Surface Carbohydrate Research Articles

In Situ Electrochemical Imaging of Membrane Glycan Expression on Micropatterned Adherent Single Cells

A scanning electrochemical microscopic (SECM) method for in situ imaging of four types of membrane glycan motifs on single adherent cells was proposed using BGC-823 human gastric carcinoma (BGC) cells as the model. These adherent cells were first micropatterned in the microwell of poly(dimethylsiloxane) membrane for precisely controlling the localized surface interaction, and the membrane glycans were then specifically recognized with corresponding lectins labeled with horseradish peroxidase (HRP). On the basis of the enzymatic oxidization of ferrocenylmethanol (FMA) by H(2)O(2) to yield FMA(+), the glycan expression level was detected by the reduction current of FMA(+) at the SECM tip. The cell-surface glycans could, thus, be in situ imaged by SECM at a single-cell level without peeling the cells from culture dish. Under the optimized conditions, four types of membrane glycan motifs showed statistically distinguishable expression levels. The SECM results for different glycan motifs on adherent single cells were consistent with those estimated by flow cytometric assay. This work provides a reliable approach for in situ evaluation of the characteristic glycopattern of single living cells and can be applied in cell biologic study based on cell surface carbohydrate expression.

Structure and Specificity of a Binary Tandem Domain F-Lectin from Striped Bass (Morone saxatilis)

The plasma of the striped bass Morone saxatilis contains a fucose-specific lectin (MsaFBP32) that consists of two F-type carbohydrate recognition domains (CRDs) in tandem. The crystal structure of the complex of MsaFBP32 with l-fucose reported here shows a cylindrical 81-Å-long and 60-Å-wide trimer divided into two globular halves: one containing N-terminal CRDs (N-CRDs) and the other containing C-terminal CRDs (C-CRDs). The resulting binding surfaces at the opposite ends of the cylindrical trimer have the potential to cross-link cell surface or humoral carbohydrate ligands. The N-CRDs and C-CRDs of MsaFBP32 exhibit significant structural differences, suggesting that they recognize different glycans. Analysis of the carbohydrate binding sites provides the structural basis for the observed specificity of MsaFBP32 for simple carbohydrates and suggests that the N-CRD recognizes more complex fucosylated oligosaccharides and with a relatively higher avidity than the C-CRD. Modeling of MsaFBP32 complexed with fucosylated glycans that are widely distributed in prokaryotes and eukaryotes rationalizes the observation that binary tandem CRD F-type lectins function as opsonins by cross-linking “non-self” carbohydrate ligands and “self” carbohydrate ligands, such as sugar structures displayed by microbial pathogens and glycans on the surface of phagocytic cells from the host.

Regulation of the 1,4-Galactosyltransferase I promoter by E2F1

Cell surface carbohydrate chains are widely known to contribute to cell migration, recognition and proliferation. beta1,4-Galactosyltransferase I (beta1,4GalT I) transfers galactose to the terminal N-acetylglucosamine of complex-type N-glycan, and contributes to cell proliferation, differentiation and migration. Here, we identified beta1,4GalT I as a novel target gene of cell cycle regulator E2F1. E2F1 proteins interact with the promoter of the beta1,4GalT I gene in vivo, and E2F1 over-expression stimulates the activity of beta1,4GalT I promoter and the mRNA and protein expression of beta1,4GalT I, and augments the level of beta1, 4-galactosyltion. Site-specific mutagenesis revealed that this region which contains two E2F1 binding site (nt -215 to -207 and +1 to +6) is necessary for beta1,4GalT I activation by E2F1. Furthermore, down-regulation of beta1,4GalT I expression attenuates E2F1-induced DNA synthesis and cell cycle progression as well as the expression of cell-cycle regulator Cyclin D1. Thus, beta1,4GalT I is an important E2F1 target gene that is required for cell cycle progression in mammalian cells, which elicits a new mechanism of cell growth and a new mechanism of beta1,4GalT I transcription.

