Glycoforms of human prostate-specific membrane antigen (PSMA) in human cells and prostate tissue.

INTRODUCTION: N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membrane-associated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy. METHODS: Mass spectrometry was used to analyze the distribution of the site-specific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment. RESULTS: While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane. CONCLUSIONS: Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.

Antiviral activities of four marine sulfated glycans against adenovirus and human cytomegalovirus.

Broad-spectrum antivirals are more needed than ever to provide treatment options for novel emerging viruses and for viruses that lack therapeutic options or have developed resistance. A large number of viruses rely on charge-dependent non-specific interactions with heparan sulfate (HS), a highly sulfated glycosaminoglycan (GAG), for attachment to cell surfaces to initiate cell entry. As such, inhibitors targeting virion-HS interactions have potential to have broad-spectrum antiviral activity. Previous research has explored organic and inorganic small molecules, peptides, and GAG mimetics to disrupt virion-HS interactions. Here we report antiviral activities against both enveloped (the herpesvirus human cytomegalovirus) and non-enveloped (adenovirus) DNA viruses for four defined marine sulfated glycans: a sulfated galactan from the red alga Botryocladia occidentalis; a sulfated fucan from the sea urchin Lytechinus variegatus, and a sulfated fucan and a fucosylated chondroitin sulfate from the sea cucumber Isostichopus badionotus. As evidenced by gene expression, time of addition, and treatment/removal assays, all four novel glycans inhibited viral attachment and entry, most likely through interactions with virions. The sulfated fucans, which both lack anticoagulant activity, had similar antiviral profiles, suggesting that their activities are not only due to sulfation content or negative charge density but also due to other physicochemical factors such as the potential conformational shapes of these carbohydrates in solution and upon interaction with virion proteins. The structural and chemical properties of these marine sulfated glycans provide unique opportunities to explore relationships between glycan structure and their antiviral activities.

Serum N-glycan profiles differ for various breast cancer subtypes.

Breast cancer is the most prevalent cancer in women. Early detection of this disease improves survival and therefore population screenings, based on mammography, are performed. However, the sensitivity of this screening modality is not optimal and new screening methods, such as blood tests, are being explored. Most of the analyses that aim for early detection focus on proteins in the bloodstream. In this study, the biomarker potential of total serum N-glycosylation analysis was explored with regard to detection of breast cancer. In an age-matched case-control setup serum protein N-glycan profiles from 145 breast cancer patients were compared to those from 171 healthy individuals. N-glycans were enzymatically released, chemically derivatized to preserve linkage-specificity of sialic acids and characterized by high resolution mass spectrometry. Logistic regression analysis was used to evaluate associations of specific N-glycan structures as well as N-glycosylation traits with breast cancer. In a case-control comparison three associations were found, namely a lower level of a two triantennary glycans and a higher level of one tetraantennary glycan in cancer patients. Of note, various other N-glycomic signatures that had previously been reported were not replicated in the current cohort. It was further evaluated whether the lack of replication of breast cancer N-glycomic signatures could be partly explained by the heterogenous character of the disease since the studies performed so far were based on cohorts that included diverging subtypes in different numbers. It was found that serum N-glycan profiles differed for the various cancer subtypes that were analyzed in this study.

Physicochemical characterization of polysaccharide from the leaf of Dendrobium officinale and effect on LPS induced damage in GES-1 cell.

