Differences In Lectin Binding Research Articles

Tamoxifen-resistant breast cancer cells exhibit reactivity with Wisteria floribunda agglutinin.

Glycosylation is one of the most important post-translational modifications of cell surface proteins involved in the proliferation, metastasis and treatment resistance of cancer cells. However, little is known about the role of glycosylation as the mechanism of breast cancer cell resistance to endocrine therapy. Herein, we aimed to identify the glycan profiles of tamoxifen-resistant human breast cancer cells, and their potential as predictive biomarkers for endocrine therapy. We established tamoxifen-resistant cells from estrogen receptor-positive human breast cancer cell lines, and their membrane-associated proteins were subjected to lectin microarray analysis. To confirm differential lectin binding to cellular glycoproteins, we performed lectin blotting analyses after electrophoretic separation of the glycoproteins. Mass spectrometry of the tryptic peptides of the lectin-bound glycoproteins was further conducted to identify glycoproteins binding to the above lectins. Finally, expression of the glycans that were recognized by a lectin was investigated using clinical samples from patients who received tamoxifen treatment after curative surgery. Lectin microarray analysis revealed that the membrane fractions of tamoxifen-resistant breast cancer cells showed increased binding to Wisteria floribunda agglutinin (WFA) compared to tamoxifen-sensitive cells. Glycoproteins seemed to be responsible for the differential WFA binding and the results of mass spectrometry revealed several membrane glycoproteins, such as CD166 and integrin beta-1, as candidates contributing to increased WFA binding. In clinical samples, strong WFA staining was more frequently observed in patients who had developed distant metastasis during tamoxifen treatment compared with non-relapsed patients. Therefore, glycans recognized by WFA are potentially useful as predictive markers to identify the tamoxifen-resistant and relapse-prone subset of estrogen receptor-positive breast cancer patients.

Altered Cell Surface N-Glycosylation of Resting and Activated T Cells in Systemic Lupus Erythematosus.

Altered cell surface glycosylation in congenital and acquired diseases has been shown to affect cell differentiation and cellular responses to external signals. Hence, it may have an important role in immune regulation; however, T cell surface glycosylation has not been studied in systemic lupus erythematosus (SLE), a prototype of autoimmune diseases. Analysis of the glycosylation of T cells from patients suffering from SLE was performed by lectin-binding assay, flow cytometry, and quantitative real-time PCR. The results showed that resting SLE T cells presented an activated-like phenotype in terms of their glycosylation pattern. Additionally, activated SLE T cells bound significantly less galectin-1 (Gal-1), an important immunoregulatory lectin, while other lectins bound similarly to the controls. Differential lectin binding, specifically Gal-1, to SLE T cells was explained by the increased gene expression ratio of sialyltransferases and neuraminidase 1 (NEU1), particularly by elevated ST6 beta-galactosamide alpha-2,6-sialyltranferase 1 (ST6GAL1)/NEU1 and ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6)/NEU1 ratios. These findings indicated an increased terminal sialylation. Indeed, neuraminidase treatment of cells resulted in the increase of Gal-1 binding. Altered T cell surface glycosylation may predispose the cells to resistance to the immunoregulatory effects of Gal-1, and may thus contribute to the pathomechanism of SLE.

Alterations in the glycome after HDAC inhibition impact oncogenic potential in epigenetically plastic SW13 cells