In Situ Scanometric Assay of Cell Surface Carbohydrate by Glyconanoparticle-Aggregation-Regulated Silver Enhancement

A convenient and label-free scanometric approach for in situ cell surface carbohydrate assay was designed by integrating the bioconjugation and aggregation of glyconanoparticles, silver signal amplification, and spot test. The novel glyconanoparticles were prepared by a one-pot procedure. In the presence of lectin, using concanavalin A and mannose as a couple of model, the glyconanoparticles exhibited fast aggregation. The aggregation process could be inhibited by the specific recognition of lectin by the carbohydrate on the cell surface. Combining the gold nanoparticle catalyzed silver enhancement and scanometric detection, the number of cell surface carbohydrate groups could be conveniently read out. The average number of mannose units on a single living intact BGC cell was detected to be (4.5 +/- 0.4) x 10(7). This largely noninstrumental method took the advantages of a nanoparticle-based recognition and an aggregation-regulated signal amplification and avoided cell pretreatment and labeling processes. It could determine cell surface densities of different carbohydrates in parallel and thus would contribute considerably to meeting the challenges in decipherment of the glycomic codes.

Characterization of a new serotype g isolate of Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is usually isolated from the oral cavity where it is associated with active periodontitis. The species can be divided into six serotypes (a-f) according to their surface carbohydrate antigens. However, some clinical isolates cannot be grouped within these six serotypes. Gram-negative, facultative anaerobic, catalase-positive coccobacilli were isolated from a patient with periodontitis and identified by employing genetic, biochemical and serological analyses. Phenotypic data identified the isolate as A. actinomycetemcomitans. Serotype-specific polysaccharide antigen from the isolate was untypeable by immunodiffusion testing in comparison with reference A. actinomycetemcomitans serotype a to f strains. Biofilm formation by the isolate was strong but cytotoxic activity was low. Gas chromatography/mass spectroscopy analysis of partially methylated alditol acetates from surface polysaccharide showed the presence of 2,4-di-O-methyl-rhamnose and 2,3,6-tri-O-methyl-glucose, with a 1 : 1 m ratio. The (1)H- and (13)C-nuclear magnetic resonance spectra of the antigen showed that both constituent glycoses had alpha-anomeric configuration. It is proposed that the untyped strain is a new A. actinomycetemcomitans serotype, designated serotype g.

Xylan sorption kinetics at industrial conditions Part 1. Experimental results

Abstract The sorption kinetics for birch black liquor xylan onto unbleached softwood kraft fibers at industrial relevant process conditions was investigated. The impact of process conditions on total sorption and rate was studied. It was found that an unbleached fiber can increase its weight up to 40% by sorption of xylan if process conditions are favorable and sufficient xylan is present in the liquor. A decrease in pH, an increase in temperature and an increase in ionic strength enhances both the initial sorption rate and the maximum sorption, as expected from the literature. Moreover, an increased xylan charge from 5 to 10 g/l increased sorption by about 20-30% on average. At the highest temperature tested some deviations from the expected results were obtained, which indicates that more than one mechanism affect sorption. Analysis of the surface carbohydrate composition suggests that xylan is preferably sorbed on the fiber surfaces. This is especially obvious at higher sorption temperatures. SEM pictographs of the cellulose fibers indicate that xylan sorbs as single xylan chains at 87 and 127°C. In contrast, at 167°C globular structures are observed on the fibers, which probably is due to precipitation of globular xylan aggregates from the liquor.

Abstract 4027: The lectin concanavalin-A induces cyclooxygenase-2 expression through an IKKgamma/NFkB-dependent pathway in U87 glioblastoma cells