The polysaccharide was first successfully isolated from the leaf of Dendrobium officinale by hot water extraction and alcohol precipitation and further purified using DEAE-52 and Sephadex G-100 chromatography. The structure of LDOP-1 was characterized by HPLC, GPC, and FT-IR and NMR spectroscopy, and its protective effect on LPS-induced GES-1 cell injury was analyzed. Results showed that LDOP was a homogeneous polysaccharide with average molecular weight of 91.8 kDa and consisted of Man, Gla, Glc, Glc acid, and Ara at a molar ratio of 2.0:1.3:1.6:1.7:0.7. LDOP had two types of residues, including 1,6-linked α-d-Glup and 1,4-linked α-d-Manp. Activity studies indicated that LDOP-1 can significantly suppress the release of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 from LPS-induced GES-1 cell injury, decreased the protein expressions of TLR4, phospho-NF-κB, ASC, NLRP3, cleaved-IL-1ß, IL-6, and Bax, increased the protein expression of Bcl-2, and downregulated the ratios of cleaved caspase-1 to pro-caspase-1, phospho-IκBα to IκBα, and phospho-NF-кB to NF-κB. These findings strongly suggested that LDOP can prevent LPS-induced GES-1 cell injury by inhibiting the release of inflammatory cytokines regulated via the TLR4/NF-κB signal pathways.

Endotracheal tube mucus as a source of airway mucus for rheological study.

Muco-obstructive lung diseases (MOLDs), like cystic fibrosis and chronic obstructive pulmonary disease, affect a spectrum of subjects globally. In MOLDs, the airway mucus becomes hyperconcentrated, increasing osmotic and viscoelastic moduli and impairing mucus clearance. MOLD research requires relevant sources of healthy airway mucus for experimental manipulation and analysis. Mucus collected from endotracheal tubes (ETT) may represent such a source with benefits, e.g., in vivo production, over canonical sample types such as sputum or human bronchial epithelial (HBE) mucus. Ionic and biochemical compositions of ETT mucus from healthy human subjects were characterized and a stock of pooled ETT samples generated. Pooled ETT mucus exhibited concentration-dependent rheologic properties that agreed across spatial scales with reported individual ETT samples and HBE mucus. We suggest that the practical benefits compared with other sample types make ETT mucus potentially useful for MOLD research.

Glycoqueuing: Isomer-Specific Quantification for Sialylation-Focused Glycomics.

Changes of α-2,3-/α-2,6-linked sialic acids (SAs) in sialylglycans have been found to be closely related with some diseases. However, accurate quantification of sialylglycans at the isomeric level remains challenging due to their instability, structural complexity, and low mass spectrometry (MS) detection sensitivity. Herein, we propose an analytical strategy named "glycoqueuing", which allows sequential chromatographic elution and high-sensitivity MS quantification of various sialylglycan isomers based on isotopic labeling followed by analysis via online reversed-phase high performance liquid chromatography coupling with MS (RP-HPLC-MS). The new method was validated by detailed structural identification and quantification of fetal bovine serum (FBS) N-linked sialylglycan isomers, during which many branching isomers were successfully differentiated, and 28 sialylglycan compositions with Neu5Gc residues were analyzed. The method was successfully applied to isomer-specific, quantitative comparison of sialylated N-glycans between bovine and rabbit immunoglobulin G (IgG) and the search for serum sialylated N-glycan biomarker candidates of hepatocellular carcinoma, during which a 55% increase of α-2,6-sialylated fucosylated N-glycans was revealed, demonstrating the great applicability and potential clinical usage of the method.

Elaboration in type, primary structure, and bioactivity of polysaccharides derived from mollusks.

Over the past decades, numerous Mollusca species have received more attention in development and utilization as valuable bio-resources. Many efforts have been focused on investigating mollusk polysaccharides because of their rich content, ease of extraction, diversified sorts, specific structure, various biofunctions and potent activities. To date, many mollusks, especially species of gastropods, bivalves, or cephalopods, have been reported containing polysaccharide compounds in tissues with abundant amount, and most of polysaccharides are obtainable through combining techniques of extraction, separation and purification. The polysaccharides isolated from mollusks appeared with various structural and physicochemical characteristics, ranged from neutral polysaccharides and sulfated polysaccharides, to GAGs series (including Hep/HS, CS/DS, HA and similarities), even to heterogeneous glycan with high molecular weight. This review article provides comprehensive knowledge of recent researches on type classification, tissue origins and possible biofunctions of various polysaccharides from mollusks. The highlights were placed in structure variation including molecular weight, sulfation pattern, linkages and monomer compositions for repeating unit, and primary molecular construction of the mollusks polysaccharides. In addition, this article covers general information on exhibition of mollusks polysaccharide extracts or preparations in the various bioactivities, such as anticoagulant, antiatherogenic, antioxidant, immunomodulatory, antivirus and antitumor activities, which would reveal their possible potentials in medical application. Furthermore, the article presents a brief overview on several challenges and future scope in this field.