BackgroundDefects in the type and degree of cellular glycosylation impact oncogenesis on multiple levels. Although the type of glycosylation is determined by protein sequence encoded by the genome, the extent and modifications of glycosylation depends on the activity of biosynthetic enzymes and recent data suggests that the glycome is also subject to epigenetic regulation. This study focuses on the ability of HDAC inhibition to alter glycosylation and to lead to pro-oncogenic alterations in the glycome as assessed by metastatic potential and chemoresistance.MethodsEpigenetically plastic SW13 adrenocortical carcinoma cells were treated with FK228, an HDAC inhibitor with high affinity for HDAC1 and, to a lesser extent, HDAC2. In comparing HDAC inhibitor treated and control cells, differential expression of glycome-related genes were assessed by microarray. Differential glycosylation was then assessed by lectin binding arrays and the ability of cellular proteins to bind to glycans was assessed by glycan binding arrays. Differential sensitivity to paclitaxel, proliferation, and MMP activity were also assessed.ResultsTreatment with FK228 alters expression of enzymes in the biosynthetic pathways for a large number of glycome related genes including enzymes in all major glycosylation pathways and several glycan binding proteins. 84% of these differentially expressed glycome-related genes are linked to cancer, some as prognostic markers and others contributing basic oncogenic functions such as metastasis or chemoresistance. Glycan binding proteins also appear to be differentially expressed as protein extracts from treated and untreated cells show differential binding to glycan arrays. The impact of differential mRNA expression of glycosylation enzymes was documented by differential lectin binding. However, the assessment of changes in the glycome is complicated by the fact that detection of differential glycosylation through lectin binding is dependent on the methods used to prepare samples as protein-rich lysates show different binding than fixed cells in several cases. Paralleling the alterations in the glycome, treatment of SW13 cells with FK228 increases metastatic potential and reduces sensitivity to paclitaxel.ConclusionsThe glycome is substantially altered by HDAC inhibition and these changes may have far-reaching impacts on oncogenesis.

Catanionic Surfactant Vesicles as a New Platform for probing Glycan-Protein Interactions.

Glycomics lags substantially behind proteomics and genomics in its ability to decipher and synthesize complex glycans. The slow progress in deciphering glycan interactions at a molecular level is in large part due to the absence of a functional system to express, on a large scale, carbohydrates of known structure, in the context of a biologically relevant assay system. Here we describe the characterization of a glycan-functionalized catanionic surfactant vesicles (CVs) as a platform for glycan synthesis, and to demonstrate that the resulting glycan-functionalized CVs can serve as a scaffold for the interrogation of protein-glycan interactions. We demonstrate that N. gonorrhoeae lipooligosaccharide (LOS) glycosyltransferase LgtE, an enzyme that catalyzes the addition of galactose onto a terminal glucose found on LOS can be used to biochemically modify LOS or glucose functionalized CVs. CVs were characterized by differential lectin binding using flow cytometry. LgtE activity was measured on whole cells and LOS functionalized vesicles and found to have approximately the same biochemical properties. We further demonstrate that CVs can be ink-jet printed. This paper presents proof-of-concept that glycan-functionalized catanionic vesicles can be used to create a high-specificity and high-throughput glycan array that will allow for the investigation of a variety of protein-glycan interactions.

Distribution of Glycan Motifs at the Surface of Midgut Cells in the Cotton Leafworm (Spodoptera littoralis) Demonstrated by Lectin Binding.

Glycans are involved in many biological phenomena, including signal transduction, cell adhesion, immune response or differentiation. Although a few papers have reported on the role of glycans in the development and proper functioning of the insect midgut, no data are available regarding the localization of the glycan structures on the surface of the cells in the gut of insects. In this paper, we analyzed the spatial distribution of glycans present on the surface of the midgut cells in larvae of the cotton leafworm Spodoptera littoralis, an important agricultural pest insect worldwide. For this purpose, we established primary midgut cell cultures, probed these individual cells that are freely suspended in liquid medium with a selection of seven fluorescently labeled lectins covering a range of different carbohydrate binding specificities [mannose oligomers (GNA and HHA), GalNAc/Gal (RSA and SSA), GlcNAc (WGA and Nictaba) and Neu5Ac(α-2,6)Gal/GalNAc (SNA-I)], and visualized the interaction of these lectins with the different zones of the midgut cells using confocal microscopy. Our analysis focused on the typical differentiated columnar cells with a microvillar brush border at their apical side, which are dominantly present in the Lepidopteran midgut and function in food digestion and absorption, and as well as on the undifferentiated stem cells that are important for midgut development and repair. Confocal microscopy analyses showed that the GalNAc/Gal-binding lectins SSA and RSA and the terminal GlcNAc-recognizing WGA bound preferentially to the apical microvillar zone of the differentiated columnar cells as compared to the basolateral pole. The reverse result was observed for the mannose-binding lectins GNA and HHA, as well as Nictaba that binds preferentially to GlcNAc oligomers. Furthermore, differences in lectin binding to the basal and lateral zones of the cell membranes of the columnar cells were apparent. In the midgut stem cells, GNA and Nictaba bound more strongly to the membrane of these undifferentiated cells compared to the microvillar pole of the columnar cells, while SSA, HHA, WGA, and SNA-I showed stronger binding to the microvilli. Our results indicated that polarization of the midgut cells is also reflected by a specific distribution of glycans, especially between the basal and microvillar pole. The data are discussed in relation to the functioning and development of the insect midgut.

