Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans.

Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-ß1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.

In vitro lectin binding to the outer surface of Spirocerca lupi at different life-stages.

Spirocerca lupi is the esophageal nematode of dogs. Early, transient eosinophilia occurs in experimentally infected dogs, but is absent in advanced cases, suggesting that the nematode evades the dog's immune system. Lectins are proteins or glycoproteins of plant or animal origin, binding different saccharides, with varying specificities and avidities, used to characterize surface haptens in plant and animal parasitic helminths. This study investigated the in vitro binding of six lectins (Concanavalin A [ConA], wheat germ agglutinin [WGA], peanut agglutinin [PNA], soybean agglutinin [SBA], Dolichus biflorus agglutinin [DBA] and Ulex earopaeus agglutinin I [UEA]) to the surface of S. lupi nematodes at different life stages, the L2 and L3 larvae (dead and alive) and to dead adult worms, with negative controls, with and without addition of the six respective inhibitory sugar haptens. Con A moderately bound to surfaces of both live and frozen L3, to the stoma and excretory pores of adult worms, and to the outer surface nematode's eggs, within a female worm, but not to L2. PNA bound only to stoma and excretory pores surfaces in both frozen and live L3. WGA bound strongly to the outer surfaces of live and dead L2 and L3, which resulted in molting of live larvae. These results suggest that the nematode's surface content change during its development. Such changes may play roles in the nematode's interactions with the intermediate and definitive hosts' tissues, and in its ability to evade the immune response, its long survival within the host, and even induce neoplastic transformation.

PNA lectin for purifying mouse acinar cells from the inflamed pancreas.

Better methods for purifying human or mouse acinar cells without the need for genetic modification are needed. Such techniques would be advantageous for the specific study of certain mechanisms, such as acinar-to-beta-cell reprogramming and pancreatitis. Ulex Europaeus Agglutinin I (UEA-I) lectin has been used to label and isolate acinar cells from the pancreas. However, the purity of the UEA-I-positive cell fraction has not been fully evaluated. Here, we screened 20 widely used lectins for their binding specificity for major pancreatic cell types, and found that UEA-I and Peanut agglutinin (PNA) have a specific affinity for acinar cells in the mouse pancreas, with minimal affinity for other major pancreatic cell types including endocrine cells, duct cells and endothelial cells. Moreover, PNA-purified acinar cells were less contaminated with mesenchymal and inflammatory cells, compared to UEA-I purified acinar cells. Thus, UEA-I and PNA appear to be excellent lectins for pancreatic acinar cell purification. PNA may be a better choice in situations where mesenchymal cells or inflammatory cells are significantly increased in the pancreas, such as type 1 diabetes, pancreatitis and pancreatic cancer.

Quantum dot based fluorometric detection of cancer TF-antigen.

Cancer is a major global health challenge that would benefit from advances in screening methods for early detection that are rapid and low cost. TF-antigen is a tumor-associated antigen displayed on cell surface proteins of a high percentage of human carcinomas. Here we present a fluorometric bioassay for TF-antigen (galactose-ß-(1→3)-N-acetyl-d-galactosamine) that utilizes quantum dot (QD) technology coupled with magnetic beads for rapid detection of TF-antigen at high sensitivity (10(-7) M range). In the competitive bioassay, 4-aminophenyl ß-d-galactopyranoside (4-APG) conjugated to QDs competes with TF-antigen for binding sites on peanut agglutinin (PNA) that is immobilized on magnetic beads. The bioassay is specific and ultrasensitive in the environment of complex protein mixtures, demonstrating its potential applicability for the screening of clinical samples.

Glyconanosomes: disk-shaped nanomaterials for the water solubilization and delivery of hydrophobic molecules.

Herein, we describe the first report on a new class of disk-shaped and quite monodisperse water-soluble nanomaterials that we named glyconanosomes (GNS). GNSs were obtained by sliding out the cylindrical structures formed upon self-organization and photopolymerization of glycolipid 1 on single-walled carbon nanotube (SWCNT) sidewalls. GNSs present a sheltered hydrophobic inner cavity formed by the carbonated tails, surrounded by PEG and lactose moieties. The amphiphilic character of GNSs allows the water solubility of insoluble hydrophobic cargos such as a perylene-bisimide derivative, [60]fullerene, or the anti-carcinogenic drug camptothecin (CPT). GNS/C60 inclusion complexes are able to establish specific interactions between peanut agglutinin (PNA) lectin and the lactose moiety surrounding the complexes, while CPT solubilized by GNS shows higher cytotoxicity toward MCF7-type breast cancer cells than CPT alone. Thus, GNS represents an attractive extension of nanoparticle-based drug delivery systems.