Abstract The lectin from Canavalia ensiformis (Concanavalin-A, ConA), one of the most abundant lectins known, enables one to mimic biological lectin/carbohydrate interactions that regulate extracellular matrix protein recognition. As such, ConA is known to induce membrane type-1 matrix metalloproteinase (MT1-MMP) and is, therefore, routinely used to assess MT1-MMP-mediated functions. Given that gliomas with increasing histological grade demonstrated MT1-MMP expression that correlated with high expression of cyclooxygenase (COX)-2, we specifically assessed the early proinflammatory cellular signaling processes triggered by ConA, a lectin known to induce MT1-MMP expression, in the regulation of COX-2. We found that treatment with ConA or direct overexpression of a recombinant MT1-MMP resulted in the induction of COX-2 expression. This increase in COX-2 was correlated with a concomitant decrease in phosphorylated AKT suggestive of cell death induction, and was independent of MT1-MMP's catalytic function. ConA and MT1-MMP-mediated intracellular signaling and induction of COX-2 was also validated in both wild-type and Nuclear Factor-kappaB (NF-κB) p65−/− mutant mouse embryonic fibroblasts (MEF), but was abrogated in NF-κB1 (p50)−/− and in I kappaB kinase (IKK) γ−/− mutant MEF cells. Collectively, our results highlight an IKK/NF-κB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of COX-2 by ConA. That signaling pathway could account for the inflammatory balance responsible for the therapy resistance phenotype of glioblastoma cells, and prompts for the design of new therapeutic strategies that target cell surface carbohydrate structures and MT1-MMP-mediated signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4027.

Design and Implementation of Electrochemical Cytosensor for Evaluation of Cell Surface Carbohydrate and Glycoprotein

A new strategy for assessing cell surface carbohydrates and P-glycoprotein (P-gp) expression status and quantifying the cell numbers with an electrochemical immunoassay was designed. In order to construct the base of the cytosensor, a novel 3-D architecture was initially fabricated by combining nitrogen-doped carbon nanotubes, thionine, and gold nanoparticles via a simple layer-by-layer method. The formed architecture provided an effective matrix for concanavalin A (Con A) binding and made the immobilized Con A hold high stability and bioactivity. On the basis of the specific recognition of cell surface mannosyl groups to Con A, the Con A/3-D architecture interface showed a predominant capability for cell capture. With another coupled signal amplification based on a enzymatic catalytic reaction of HRP toward the oxidation of thionine by the H(2)O(2), which was induced by two-step immunoreactions, the proposed cytosensor showed an excellent analytical performance for the detection of HeLa cells ranging from 8.0 x 10(2) to 2.0 x 10(7) cells mL(-1) with a limit of detection of 500 cells mL(-1). Moreover, with the use of preblocking procedures, the mannosyl groups and P-gp on single HeLa cell could be further detected to be (4 +/- 2) x 10(10) molecules of mannose moieties and 8.47 x 10(6) molecules of P-gp. This strategy offers great promise for sensitive detection of cancer cells and cell surface receptors and thus may help improve cancer diagnosis and treatment.

Carbohydrate vaccines: developing sweet solutions to sticky situations?

Recent technological advances in glycobiology and glycochemistry are paving the way for a new era in carbohydrate vaccine design. This is enabling greater efficiency in the identification, synthesis and evaluation of unique glycan epitopes found on a plethora of pathogens and malignant cells. Here, we review the progress being made in addressing challenges posed by targeting the surface carbohydrates of bacteria, protozoa, helminths, viruses, fungi and cancer cells for vaccine purposes.

Carbohydrate Oxidation Acidifies Endosomes, Regulating Antigen Processing and TLR9 Signaling

Phagocytes kill encapsulated microbes through oxidative cleavage of surface carbohydrates, releasing glycan fragments and microbial contents that serve as ligands for immune receptors, which tailor the immune response against the offending pathogen. The glycan fragments serve as MHC class II (MHC II) ligands and innate receptor agonists, whereas microbial proteins serve as substrates for proteolytic cleavage and MHC II presentation, and released nucleic acids activate innate pattern-recognition receptors (e.g., TLR9). In the current study, confocal microscopy of live macrophages and dendritic cells revealed that endocytosis of carbohydrates lead to vesicular acidification independent of proton pump activity. Acidification was dependent on NO-mediated oxidation in the presence of the ingested carbohydrate and was sufficient to negatively regulate T cell-dependent polysaccharide Ag cleavage, promote acid-dependent protein Ag processing, and facilitate CpG-mediated TLR9 signaling. Our findings lead to a model in which oxidation of carbohydrates from encapsulated microbes facilitates adaptive immune responses against microbial protein and carbohydrate Ags through promoting Ag processing for MHC II-mediated presentation as well as innate responses against released microbial DNA via TLR9 signaling.