Вплив гіпотирозу материнського орґанізму на постнатальний морфоґенез яєчок щурів за даними морфометрії та лектинової гістохімії

Thyroid disorders are currently among the most widespread endocrine pathologies, influencing multiple organs, testes among others. However, the impact of maternal hypothyroidism on postnatal development of male reproductive system including its glycome currently remains obscure. Aim. By means of morphometric and lectin histochemistry analysis to investigate the influence of maternal hypothyroidism on postnatal morphogenesis of rat progeny testes. Material and Methods. Experimental hypothyroidism was induced in 15 female Wistar rats by supplementation of their daily food allowance with 5 mg/kg of mercazolil during 14 days prior to fertilization and during whole gestational period. Testes of their progeny on prenatal day 20th, postnatal days 1st, 20th, 40th and 120th were excised, fixed in Bouin's fluid, embedded in paraffin and subjected to morphometric and lectin histochemistry investigation. The obtained data was compared with the same parameters of progeny from control group rats.  Results and Discussion. Maternal hypothyroidism in prenatal day 20th rats was associated with decreased size and enhanced count of Sertoli cells within the seminiferous cords; decreased amount of interstitial connective tissue combined with significant elevation of Leydig cells count and reduced lectin binding to all testicular components. On postnatal day 1st it was detected the increased Leydig cells content, though less prominent in comparison with prenatal period. On postnatal day 20th size of seminiferous tubules of hypothyroid rats progeny strongly exceeded control parameters due to the increased spermatogenic cells proliferation rate. On postnatal day 40th seminiferous tubules of experimental group rats contained large conglomerates of degenerating and apoptotic spermatogenic cells. In the adult rats, the decreased size of seminiferous tubules coincided with the increased spermatogenic index and content of Leydig cells. The latter were selectively labeled with PSA, GNA and CCRA, mast cells - with WGA, early acrosomes - with SBA, cap stage and wedged acrosomes - with PNA and SNA lectins.  Conclusions. Maternal hypothyroidism has significant impact on postnatal morphogenesis of the rat testes. Differences in lectin binding to tissue samples of control and experimental rat testes were most significant in prenatal animals with certain levelling until adulthood, while morphometric indices were clearly distinct in all investigated progeny groups. Documented selective lectin labeling of Leydig cells with lectins PSA, GNA and CCRA, of mast cells with WGA, as well as different stages of acrosomal maturation with SBA, PNA and SNA can be recommended for further morphometric investigations.

Glyco-centric lectin magnetic bead array (LeMBA) - proteomics dataset of human serum samples from healthy, Barrett׳s esophagus and esophageal adenocarcinoma individuals.