Characterization of membrane-associated glycoproteins using lectin affinity chromatography and mass spectrometry.

Membrane-associated glycoproteins play critical roles in many biological processes and are often the therapeutic targets for drug discovery. Lectin affinity chromatography is one of the most widely used approaches for enrichment of glycoproteins at the protein level. Here, we describe a strategy for the characterization of membrane glycoproteins including membrane protein extraction, lectin affinity chromatography, protein digestion, and analysis by LC-MS/MS.

Engineering of near IR fluorescent albumin nanoparticles for in vivo detection of colon cancer.

BACKGROUND: The use of near-infrared (NIR) fluorescence imaging techniques has gained great interest for early detection of cancer because water and other intrinsic biomolecules display negligible absorption or autofluorescence in this region. Novel fluorescent nanoparticles with potential to improve neoplasm detection sensitivity may prove to be a valuable tool in early detection of colon tumors. METHODS: The present study describes the synthesis and use of NIR fluorescent albumin nanoparticles as a diagnostic tool for detection of colon cancer. These fluorescent nanoparticles were prepared by a precipitation process of human serum albumin (HSA) in aqueous solution in the presence of a carboxylic acid derivative of the NIR dye IR-783 (CANIR). Tumor-targeting ligands such as peanut agglutinin (PNA), anti-carcinoembryonic antigen antibodies (anti-CEA) and tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72) were covalently conjugated to the albumin nanoparticles via the surface carboxylate groups by using the carbodiimide activation method. RESULTS AND DISCUSSION: Leakage of the encapsulated dye into PBS containing 4% HSA or human bowel juice was not detected. This study also demonstrates that the encapsulation of the NIR fluorescent dye within the HSA nanoparticles reduces the photobleaching of the dye significantly. Specific colon tumor detection in a mouse model was demonstrated for PNA, anti-CEA and anti-TAG-72 conjugated NIR fluorescent HSA nanoparticles. These bioactive NIR fluorescent albumin nanoparticles also detected invisible tumors that were revealed as pathological only subsequent to histological analysis. CONCLUSIONS: These results may suggest a significant advantage of NIR fluorescence imaging using NIR fluorescent nanoparticles over regular colonoscopy. In future work we plan to broaden this study by encapsulating cancer drugs, such as paclitaxel and doxorubicin, within these biodegradable NIR fluorescent HSA nanoparticles, in order to use them for both detection as well as therapy of colon cancer and others.

Microfluidic platform for the evaluation of multi-glycan expressions on living cells using electrochemical impedance spectroscopy and optical microscope.

A microfluidic platform to evaluate the expression of multi-glycans on a cell surface was developed using electrochemical impedance spectroscopy (EIS) and optical microscope technique. In the microfluidic channel, four indium tin oxide (ITO) electrodes were modified with three lectins and one passivation agent, respectively, to selectively recognize the corresponding carbohydrate epitopes on the cell surface. The binding of the cells on the electrode array was monitored by the electrochemical impedance to evaluate the expression of cell surface glycans. The excellent optical transparency of ITO electrode permitted the microscopic observation of the cell binding simultaneously to substantiate the impedance measurement. Compared with the individual technology, the double-check mode increased the sensitivity and accuracy of the assay. The experimental results using these two techniques indicated that the cell binding ability decreased in the order WGA > Con A > PNA, which was consistent with the expression difference of carbohydrate epitopes on K562 cell surface. The proposed strategy was further used for facile evaluating the variations of glycan expression on living cells in response to drugs. The consumption of cell sample for each sensing interface in the whole experiments is merely 5 × 10(3) cells. This platform offers great promise for cancer-associated glycol-biomarkers screening and further helps cancer diagnosis and treatment.

Relevance of exocytotic glutamate release from retinal glia.

Glial cells release molecules that influence brain development, function, and disease. Calcium-dependent exocytosis has been proposed as potential release mechanism in astroglia, but the physiological relevance of "gliotransmission" in vivo remains controversial. We focused on the impact of glial exocytosis on sensory transduction in the retina. To this end, we generated transgenic mice to block exocytosis by Cre recombinase-dependent expression of the clostridial botulinum neurotoxin serotype B light chain, which cleaves vesicle-associated membrane protein 1-3. Ubiquitous and neuronal toxin expression caused perinatal lethality and a reduction of synaptic transmission thus validating transgene function. Toxin expression in Müller cells inhibited vesicular glutamate release and impaired glial volume regulation but left retinal histology and visual processing unaffected. Our model to study gliotransmission in vivo reveals specific functions of exocytotic glutamate release in retinal glia.