Lectin Labeling of Surface Carbohydrates on Gametes of Three Bivalves:Crassostrea virginica, Mytilus galloprovincialis, andDreissena bugensis

The distribution of carbohydrates on the surface of freshwater and marine bivalve gametes was examined based on labeling by 5 lectins. The 3 species of interest were Dreissena bugensis, Mytilus galloprovincialis, and Crassostrea virginica. The lectins used to identify the surface carbohydrates are Arachis hypogaea (PNA), concanavalin A, Griffonia simplicifolia, Lens culinaris (LcH), and wheat germ agglutinin. Live eggs and sperm from each species were examined for lectin affinity. Lectin labeling differed greatly between the 3 species, with very few, if any, conserved trends. Furthermore, there was no obvious difference in labeling between marine and dreissenid species. Of particular interest were LcH labeling of M. galloprovincialis sperm that labeled the tip of the outer sperm acrosome and PNA labeling of C. virginica gametes that was specific only to the sperm inner acrosomal membrane. Fertilization series looking at the distribution of PNA labeling of the sperm acrosome showed localization to the basal ring associated with sperm-egg binding.

BAAV Transcytosis Requires an Interaction with β-1-4 Linked- Glucosamine and gp96

Cell surface carbohydrates play an important role in virus entry and intracellular trafficking. Bovine Adeno-Associated Virus (BAAV) uses plasma membrane gangliosides for transduction and infection. In addition, independent of the infectious pathway, BAAV also has the ability to pass through barrier epithelia and endothelia using a transcytosis pathway dependent upon the presence of cell surface carbohydrates. Thus, in order to better define the carbohydrate interactions that are necessary for BAAV infection or transcytosis, a glycan microarray composed of both natural and synthetic carbohydrates was probed with HA-tagged BAAV particles. This identified chitotriose, a trimer of β-1-4-linked N-acetyl glucosamine, as having an interaction with BAAV. Competition experiments showed that the BAAV interaction with this carbohydrate is not necessary for infection but is instead important in the transcytosis pathway. The β-1-4-linked N-acetyl glucosamine modification has been reported on gp96, a glycoprotein involved in the transcytosis of bacteria and toxins. Significantly, immunoprecipitation and competition experiments with an anti-gp96 antibody and a soluble form of gp96, respectively, showed this glycoprotein can also interact with BAAV to serve as a receptor for its transcytosis.

Determination of Glycoconjugate Residues of Erythrocytes at Different Age Groups of Rats.

Aging is a time-dependent process that contains cell injury caused by molecular damages and eventually functional impairment of tissues and organs. The possible roles of cell surface carbohydrates, which are very important molecules in cell to cell and/or cell to extracellular matrix recognition, on aging process are not yet clear. In this study, glycoconjugate alterations of membrane glycoproteins of erythrocytes in aging organism were evaluated with lectin histochemistry and lectin blotting studies in 1, 4 and 7 months old rats. Lectin histochemistry results indicated that α(2→3) and α(2→6)-linked sialic acids are intensively found in erythrocyte membranes, but this intensity of sialic acids decreases with age. Similar evidences were obtained from lectin blotting studies which performed with same lectins. These results suggest that sialic acid reactivity alters with the age of organism. We thought that (a) sialic acid containing glycoconjugates altered not only with erythrocyte senescence, but also aging process of organism or (b) the number of sialic acid containing erythrocytes decreased by age.

(Pseudo)amide-linked oligosaccharide mimetics: molecular recognition and supramolecular properties.