This data article describes serum glycoprotein biomarker discovery and qualification datasets generated using lectin magnetic bead array (LeMBA) – mass spectrometry techniques, “Serum glycoprotein biomarker discovery and qualification pipeline reveals novel diagnostic biomarker candidates for esophageal adenocarcinoma” [1]. Serum samples collected from healthy, metaplastic Barrett׳s esophagus (BE) and esophageal adenocarcinoma (EAC) individuals were profiled for glycoprotein subsets via differential lectin binding. The biomarker discovery proteomics dataset consisting of 20 individual lectin pull-downs for 29 serum samples with a spiked-in internal standard chicken ovalbumin protein has been deposited in the PRIDE partner repository of the ProteomeXchange Consortium with the data set identifier PRIDE: PXD002442. Annotated MS/MS spectra for the peptide identifications can be viewed using MS-Viewer (〈http://prospector2.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer〉) using search key “jn7qafftux”. The qualification dataset contained 6-lectin pulldown-coupled multiple reaction monitoring-mass spectrometry (MRM-MS) data for 41 protein candidates, from 60 serum samples. This dataset is available as a supplemental files with the original publication [1].

Differential Lectin Binding and Coronary Angiography in a Danio and non‐Model Fish Species

Lectins are carbohydrate‐binding proteins commonly used in the study of mammalian cells and tissues. However lectins have received little use in fish models and non‐models species. Here, we determine the binding patterns of commonly used lectins, including wheat germ agglutinin (WGA), Ulex europaeus agglutinin (UEA lectin), Bandeiraea simplicifolia lectin (BS lectin), Concanavalin A (Con A), Ricinus communis (RCA), and Lycopersicon esculentum lectin (tomato lectin) in zebrafish and giant danio hearts. Our results show that WGA stained fish cardiac myocyte borders in patterns similar to that seen in mammalian hearts, with staining markedly stronger in the fish compact hearts. Con A showed broad and strong staining in fish ventricles. Interestingly, BS lectin reacted poorly to a number of fish species including zebrafish, pearl danio, common koi, goldfish, and Buenos Aires tetra. By contrast BS lectin strongly stained coronary vessels of giant danio, and several pleco and gourami species. Importantly, enhanced BS lectin binding to vascular endothelium compared to endocardium allowed for 3D coronary angiography in the giant danio by confocal and lightsheet microscopy, and for quantitation of vascular reconstruction during regeneration. In conclusion, our study demonstrates that lectins are simple but important tools for studies in models and non‐models fish species, and in adult and regenerating giant danio heart.Support or Funding InformationR15 HD084262‐01, FDC‐PDF‐DePauw University

Histochemical and ultrastructural analyses of the lubrication systems in the olfactory organs of soft-shelled turtle.

In general, the nasal cavity of turtles is divided into two chambers: the upper chamber, lined with the olfactory epithelium containing ciliated olfactory receptor cells, and the lower chamber, lined with the vomeronasal epithelium containing microvillous receptor cells. In the nasal cavity of soft-shelled turtles, however, differences between the upper and lower chamber epithelia are unclear due to the presence of ciliated receptor cells in both epithelia. In the olfactory organ of vertebrates, the surface of sensory epithelium is covered with secretory products of associated glands and supporting cells, playing important roles in the olfaction by dissolving odorants and transporting them to the olfactory receptors. Here, the associated glands and supporting cells in the olfactory organ of soft-shelled turtles were analyzed histochemically and ultrastructurally. The upper chamber epithelium possessed associated glands, constituted by cells containing serous secretory granules; whereas, the lower chamber epithelium did not. In the upper chamber epithelium, secretory granules filled the supranuclear region of supporting cells, while most of the granules were distributed near the free border of supporting cells in the lower chamber epithelium. The secretory granules in the supporting cells of both epithelia were seromucous, but alcian blue stained them differently from each other. In addition, distinct expression of carbohydrates was suggested by the differences in lectin binding. These data indicate the quantitative and qualitative differences in the secretory properties between the upper and lower chamber epithelia, suggesting their distinct roles in the olfaction.

