement and antibody-mediated eosinophil adherence assays. Attachment of eosinophils was maximal with day 5 larvae, although even at this stage not all of the parasite surface was covered with attached cells. Despite adherence, eosinophils were unable to effect parasite killing, even after 48 h of incubation. These studies showed that eosinophil adherence-promoting antibodies were present in immune sera; that the larval parasite surface was able to activate complement by the alternate pathway with subsequent generation of C3b molecules, and that the parasite was able to withstand eosinophil adherence and secretion by an as yet unidentified evasive stratagem. Studies with fluorescent-labelled lectins showed that all larval stages (days 5-25 after infection) were positive for Con A binding. Early larval stages (days 5-10 after infection) also bound PNA and WGA. Interestingly, recently moulted individuals rarely exhibited fluorescence, but cast cuticles fluoresced brightly.

Effect of Fluorescent-Labeled Lectins on Visualization of Decay Fungi in Wood Sections

Effect of Fluorescent-Labeled Lectins on Visualization of Decay Fungi in Wood Sections