Differential glycosylation of MUC1 and CEACAM5 between normal mucosa and tumour tissue of colon cancer patients.

Altered glycosylation in epithelial cancers may play an important role in tumour progression, as it may affect tumour cell migration and antigen presentation by antigen presenting cells. We specifically characterise the glycosylation patterns of two tumour antigens that are highly expressed in cancer tissue and often detected in their secreted form in serum: the epithelial mucin MUC1 and carcinoembryonic antigen (CEA, also called CEACAM5). We analysed 48 colorectal cancer patients, comparing normal colon and tumour epithelium within each patient. Lectin binding was studied by a standardised CEA/MUC1 capture ELISA, using several plant lectins, and the human C-type lectins MGL and DC-SIGN, and Galectin-3. Peanut agglutinin (PNA) bound to MUC1 from tumour tissue in particular, suggests increased expression of the Thomsen-Friedenreich antigen (TF-antigen) (Core 1, Galß1-3GalNAc-Ser/Thr). Only small amounts of Tn-antigen (GalNAcα-Ser/Thr) expression was observed, but the human C-type lectin MGL showed increased binding to tumour-associated MUC1. Furthermore, sialylation was greatly enhanced. In sharp contrast, tumour-associated CEA (CEACAM5) contained high levels of the blood-group related carbohydrates, Lewis X and Lewis Y. This correlated strongly with the interaction of the human C-type lectin DC-SIGN to tumour-associated CEA, suggesting that CEA can be recognized and taken up by antigen presenting cells. In addition, increased mannose expression was observed and branched N-glycans were prominent, and this correlated well with human Galectin-3 binding. These data demonstrate that individual tumour antigens contain distinct glycan structures associated with cancer and, since glycans affect cellular interactions with its microenvironment, this may have consequences for progression of the disease.

Chemiluminescent detection of carbohydrates in the tumoral breast diseases.

Nowadays, there is an increase of investigations into the fibroadenoma, mainly because some studies have shown that the occurrence of fibroadenoma is linked to an increased risk of developing breast carcinoma. Currently, the chemiluminescence biomarkers are applied for validation methods and screening. Here, a lectin chemiluminescence is proposed as new histochemistry method to identify carbohydrates in mammary tumoral tissues. The lectins concanavalin A (Con A) and peanut agglutinin (PNA) conjugated to acridinium ester were used to characterize the glycocode of breast tissues: normal, fibroadenoma, and invasive duct carcinoma (IDC). The lectin chemiluminescence expressed in relative light units (RLU) was higher in fibroadenoma and IDC than in normal tissue for both lectins tested. The relationship RLU emission versus tissue area described a linear and hyperbolic curve for IDC and fibroadenoma, respectively, using Con A whereas hyperbolic curves for both transformed tissues using PNA. RLU was abolished by inhibiting the interaction between tissues and lectins using their specific carbohydrates: methyl-α-D: -mannoside (Con A) and galactose (PNA). The intrinsic fluorescence emission did not change with combination of the lectins (Con A/PNA) to the acridinium ester for hydrophobic residues. These results represent the lectin chemiluminescence as an alternative of histochemistry method for tumoral diagnosis in the breast.

In vitro growth kinetics, differentiation and morphological characterisation of Tunisian Leishmania infantum parasites.

In this study, a negative peanut agglutinin (PNA) selection was used as a marker for promastigote differentiation to compare the in vitro growth and differentiation kinetics of two visceral and two cutaneous Leishmania (Leishmania) infantum parasites. All parasites had different growth and differentiation kinetics. Cultures initiated with PNA(+) parasites purified during the early stationary phase (Day 4), when PNA(-) (non-agglutinating) parasites peaked, yielded a high PNA(-) percent. Further morphological analysis at this time point showed that 60-86% of PNA(+) forms were procyclics, whilst PNA(-) forms were composed of 53-71% leptomonads. Nectomonads were present both in PNA(-) and PNA(+) promastigote fractions at nearly equivalent proportions, suggesting that they constitute a transition state in the Leishmania development process, with a fraction of them sharing common constituents of the surface coat with procyclics and the other with leptomonads. Obtaining a high density of promastigotes undergoing developmental differentiation may be useful for further molecular and biochemical identification of developmental stage-specific markers.

Essence of affinity and specificity of peanut agglutinin-immobilized fluorescent nanospheres with surface poly(N-vinylacetamide) chains for colorectal cancer.