Differential Lectin Binding in Giant Danio, Goldfish, and Zebrafish hearts

Lectins are carbohydrate‐binding proteins commonly used in the study of mammalian cells and tissues. However lectins have received little use in fish models. Here, we determine the binding patterns of three commonly used lectins: wheat germ agglutinin (WGA), Ulex europaeus agglutinin (UEA lectin), Bandeiraea simplicifolia lectin (BS lectin), in giant danio (GD), zebrafish (ZF), and goldfish (GF) hearts. Our results show that WGA stained cardiac myocyte borders of all three fish in patterns similar to that seen in mammalian hearts, with staining markedly stronger in the fish compact hearts. By contrast, UEA lectin, an endothelial cell marker in mammalian vascular beds, showed little to no binding to the coronary vasculature of these fish. Interestingly, BS lectin reacted poorly to the zebrafish coronary vasculature similar to our previously reported study in the goldfish. However BS lectin strongly stained coronary vessels of the Giant danio. Importantly, BS lectin biding was particularly strong in the vascular endothelium compared to endocardium of the Giant danio. This differential regional staining allowed for coronary angiography of the Giant danio heart and 3D reconstruction by confocal and light sheet fluorescence microscopy. In addition we were able to quantify coronary revascularization following cautery injury and during repair and regeneration of the Giant danio heart. Our results demonstrate that lectins differentially bind to coronary vasculature of fish and can be used as simple but powerful tools for the study of adult and regenerating fish hearts.

Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and haemolymph-derived cells of the entomopathogenic fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana and its insect host target represent a model system with which to examine host-pathogen interactions. Carbohydrate epitopes on the surfaces of fungal cells play diverse roles in processes that include adhesion, non-self recognition and immune invasion with respect to invertebrate hosts. B. bassiana produces a number of distinct cell types that include aerial conidia, submerged conidia, blastospores and haemolymph-derived cells termed in vivo blastospores or hyphal bodies. In order to characterize variations in the surface carbohydrate epitopes among these cells, a series of fluorescently labelled lectins, combined with confocal microscopy and flow cytometry to quantify the response, was used. Aerial conidia displayed the most diverse lectin binding characteristics, showing reactivity against concanavalin A (ConA), Galanthus nivalis (GNL), Griffonia simplicifolia (GSII), Helix pomatia (HPA), Griffonia simplicifolia isolectin (GSI), peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEAI) and wheatgerm agglutinin (WGA), and weak reactivity against Ricinus communis I (RCA), Sambucus nigra (SNA), Limax flavus (LFA) and Sophora japonica (SJA) lectins. Lectin binding to submerged conidia was similar to that to aerial conidia, except that no reactivity against UEAI, HPA and SJA was noted, and WGA appeared to bind strongly at specific polar spots. In contrast, the majority of in vitro blastospores were not bound by ConA, GNL, GSII, GSI, SNA, UEAI, LFA or SJA, with PNA binding in large patches, and some polarity in WGA binding noted. Significant changes in lectin binding also occurred after aerial conidial germination and in cells grown on either lactose or trehalose. For germinated conidia, differential lectin binding was noted between the conidial base, the germ tube and the hyphal tip. Fungal cells isolated from the haemolymph of the infected insect hosts Manduca sexta and Heliothis virescens appeared to shed most carbohydrate epitopes, displaying binding only to the GNL, PNA and WGA lectins. Ultrastructural examination of the haemolymph-derived cells revealed the presence of a highly ordered outermost brush-like structure not present on any of the in vitro cells. Haemolymph-derived hyphal bodies placed into rich broth medium showed expression of several surface carbohydrate epitopes, most notably showing increased PNA binding and strong binding by the RCA lectin. These data indicate robust and diverse production of carbohydrate epitopes on different developmental stages of fungal cells and provide evidence that surface carbohydrates are elaborated in infection-specific patterns.

GLYCOCONJUGATE ORGANIZATION OF ENTEROMORPHA (=ULVA) FLEXUOSA AND ULVA FASCIATA (CHLOROPHYTA) ZOOSPORES(1).