Optimization of the microwave-assisted enzymatic extraction of Rosa roxburghii Tratt. polysaccharides using response surface methodology and its antioxidant and α-d-glucosidase inhibitory activity.

An extraction assay applying microwave-assisted enzymatic treatment for polysaccharides in Rosa roxburghii was developed using response surface methodology. The process parameters were optimized using Plackett-Burman (PB) design and central composite design to enhance the Rosa roxburghii polysaccharide extraction yield. Specific conditions (microwave power, 575W; microwave time, 18min; liquid-to-material ratio, 13.5:1mL/g; and enzyme dose, 6.5g/mL) generated an experimental yield of 36.21±0.62%, which closely agreed with the predicted value of 35.75%. Purification with a DEAE-52 cellulose column generated two fractions, PR-1 (from 6.2×103 to 7.4KDa) and PR-2 (from 559.8 to 106.6KDa). Subsequently, the antioxidant activity and α-d-glucosidase inhibitory activity of the two polysaccharide fractions were assessed; PR-1 exhibited stronger antioxidant activity and α-d-glucosidase inhibitory activity than PR-2. Finally, the monosaccharide composition of PR-1 was determined by HPLC using a 1-phenyl-3-methyl-5-pyrazolone precolumn derivatization method. The result showed that PR-1 contained mannose, ribose, rhamnose, glucosamine hydrochloride, glucuronic acid, galacturonic acid, glucose, galactose, arabinose and fucose with molar percentages of 2.1%, 0.54%, 2.1%, 0.26%, 1.5%, 22.7%, 24.0%, 26.4%, 19.6% and 0.89%, respectively.

Pullulan as a stabilizer agent of polymeric nanocapsules for drug delivery

ABSTRACT Polymeric stabilizers have received attention in the preparation of nanostructured systems due to their ability to enhance formulation stability. Considering this, the objective of this work was to prepare poly(ε-caprolactone) nanocapsules using the pullulan as a polymeric stabilizer. The nanocapsules were prepared using the interfacial deposition method of preformed polymers and they were characterized in terms of pH, average diameter, polydispersity index, zeta potential, beclomethasone dipropionate content, encapsulation efficiency, photostability and drug release profiles. The formulations showed physicochemical characteristics consistent with nanocarriers for drug delivery such as: average diameter lower than 270 nm, polydispersity indexes lower than 0.2, negative zeta potential (-22.7 to -26.3 mV) and encapsulation efficiencies close to 100%. In addition, the nanocapsules were able to delay the beclomethasone dipropionate photodegradation under UVC radiation and by the dialysis bag diffusion technique, the nanocapsules were able to prolong the drug release. Thus, pullulan could be considered an interesting excipient to formulate polymeric nanocapsules.

Orthogonal Assessment of Biotherapeutic Glycosylation: A Case Study Correlating N-Glycan Core Afucosylation of Herceptin with Mechanism of Action.