We have designed a novel colonoscopic imaging agent that is composed of submicron-sized fluorescent polystyrene nanospheres with two functional groups - peanut agglutinin (PNA) and poly(N-vinylaceamide) (PNVA) - on their surfaces. PNA is a targeting moiety that binds to ß-d-galactosyl-(1-3)-N-acetyl-d-galactosamine (Gal-ß(1-3)GalNAc), which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells; it is anchored on the nanosphere surface via a poly(methacrylic) acid (PMAA) linker. PNVA is immobilized to enhance the specificity of PNA by reducing nonspecific interactions between the imaging agent and normal tissues. The essential nature of both functional groups was evaluated through in vivo experiments using PNA-free and PNVA-free nanospheres. The imaging agent recognized specifically tumors on the cecal mucosa of immune-deficient mice in which human colorectal cancer cells had been implanted; however, the recognition capability disappeared when PNA was replaced with wheat germ agglutinin, which has no affinity for Gal-ß(1-3)GalNAc. PNA-free nanospheres with exclusively surface PNVA chains rarely adhered to the cecal mucosa of normal mice that did not undergo the cancer cell implantation. In contrast, there were strong nonspecific interactions between normal tissues and PNA-free nanospheres with exclusively surface PMAA chains. In vivo data proved that PNA and PNVA were essential for biorecognition for tumor tissues and a reduction of nonspecific interactions with normal tissues, respectively.

PpGalNacT2 participating in vanadium-induced HL-60 cell differentiation.

The current study demonstrates vanadium plays the role of antitumor, and its antitumor effect is dosage-dependent. N-acetyl-galactosamine-transferase 2 (polypeptide: N-acetyl-α-galactosaminyl-transferases 2, ppGalNAc-T2) is a member of ppGalNAcTs (polypeptide: N-acetyl-α-galactosaminyl-transferases) family, which proves to play a vital role in the tumor emergence and development process. In this study, we focused on ppGalNAc-T2 and vanadium and aimed to determine whether ppGalNAc-T2 is correlated with vanadium's antitumor effect. We discovered that ppGalNAc-T2 changed with the variation of HL-60 cell growth induced by vanadium at mRNA level. Peanut agglutinin (PNA) is an exogenous lectin. PpGalNacT2 can be indirectly recognized by PNA. By means of flow cytometry and immunofluorescent staining, we found the deviation of PNA binding increased significantly at high concentration vanadium. Then we docked one of the possible compound substances of vanadium onto the body, VO(3) (molecular formula O(13)V(4), partial vanadate tetramer) and ppGalNAcT2, and simulated them via molecular dynamics, which showed that VO(3) may inhibit the activity of the enzyme by stemming conformational changes of a key loop of ppGalNAcT2. To sum up, our results suggested that ppGalNacT2 participated in vanadium induced HL-60 cell differentiation, which might be able to provide a new mechanism of vanadium's antitumor effect.

MicroRNA-488 suppresses cell migration through modulation of the focal adhesion activity during chondrogenic differentiation of chick limb mesenchymal cells.

miRNAs (microRNAs) have proven to play essential roles in diverse biological processes including early development, cell proliferation and cell death, and cell differentiation. However, there is only limited amount of information about their potential role in chondrogenesis. In the present study, we investigated the role of miRNA-488 in the cellular condensation, which is essential initiation for chondrogenic differentiation. We found that miRNA-488 expression is up-regulated at the precondensation stage and then down-regulated at the postcondensation stage. Blockade of miRNA-488 via the use of PNA (peanut agglutinin)-based ASOs (antisense oligonucleotides) decreased the protein level of integrins ß1 and phosphorylated FAK (focal adhesion kinase) and resulted in the suppression of cell motility and migration. Moreover, in parallel with theses observation, treatment of anti-miRNA-488 oligonucleotides up-regulated the level of MMP (matrix metalloprotease)-2 activity, and co-treatment with GM6001, an MMP inhibitor, induced recovery of cellular condensation inhibited by blockade of miRNA-488. Collectively, our results suggest that miRNA-488 is one of regulator in cell to ECM (extracellular matrix) interaction through modulation of focal adhesion activity by MMP-2 during chondrogenesis of limb mesenchymal cells.

Alterations in PNA binding of keratinocytes in oral keratosis.