Ecologically successful algae that colonize natural and artificial substrates in the marine environment have distinct strategies for opportunistic dispersal and settlement. The objective of this research was to visualize molecular architecture of zoospores from Enteromorpha (=Ulva) flexuosa (Wulfen) J. Agardh and Ulva fasciata Delile that coexist but alternate in dominance on an intertidal bench. Multiple fluorescent lectins were used to stabilize and probe for diverse zoospore glycoconjugates (GC) that could be involved in cell and substrate interactions. Results from epifluorescence microscopy showed distinct cellular and extracellular polymeric substance (EPS) domains of GC relative to settlement morphologies. Glycoconjugates were similar for both species with (1) α-d mannose and/or glucose moieties localized on flagella, the anterior domes and anterior regions, the plasma membranes, and EPS; (2) α-fucose localized on flagella and anterior regions; (3) N or α,ß-N acetylglucosamine localized on flagella, the anterior regions, and EPS; and (4) varied N-acetylgalactosamine and/or galactose moieties localized on each domain for both species excluding the plasma membranes. Some differences in lectin binding were observed for each species at the flagella, the anterior domes, and the plasma membranes. Glycoconjugate distributions shifted with morphological changes that followed initial adhesion. TEM of E.flexuosa zoospore stages following carbohydrate-stabilizing fixations and gold-conjugated lectin probes resolved GC with α-d mannose and/or glucose, and/or N-acetylglucosamine at the plasma membrane, ER and diverse vesicles of the anterior pole, EPS, and discontinuous regions or knobs associated with flagellar surfaces. The distinct distribution and diversity of zoospore GC may be central to recognition and attachment on diverse substrata by these algae.

Intracellular Glycoproteins Binding Galectin-1 in Thyroid Lesions

The increased expression of galectin-1 on the mRNA and protein level observed in malignant thyroid tumors in comparison with benign lesions suggests that this protein may be associated with malignant transformation of thyroid epithelium. Extracellular and membrane glycoproteins are the main known ligands for this galactose-binding lectin. However, immunofluorescence studies have shown that galectin-1 is found predominantly in the intracellular compartment. The aim of this study was to examine intracellular carbohydrate ligands of galectin-1 in the thyroid, with particular attention to potential differences in their expression levels between benign and malignant lesions. Identification of cytosolic and nuclear glycoproteins binding galectin-1 was performed by affinoblotting after separation of proteins in 8% polyacrylamide slab gels and electrotransfer onto Immobilon-P membranes. For semiquantitative analysis of glycoproteins binding galectin-1, an enzyme-linked lectino-solid-phase assay (ELLSA) was used. The predominant cytosolic glycoproteins binding galectin-1 had molecular masses of 50, 55, 59, 64, 85-87, 100, and 133 kDa and nuclear glycoprotein had a molecular mass of 75 kDa. There were no evident differences in glycoprotein patterns between benign and malignant thyroid lesions. The results obtained by ELLSA did not show any significant differences in lectin binding by cytosolic and nuclear proteins of thyroid lesions either. On the basis of these results it is tempting to suggest that interactions between galectin-1 and intracellular glycoconjugates are not critical for malignant transformation in the thyroid gland.

Effects of Differential Glycosylation of Glycodelins on Lymphocyte Survival

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetra-antennary complex-type glycans carrying Galbeta1-4GlcNAc (lacNAc) and/or GalNAcbeta1-4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcalpha2-3(GalNAcbeta1-4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.