In the development of therapeutic antibodies and biosimilars, an appropriate biopharmaceutical CMC control strategy that connects critical quality attributes with mechanism of action should enable product assessment at an early stage of development in order to mitigate risk. Here we demonstrate a new analytical workflow using trastuzumab which comprises "middle-up" analysis using a combination of IdeS and the endoglycosidases EndoS and EndoS2 to comprehensively map the glycan content. Enzymatic cleavage between the two N-acetyl glucosamine residues of the chitobiose core of N-glycans significantly simplifies the oligosaccharide component enabling facile distinction of GlcNAc from GlcNAc with core fucose. This approach facilitates quantitative determination of total Fc-glycan core-afucosylation, which was in turn correlated with receptor binding affinity by surface plasmon resonance and in vitro ADCC potency with a cell based bioassay. The strategy also quantifies Fc-glycan occupancy and the relative contribution from high mannose glycans.

Pullulan: an advantageous natural polysaccharide excipient to formulate tablets of alendronate-loaded microparticles

This work reports the preparation of tablets by direct compression of sodium alendronate-loaded microparticles, using pullulan as filler. The tableting properties of pullulan were compared with those of microcrystalline cellulose and lactose. Pullulan tablets showed low variations in average weight, thickness and drug content. Moreover, these tablets exhibited a higher hardness compared to the other excipients. In vitro release studies showed that only pullulan was capable to maintain gastroresistance and release properties of microparticles, due to its ability to protect particles against damage caused by compression force. Thus, pullulan was considered an advantageous excipient to prepare tableted microparticles.

Neste trabalho relata-se a preparação de comprimidos pela compressão direta de micropartículas contendo alendronato de sódio, utilizando o pullulan como diluente. As propriedades dos comprimidos de pullulan foram comparadas com as de comprimidos de celulose microcristalina e de lactose. Os comprimidos de pullulan mostraram baixa variação no peso médio, espessura e teor. Por outro lado, estes apresentaram altos valores de dureza comparados aos preparados com os outros excipientes. Através dos estudos de liberação in vitro pode-se observar que apenas o pullulan foi capaz de manter a gastrorresistência e as propriedades de liberação das micropartículas, o que se deve à sua capacidade de proteger as partículas do dano causado pela força de compressão. Dessa forma, o pullulan foi considerado um excipiente vantajoso para a preparação de comprimidos microparticulados.

Association of N-glycosylation with breast carcinoma and systemic features using high-resolution quantitative UPLC.

An improved separation of the human serum N-glycome using hydrophilic interaction chromatography technology with UPLC is described, where more than 140 N-glycans were assigned. Using this technique, serum samples from 107 healthy controls and 62 newly diagnosed breast cancer patients were profiled. The most statistically significant alterations were observed in cancer patients compared with healthy controls: an increase in sialylation, branching, and outer-arm fucosylation and a decrease in high-mannosylated and biantennary core-fucosylated glycans. In the controls and cases combined systemic features were analyzed; serum estradiol was associated with increase in digalactosylated glycans, and higher mammographic density was associated with increase in biantennary digalactosylated glycans and with decrease in trisialylated and in outer-arm fucosylated glycans. Furthermore, particular glycans were altered in some features of the breast carcinomas; bisected biantennary nonfucosylated glycans were decreased in patients with progesterone receptor positive tumors, and core-fucosylated biantennary bisected monogalactosylated glycans were decreased in patients with the TP53 mutation. Systemic features show more significant associations with the serum N-glycome than do the features of the breast carcinomas. In conclusion, the UPLC-based glycan analysis technique described here reveals highly significant differences between healthy women and breast cancer patients. Significant associations with breast carcinoma and systemic features are described.

Emerging principles for the therapeutic exploitation of glycosylation.

Glycosylation plays a key role in a wide range of biological processes. Specific modification to a glycan's structure can directly modulate its biological function. Glycans are not only essential to glycoprotein folding, cellular homeostasis, and immune regulation but are involved in multiple disease conditions. An increased molecular and structural understanding of the mechanistic role that glycans play in these pathological processes has driven the development of therapeutics and illuminated novel targets for drug design. This knowledge has enabled the treatment of metabolic disorders and the development of antivirals and shaped cancer and viral vaccine strategies. Furthermore, an understanding of glycosylation has led to the development of specific drug glycoforms, for example, monoclonal antibodies, with enhanced potency.