Changes in the expression of peanut lectin (PNA) were examined in keratinocytes of oral keratosis showing a mixture of hyperortho- and hyperparakeratinized epithelium. In the hyperorthokeratinized epithelium, which was reacted with anti-filaggrin antibody in both granular and cornified cells, PNA bound to the surface of keratinocytes from the spinous layer to the granular layer. Neither anti-filaggrin nor PNA reactions were detected in keratinocytes of the hyperparakeratinized epithelium. After neuraminidase pretreatment, however, PNA staining appeared in all cells, except cornified cells, of both hyperortho- and hyperparakeratinized epithelia. These findings suggest that PNA-binding epitopes in keratinocytes were modified by sialic acid during the hyperparakeratotic process of oral keratosis.

Histochemical evaluation of human prostatic tissues with Cratylia mollis seed lectin.

Lectins, proteins which selectively recognize carbohydrates, have been used in histochemistry for the evaluation of changes in glycosylation in processes of cellular differentiation and/or dedifferentiation. Cratylia mollis seed lectins (Cramoll 1,4 and Cramoll 3), conjugated to horseradish peroxidase, were used as histochemical probes in human prostate tissues: normal (NP), hyperplasia (BPH), and prostate carcinoma (PCa). The staining pattern of Con-A and Cramoll 1,4 in BPH was more intense than in NP. These lectins also showed staining differences between BPH and PCa; the latter showing decreased staining intensity with an increased degree of malignancy. PNA and Cramoll 3 stained epithelial cells similarly in all diagnoses although they did present intense staining of PCa glands lumen. Corpora amylacea were not differentially recognized by any of the lectins. Cramoll 1,4 and Cramoll 3 seed lectins present themselves as candidates for histochemical probes for prostate pathologies when compared to commercial lectins such as Con-A and PNA.

[Characteristic of carbohydrate components of chickens and human's fibronectins].

The paper presents new information about the carbohydrate structures of 39-days chicken's fibronectin. It is found out that chicken fibronectin contains mainly biantennary N-glycans with a core fucose and fucosylated O-glycans. It is shown that N-glycans of chicken fibronectin are poorly sialated, since this protein exhibits affinity for the PNA and weak binding to sialospecific SNA. A comparative analysis of lectin-binding activity of chicken and human fibronectins has shown that both glycoproteins differ in glycan composition.

Temperature-dependent ultrastructural changes in the cone interphotoreceptor matrix.

PURPOSE: To determine the temperature-dependent ultrastructural changes of the cone matrix sheath, a possible critical factor in the adhesive strength binding photoreceptors and pigment epithelial cells. METHODS: Aqueous-insoluble interphotoreceptor matrix (IPM) was extracted by soaking fresh bovine retinas in acidic HEPES-buffered solution followed by treatment with 1% Triton X-100 to remove cone membrane fragments. The IPM preparations were then incubated in the same buffer solution at either 34 degrees C, 37 degrees C, or 40 degrees C for 60 min. After labeling with peanut agglutinin, the samples were prepared for electron microscopy. RESULTS: The cone matrix sheath, specifically stained with peanut agglutinin, was markedly condensed at 34 degrees C. It became more dispersed and composed of granular elements at 37 degrees C. At 40 degrees C, the matrix appeared as a network of fine filamentous structures. CONCLUSIONS: These findings suggest changing temperatures can alter the molecular organization of the IPM and thus influence its retinal adhesion.

Improved methodology for the detection and quantification of the acrosome reaction in mouse spermatozoa.

This study evaluates the use of two fluorescein-labelled (FITC) plant lectins, Pisum sativum (edible pea) agglutinin (PSA) and Arachis hypogaea (peanut) agglutinin (PNA), in order to determine the most accurate and reliable method to experimentally detect and assess the acrosome reaction in mouse spermatozoa. PNA-FITC labelling was restricted to the acrosome and was not influenced by the fixation procedure; either absolute methanol or paraformaldehyde. In contrast, PSA-FITC not only labelled the acrosome, but also the whole head and the flagellum. This aspect was especially marked after methanol fixation. The cytoplasmic droplet, when present, was also stained by PSA-FITC. Incubation with the calcium ionophore ionomycin induced a concentration and time-dependent increase in the number of acrosome reactions. Compared to spotted preparations, smear samples exhibited a high proportion of spermatozoa with damaged acrosome. In conclusion, PNA-FITC labelling was more accurate than PSA-FITC labelling to detect the acrosome of mouse spermatozoa. The fixation method (methanol vs. paraformaldehyde) had no influence on the staining pattern of PNA-FITC labelling, but spotted preparations are recommended to avoid mechanical damage to the acrosome. Ionophore challenge confirmed the existence of a calcium-dependent acrosome reaction in mouse spermatozoa and validated the use of PNA-FITC to quantify this physiological process. The present study illustrates important methodological considerations which need to be taken into account in order to design a reliable and reproducible protocol for the study of the acrosome reaction.