Differential antibody binding to the surface TCR{middle dot}CD3 complex of CD4+ and CD8+ T lymphocytes is conserved in mammals and associated with differential glycosylation

We have previously shown that the surface alphabeta T cell antigen receptor (TCR).CD3 complex borne by human CD4(+) and CD8(+) T lymphocytes can be distinguished using mAbs. Using two unrelated sets of antibodies, we have now extended this finding to the surface alphabetaTCR.CD3 of seven additional mammalian species (six non-human primates and the mouse). We have also produced data supporting that differential glycosylation of the two main T cell subsets is involved in the observed TCR.CD3 antibody-binding differences in humans. First, we show differential lectin binding to human CD4(+) versus CD8(+) T lymphocytes, particularly with galectin 7. Second, we show that certain lectins can compete differentially with CD3 mAb binding to human primary CD4(+) and CD8(+) T lymphocytes. Third, N-glycan disruption using swainsonine was shown to increase mAb binding to the alphabetaTCR.CD3. We conclude that the differential antibody binding to the surface alphabetaTCR.CD3 complex of primary CD4(+) and CD8(+) T lymphocytes is phylogenetically conserved and associated with differential glycosylation. The differences may be exploited for therapeutic purposes, such as T cell lineage-specific immunosuppression of graft rejection. Also, the impact of glycosylation on CD3 antibody binding requires a cautious interpretation of CD3 expression levels and T cell numbers in clinical diagnosis.

Biology of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a common cancer in the world due to high prevalence of hepatitis B or C virus infection. Research in recent years has uncovered important molecular pathways involved in development and progression of HCC. Several genetic aberrations and molecular mechanisms responsible for initiation of hepatocarcinogenesis have been identified. Novel biomarkers for HCC are being developed for better detection and prognostication. Alpha-fetoprotein, the conventional marker of HCC, has limited sensitivity and specificity. Serum levels of isoforms of AFP based on differential lectin binding of the glycan moiety appear to be more sensitive and specific than total AFP level in early detection of HCC. The clinical usefulness of other HCC biomarkers such as des-gamma-carboxy prothrombin and glypican-3 are under investigation. HCC is an aggressive tumor with early vascular invasion and metastasis. Studies over the past two decades have elucidated the clinical predictors of outcome, leading to several staging systems for HCC based on clinical parameters. However, the predictive accuracy of clinical staging systems is limited. Recent studies suggested that biological factors may provide additional prognostic information. In particular, gene expression profiling appears to be a promising approach. Study of tumor angiogenesis in HCC reveals that the expression of angiogenic factors such as vascular endothelial growth factor and angiopoietins may also predict prognosis. The elucidation of tumor biology of HCC is of particular importance in the current era of rapid development of anti-cancer molecular targeting agents, which provide hope for an effective systemic therapy for HCC.

In situ evidence for microdomains in the polymer matrix of bacterial microcolonies

Confocal laser scanning microscopy and fluorescent lectin-binding analyses (FLBA) were used to study the form, arrangement, and composition of exopolymeric substances (EPS) surrounding naturally occurring microcolonies in biofilms. FLBA, using multiple lectin staining and multichannel imaging, indicated that the EPS of many microcolonies exhibit distinct multiple binding regions. A common pattern in the microcolonies is a three zone arrangement with cell-associated, intercellular, and an outer layer of EPS covering the exterior of the colony. Differential binding of lectins suggests that there are differences in the glycoconjugate composition or their arrangement in the EPS of microcolonies. The combination of FLBA with fluorescent in situ hybridization (FISH) indicates that the colonies consist of the major groups, alpha- and beta-Proteobacteria. It is suggested that the EPS arrangement observed provides a physical structuring mechanism that can segregate extracellular activities at the microscale.

Differential lectin binding patterns in the oviductal ampulla of the horse during oestrus.