Oligosaccharides are currently recognised as having functions that influence the entire spectrum of cell activities. However, a distinct disadvantage of naturally occurring oligosaccharides is their metabolic instability in biological systems. Therefore, much effort has been spent in the past two decades on the development of feasible routes to carbohydrate mimetics which can compete with their O-glycosidic counterparts in cell surface adhesion, inhibit carbohydrate processing enzymes, and interfere in the biosynthesis of specific cell surface carbohydrates. Such oligosaccharide mimetics are potential therapeutic agents against HIV and other infections, against cancer, diabetes and other metabolic diseases. An efficient strategy to access this type of compounds is the replacement of the glycosidic linkage by amide or pseudoamide functions such as thiourea, urea and guanidine. In this review we summarise the advances over the last decade in the synthesis of oligosaccharide mimetics that possess amide and pseudoamide linkages, as well as studies focussing on their supramolecular and recognition properties.

Microalgal Cell Surface Carbohydrates as Recognition Sites for Particle Sorting in Suspension-Feeding Bivalves

Cell surface carbohydrates play important roles in cell recognition mechanisms. Recently, we provided evidence that particle selection by suspension-feeding bivalves can be mediated by interactions between carbohydrates associated with the particle surface and lectins present in mucus covering bivalve feeding organs. In this study, we used lectins tagged with fluorescein isothiocyanate (FITC) to characterize carbohydrate moieties on the surface of microalgal species and evaluate the effect of oyster mucus on lectin binding. These analyses revealed that concanavalin A (Con A), one of six lectins tested, bound to Isochrysis sp., while Nitzschia closterium reacted with Pisum sativum agglutinin (PNA) and peanut agglutinin (PEA). The cell surface of Rhodomonas salina bound with PNA and Con A, and Tetraselmis maculata cell surface was characterized by binding with PNA, PEA, and Con A. Pre-incubation of microalgae with oyster pallial mucus significantly decreased the binding of FITC-labeled lectins, revealing that lectins present in mucus competitively blocked binding sites. This decrease was reversed by washing mucus-coated microalgae with specific carbohydrates. These results were used to design a feeding experiment to evaluate the effect of lectins on sorting of microalgae by oysters. Crassostrea virginica fed with an equal ratio of Con A-labeled Isochrysis sp. and unlabeled Isochrysis sp. produced pseudofeces that were significantly enriched in Con A-labeled Isochrysis sp. and depleted in unlabeled microalgae. Selection occurred even though two physical-chemical surface characteristics of the cells in each treatment did not differ significantly. This work confirms the involvement of carbohydrate-lectin interaction in the particle sorting mechanism in oysters, and provides insights into the carbohydrate specificity of lectins implicated in the selection of microalgal species.

Identification and Role of Carbohydrates on the Surface of Gametes in the Zebra Mussel, Dreissena polymorpha

The objective of this study is to identify surface carbohydrates on zebra mussel, Dreissena polymorpha, eggs and sperm and to analyze their potential role in fertilization. The lectins WGA, Con A, LcH, LTA, SBA, PNA, and GSII were tested for affinity to both eggs and sperm. WGA, Con A, and LcH uniformly labeled eggs. LTA, SBA, PNA, and GSII did not. WGA labeled the entire sperm surface including the unreacted acrosome. Labeling by Con A, LcH, LTA, SBA, PNA, and GSII was restricted to the inner acrosomal region of acrosome-reacted sperm. GSII labeling suggests the presence of N-acetyl-d-glucosamine (GlcNAc) only in the inner acrosomal membrane and not on eggs. GlcNAc blocked sperm-egg binding. GSII labeling was associated with a ring-like structure at the site of sperm entry intimately associated with sperm-egg binding. Nonfertilizing sperm were detached from the egg surface along with the GSII basal ring about 15 min postinsemination in a process blocked by trypsin inhibitors.