A new method for the rapid diagnosis of protein N-linked congenital disorders of glycosylation.

The Congenital Disorders of Glycosylation (CDG) are a devastating group of genetic disorders that encompass a spectrum of glycosylation defects and are characterized by the underglycosylation of or the presence of abnormal glycans on glycoproteins. The N-linked CDG disorders (Type I and II) are usually diagnosed in chemical pathology laboratories by an abnormal serum transferrin isoelectric focusing (IEF) pattern. Transferrin has been the protein of choice for CDG analysis because it is well characterized, highly abundant, and easily detected in plasma. However, IEF provides limited information on the glycosylation defect and requires a separate and extensive glycan analysis to diagnose CDG Type II. We have therefore developed a simple bead-based immunoaffinity and mass spectrometry-based assay to address these issues. Our method uses immuno-purified transferrin and proteolytic digestion followed by a rapid 30 min mass spectral analysis and allows us to identify both micro- and macroheterogeneity of transferrin by sequencing of peptides and glycopeptides. In summary, we have developed a simple, rapid test for N-linked glycosylation disorders that is a significant improvement on existing laboratory tests currently used for investigating defective N-linked glycosylation.

Delineating diseases by IMS-MS profiling of serum N-linked glycans.

Altered branching and aberrant expression of N-linked glycans is known to be associated with disease states such as cancer. However, the complexity of determining such variations hinders the development of specific glycomic approaches for assessing disease states. Here, we examine a combination of ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements, with principal component analysis (PCA) for characterizing serum N-linked glycans from 81 individuals: 28 with cirrhosis of the liver, 25 with liver cancer, and 28 apparently healthy. Supervised PCA of combined ion-mobility profiles for several, to as many as 10 different mass-to-charge ratios for glycan ions, improves the delineation of diseased states. This extends an earlier study [J. Proteome Res.2008, 7, 1109-1117] of isomers associated with a single glycan (S(1)H(5)N(4)) in which PCA analysis of the IMS profiles appeared to differentiate the liver cancer group from the other samples. Although performed on a limited number of test subjects, the combination of IMS-MS for different combinations of ions and multivariate PCA analysis shows promise for characterizing disease states.

Phlorotannins and fucoidans from marine macroalgae as matrix metalloproteinase inhibitory substances and their possible application as medicinal foods.

Metalloproteinases especially matrix metalloproteinases are a group of endopeptidases that contribute for the extracellular matrix degradation, and several tissue remodeling processes. Improper regulation of these endopeptidases could lead to several severe pathological problems that include cardiac, cartilage, and cancer-related diseases. Until now, many synthetic matrix metalloproteinase inhibitory substances (MMPIs) have been reported; however, many of them could not make to the final clinical trials. Hence, the emphasis on screening of MMPIs from different natural resources has gained much importance and marine resources are one among them. As marine organisms have been contributing with several biologically active compounds that have profound applications in nutraceuticals, cosmeceuticals, and pharmaceuticals; in this chapter, an attempt has been made to discuss the various MMPIs from edible seaweeds, which could be considered as medicinal foods.

Functional network of glycan-related molecules: glyco-net in glycoconjugate data bank.