We investigated the oligosaccharide sequence of glycoconjugates, mainly sialoglycoconjugates, in the horse oviductal ampulla during oestrus by means of lectin and pre-lectin methods such as the KOH-neuraminidase procedure to remove sialic acid residues and incubation with N-glycosidase F to cleave N-linked glycans. Ciliated cells displayed N-linked oligosaccharides throughout the cytoplasm. The cilia glycocalyx expressed both N- and O-linked (mucin-type) oligosaccharides, both showing a high variety of terminal sequences. In the most non-ciliated cells, the whole cytoplasm contained N-linked oligosaccharides with terminal alphaGal as well as mucin-type glycans with terminal Forssman pentasaccharides. In a few scattered non-ciliated cells, the whole cytoplasm displayed sialylated N-linked oligosaccharides with terminal Neu5Ac-GalNAc and O-linked glycans terminating with neutral and/or alphaGalNAc, Neu5Ac alpha2,6Gal/GalNAc, Neu5AcGal beta1,3GalNAc. Supra-nuclear granules, probably Golgi zones, of non-ciliated cells showed mainly O-linked glycans rich in sialic acid residues. The luminal surface of non-ciliated cells showed N-linked oligosaccharides, containing terminal/internal alphaMan/alphaGlc, betaGlcNAc and terminal alphaGal, as well as mucin-type oligosaccharides terminating with a large variety of either neutral saccharides or sialylated sequences. Apical protrusions containing O-linked oligosaccharides with terminal Forssman pentasaccharide, Neu5Ac-Gal beta1,4GlcNAc, Neu5Ac-GalNAc were seen in non-ciliated cells scattered along the epithelium. These findings show the presence of sialoglycoconjugates in the oviductal ampulla epithelium of the mare and the existence of different lectin binding profiles between ciliated and non-ciliated (secretory) cells, as well as the presence of non-ciliated cell sub-types which might determine functional differences along the ampullary epithelium of mare oviduct.

The two envelope membrane glycoproteins of Tomato spotted wilt virus show differences in lectin-binding properties and sensitivities to glycosidases

Tomato spotted wilt virus (TSWV, Genus: Tospovirus, Family: Bunyaviridae) is a major constraint to the production of several different crops of agronomic and horticultural importance worldwide. The amino acid sequence of the two envelope membrane glycoproteins, designated as G N (N-terminal) and G C (C-terminal), of TSWV contain several tripeptide sequences, Asn-Xaa-Ser/Thr, suggesting that the proteins are N-glycosylated. In this study, the lectin-binding properties of the viral glycoproteins and their sensitivities to glycosidases were examined to obtain information on the nature of potential oligosaccharide moieties present on G N and G C. The viral proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed by affinoblotting using a battery of biotinylated lectins with specificity to different oligosaccharide structures. G C showed strong binding with five mannose-binding lectins, four N-acetyllactosamine-binding lectins and one fucose-binding lectin. G N was resolved into two molecular masses and only the slow migrating form showed binding, albeit to a lesser extent than G C, with three of the five mannose-binding lectins. The N-acetyllactosamine- and fucose-specific lectins did not bind to either molecular mass form of G N. None of the galactose-, N-acetylgalactosamine-, or sialic acid-binding lectins tested showed binding specificity to G C or G N. Treatment of the denatured virions with endoglycosidase H and peptide: N-glycosidase F (PNGase F) resulted in a significant decrease in the binding of G C to high mannose- and N-acetyllactosamine-specific lectins. However, no such differences in lectin binding were apparent with G N. These results indicate the presence of N-linked oligosaccharides of high mannose- and complex-type on G C and possibly high mannose-type on G N. Differences in the extent of binding of the two envelope glycoproteins to different lectins suggest that G C is likely to be more heavily N-glycosylated than G N. No evidence was observed for the presence of O-linked oligosaccharides on G N or G C.

Differences in glycosylation of intracellular proteins between benign and malignant thyroid neoplasms

Differences in glycosylation of nuclear and cytosolic proteins isolated from benign and malignant human thyroid neoplasms were analyzed by lectin blotting and enzyme linked lectino-solid-phase assay using Erythrina cristagalli and Ricinus communis agglutinins. The results reported in this study have not shown any significant differences in lectin binding by nuclear proteins of benign and malignant tumors, however, quantitative and qualitative differences were observed in the patterns of cytosolic glycoproteins. In the majority of carcinomas samples lectin binding to cytosolic proteins was definitely weaker in comparison with adenomas and non-neoplastic specimens, which suggested alterations in glycosylation of cytosolic proteins in malignant tumors.