The lectin concanavalin‐A signals MT1‐MMP catalytic independent induction of COX‐2 through an IKKγ/NF‐κB‐dependent pathway

The lectin from Canavalia ensiformis (Concanavalin-A, ConA), one of the most abundant lectins known, enables one to mimic biological lectin/carbohydrate interactions that regulate extracellular matrix protein recognition. As such, ConA is known to induce membrane type-1 matrix metalloproteinase (MT1-MMP) which expression is increased in brain cancer. Given that MT1-MMP correlated to high expression of cyclooxygenase (COX)-2 in gliomas with increasing histological grade, we specifically assessed the early proinflammatory cellular signaling processes triggered by ConA in the regulation of COX-2. We found that treatment with ConA or direct overexpression of a recombinant MT1-MMP resulted in the induction of COX-2 expression. This increase in COX-2 was correlated with a concomitant decrease in phosphorylated AKT suggestive of cell death induction, and was independent of MT1-MMP’s catalytic function. ConA- and MT1-MMP-mediated intracellular signaling of COX-2 was also confirmed in wild-type and in Nuclear Factor-kappaB (NF-κB) p65−/− mutant mouse embryonic fibroblasts (MEF), but was abrogated in NF-κB1 (p50)−/− and in I kappaB kinase (IKK) γ−/− mutant MEF cells. Collectively, our results highlight an IKK/NF-κB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of COX-2. That signaling pathway could account for the inflammatory balance responsible for the therapy resistance phenotype of glioblastoma cells, and prompts for the design of new therapeutic strategies that target cell surface carbohydrate structures and MT1-MMP-mediated signaling. Concise summary Concanavalin-A (ConA) mimics biological lectin/carbohydrate interactions that regulate the proinflammatory phenotype of cancer cells through yet undefined signaling. Here we highlight an IKK/NF-κB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of cyclooxygenase-2, and that could be responsible for the therapy resistance phenotype of glioblastoma cells.

The Recognition Unit of FIBCD1 Organizes into a Noncovalently Linked Tetrameric Structure and Uses a Hydrophobic Funnel (S1) for Acetyl Group Recognition

We have recently identified FIBCD1 (Fibrinogen C domain containing 1) as a type II transmembrane endocytic receptor located primarily in the intestinal brush border. The ectodomain of FIBCD1 comprises a coiled coil, a polycationic region, and a C-terminal FReD (fibrinogen-related domain) that assembles into disulfide-linked homotetramers. The FIBCD1-FReD binds Ca(2+) dependently to acetylated structures like chitin, N-acetylated carbohydrates, and amino acids. FReDs are present in diverse innate immune pattern recognition proteins including the ficolins and horseshoe crab TL5A. Here, we use chemical cross-linking, combined with analytical ultracentrifugation and electron microscopy of the negatively stained recombinant FIBCD1-FReD to show that it assembles into noncovalent tetramers in the absence of the coiled coil. We use surface plasmon resonance, carbohydrate binding, and pulldown assays combined with site-directed mutagenesis to define the binding site involved in the interaction of FIBCD1 with acetylated structures. We show that mutations of central residues (A432V and H415G) in the hydrophobic funnel (S1) abolish the binding of FIBCD1 to acetylated bovine serum albumin and chitin. The double mutations (D393N/D395A) at the putative calcium-binding site reduce the ability of FIBCD1 to bind ligands. We conclude that the FReDs of FIBCD1 forms noncovalent tetramers and that the acetyl-binding site of FReDs of FIBCD1 is homologous to that of tachylectin 5A and M-ficolin but not to the FReD of L-ficolin. We suggest that the spatial organization of the FIBCD1-FReDs determine the molecular pattern recognition specificity and subsequent biological functions.

A simple fluorescent strategy for in situ evaluation of cell surface carbohydrate with a quantum dot–lectin nanoprobe

A simple and rapid fluorescent method was developed for the in situ evaluation of cell surface carbohydrate by homogeneous specific recognition of a quantum dot-lectin nanoprobe to mannosyl groups on the cell surface. The strategy was further used for dynamically monitoring the alteration of cell surface carbohydrate expression in response to drugs.

Cytosensing and dynamic monitoring of cell surface carbohydrate expression by electrochemiluminescence of quantum dots

A novel electrochemiluminescent (ECL) cytosensing strategy for sensitive dynamic monitoring of carbohydrate expression on living cells was designed by combining the specific recognition of lectin to carbohydrate groups with the functionalization of immobilized CdSe quantum dots, which acted as ECL emitting species.