BACKGROUND: Glycans are involved in a wide range of biological process, and they play an essential role in functions such as cell differentiation, cell adhesion, pathogen-host recognition, toxin-receptor interactions, signal transduction, cancer metastasis, and immune responses. Elucidating pathways related to post-translational modifications (PTMs) such as glycosylation are of growing importance in post-genome science and technology. Graphical networks describing the relationships among glycan-related molecules, including genes, proteins, lipids and various biological events are considered extremely valuable and convenient tools for the systematic investigation of PTMs. However, there is no database which dynamically draws functional networks related to glycans. DESCRIPTION: We have created a database called Glyco-Net http://www.glycoconjugate.jp/functions/, with many binary relationships among glycan-related molecules. Using search results, we can dynamically draw figures of the functional relationships among these components with nodes and arrows. A certain molecule or event corresponds to a node in the network figures, and the relationship between the molecule and the event are indicated by arrows. Since all components are treated equally, an arrow is also a node. CONCLUSIONS: In this paper, we describe our new database, Glyco-Net, which is the first database to dynamically show networks of the functional profiles of glycan related molecules. The graphical networks will assist in the understanding of the role of the PTMs. In addition, since various kinds of bio-objects such as genes, proteins, and inhibitors are equally treated in Glyco-Net, we can obtain a large amount of information on the PTMs.

Genotoxicity studies on fucoidan from Sporophyll of Undaria pinnatifida.

The sulfated seaweed extract, fucoidan, has anticoagulant, antithrombotic, and antiviral activities. Despite extensive work on the biological activities of fucoidan, detailed studies on the genotoxicity of fucoidan from Sporophyll of Undaria pinnatifida sources have not been tested before. The objective of this study was to investigate the genotoxicity effects of fucoidan extracted from Sporophyll of U. pinnatifida using a test battery of three different methods. In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, fucoidan did not increase the number of revertant colonies in any tester strain regardless of metabolic activation by S9 mix, and did not cause chromosomal aberration in short tests with S9 mix or in the continuous (24 h) test. A bone marrow micronucleus test in ICR mice dosed by oral gavage at doses up to 2000 mg/kg bw/day showed no significant or dose-dependent increases in the frequency of micronucleated polychromatic erythrocytes (MNPCE), and the high dose suppressed the ratio of polychromatic erythrocytes (PCE) to total erythrocytes. We conclude that fucoidan presents no significant genotoxic concern under the anticipated conditions of use.

BlotGlycoABCTM, an integrated glycoblotting technique for rapid and large scale clinical glycomics.

Recent progress in mass spectrometry has led to new challenges in glycomics, including the development of rapid glycan enrichment techniques. A facile technique for exploration of a carbohydrate-related biomarker is important because proteomics research targets glycosylation, a posttranslational modification. Here we report an "all-in-one" protocol for high throughput clinical glycomics. This new technique integrates glycoblotting-based glycan enrichment onto the BlotGlycoABC bead, on-bead stabilization of sialic acids, and fluorescent labeling of oligosaccharides in a single workflow on a multiwell filter plate. The advantage of this protocol and MALDI-TOF MS was demonstrated through differentiation of serum N-glycan profiles of subjects with congenital disorders of glycosylation and hepatocellular carcinoma and healthy donors. The method also permitted total cellular glycomics analysis of human prostate cancer cells and normal human prostate epithelial cells. These results demonstrate the potentials of glycan enrichment/processing for biomarker discovery.

Glycan classification with tree kernels.

MOTIVATION: Glycans are covalent assemblies of sugar that play crucial roles in many cellular processes. Recently, comprehensive data about the structure and function of glycans have been accumulated, therefore the need for methods and algorithms to analyze these data is growing fast. RESULTS: This article presents novel methods for classifying glycans and detecting discriminative glycan motifs with support vector machines (SVM). We propose a new class of tree kernels to measure the similarity between glycans. These kernels are based on the comparison of tree substructures, and take into account several glycan features such as the sugar type, the sugar bound type or layer depth. The proposed methods are tested on their ability to classify human glycans into four blood components: leukemia cells, erythrocytes, plasma and serum. They are shown to outperform a previously published method. We also applied a feature selection approach to extract glycan motifs which are characteristic of each blood component. We confirmed that some leukemia-specific glycan motifs detected by our method corresponded to several results in the literature. AVAILABILITY: Softwares are available upon request. SUPPLEMENTARY INFORMATION: Datasets are available at the following website: http://web.kuicr.kyoto-u.ac.jp/supp/yoshi/glycankernel/