Synthesis, evaluation and molecular docking studies of amino acid derived N-glycoconjugates as antibacterial agents.

PubMed

Baig, Noorullah; Singh, Rajnish Prakash; Chander, Subhash; Jha, Prabhat Nath; Murugesan, Sankaranarayanan; Sah, Ajay K

2015-12-01

Six amino acid derived N-glycoconjugates of d-glucose were synthesized, characterized and tested for antibacterial activity against G(+)ve (Bacillus cereus) as well as G(-)ve (Escherichia coli and Klebsiella pneumoniae) bacterial strains. All the tested compounds exhibited moderate to good antibacterial activity against these bacterial strains. The results were compared with the antibacterial activity of standard drug Chloramphenicol, where results of A5 (Tryptophan derived glycoconjugates) against E. coli and A4 (Isoleucine derived glycoconjugates) against K. pneumoniae bacterial strains are comparable with the standard drug molecule. In silico docking studies were also performed in order to understand the mode of action and binding interactions of these molecules. The docking studies revealed that, occupation of compound A5 at the ATP binding site of subunit GyrB (DNA gyrase, PDB ID: 3TTZ) via hydrophobic and hydrogen bonding interactions may be the reason for its significant in vitro antibacterial activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Changing patterns of peanut agglutinin labelling in the dorsal cochlear nucleus correspond to axonal ingrowth.

PubMed Central

Riggs, G H; Schweitzer, L

1994-01-01

Various studies have suggested that glycoconjugates may influence connectivity and lamination in the developing central nervous system and may function as barriers to neuritic extension. It has been proposed that the peanut agglutinin lectin labels a glycoconjugate subserving a barrier function. We chose to investigate the distribution of this peanut-agglutinin-labelled glycoconjugate in the dorsal cochlear nucleus of the developing hamster since the development of the dorsal cochlear nucleus is well characterised and its axons obey laminar boundaries. The distribution of peanut agglutinin label throughout the cochlear nucleus delineated zones that cochlear axons fail to invade. In the dorsal cochlear nucleus, laminar differences were reduced on postnatal d 13 and virtually disappearing by postnatal d 23. Label in the molecular layer dissipated as axons and dendrites grew into this layer. These patterns of peanut agglutinin binding correspond to axonal ingrowth and are consistent with a barrier function for glycoconjugates in the molecular layer. Images Fig. 1 Fig. 2 Fig. 4 PMID:7961144

Determination of the importance of in-mouth release of volatile phenol glycoconjugates to the flavor of smoke-tainted wines.

PubMed

Mayr, Christine M; Parker, Mango; Baldock, Gayle A; Black, Cory A; Pardon, Kevin H; Williamson, Patricia O; Herderich, Markus J; Francis, I Leigh

2014-03-19

The volatile phenols guaiacol, 4-methylguaiacol, syringol, 4-methylsyringol, o-, m-, and p-cresol, as well as their glycoconjugates, have previously been shown to be present in elevated concentrations in smoke-tainted wine. Sensory descriptive analysis experiments, with addition of free volatile phenols in combination with their glycosidically bound forms, were used to mimic smoke taint in red wines. The addition of volatile phenols together with glycoconjugates gave the strongest off-flavor. The hydrolysis of glycosidically bound flavor compounds in-mouth was further investigated by in vitro and in vivo experiments. The results indicate that enzymes present in human saliva are able to release the volatile aglycones from their glycoconjugates even under low pH and elevated ethanol conditions, confirming that in-mouth breakdown of monosaccharide and disaccharide glycosides is an important mechanism for smoke flavor from smoke affected wines, and that this mechanism may play an important general role in the flavor and aftertaste of wine.

Naegleria fowleri glycoconjugates with residues of α-D-mannose are involved in adherence of trophozoites to mouse nasal mucosa.

PubMed

Carrasco-Yepez, Maricela; Campos-Rodriguez, Rafael; Godinez-Victoria, Marycarmen; Rodriguez-Monroy, Marco Aurelio; Jarillo-Luna, Adriana; Bonilla-Lemus, Patricia; De Oca, Arturo Contis-Montes; Rojas-Hernandez, Saul

2013-10-01

We analyzed the possible role of glycoconjugates containing α-D-mannose and α-D-glucose residues in adherence of trophozoites to mouse nasal epithelium. Trophozoites incubated with 20 μg of one of three different lectins which preferentially recognized these residues were inoculated intranasally in Balb/c mice. Mouse survival was 40% with Pisum sativum and Canavalia ensiformis and 20% with Galanthus nivalis amebic pretreatment, compared with 0% survival for control animals administered trophozoites without pretreatment. Possibly some of the glycoproteins found in Naegleria fowleri represent an adherence factor. Differences in the saccharide sequences of the Naegleria species, even on the same glycoconjugate structure, could explain the different results corresponding to the distinct pretreatments (C. ensiformis, G. nivalis, and P. sativum). We found a higher expression of glycoconjugates recognized by P. sativum in Naegleria lovaniensis than N. fowleri, probably due to the higher number of oligosaccharides containing an α-1,6-linked fucose moiety expressed on the former species.

Histochemistry of glycoconjugates in the skin of the bovine muzzle, with special reference to glandular structures.

PubMed

Meyer, W; Tsukise, A

1989-01-01

The distribution of glycoconjugates in the muzzle of young adult Holstein cows has been studied by means of selected light-microscopic histochemical methods, including lectin histochemistry. In the skin layers, strong reactions were confined to intercellular substances in between the cells of the vital epidermis, exhibiting neutral glycoconjugates mainly with alpha-D-galactosyl and N-acetyl-D-galactosaminyl residues. In the nasolabial glands, distinctly positive staining for neutral glycoproteins with various saccharide residues (alpha-D-galactose, alpha-N-acetylgalactosamine, D-galactose-beta(1----3)D-N-acetylgalactosamine, beta-D-galactose), and for smaller amounts of acidic glycoconjugates, was found in the secretory cells and the luminal secretion. The cells of the excretory duct system showed weak to moderate reactions (alpha-D-galactose, beta-D-galactose), only the collecting ducts reacted positively for acidic glycoproteins with sialyl residues. The results obtained are discussed in view of muzzle function, with special reference to the salivary nature of the secretion of bovine nasolabial glands.

N-O linkage in carbohydrates and glycoconjugates.

PubMed

Chen, N; Xie, J

2016-11-29

The importance of oligosaccharides and their conjugates in various biological and pathological processes has stimulated growing interest in the development of (neo)glycoconjugates. Thanks to its high nucleophilicity, hydroxylamine has been employed as a powerful chemoselective ligation tool. Great effort has been focused on carbohydrates bearing aminooxy or N-hydroxy amino groups for organic synthesis, glycobiology and drug discovery. This review provides an overview of N-O linked carbohydrates and glycoconjugates, focusing particularly on the synthetic methodologies and chemical and physicochemical properties as well as biological and medical applications of N-glycosyl and O-glycosyl hydroxylamines, N-hydroxy amino and O-amino sugar as well as sugar aminooxy acid derivatives.

Glycoconjugates in Host-Helminth Interactions

PubMed Central

Prasanphanich, Nina Salinger; Mickum, Megan L.; Heimburg-Molinaro, Jamie; Cummings, Richard D.

2013-01-01

Helminths are multicellular parasitic worms that comprise a major class of human pathogens and cause an immense amount of suffering worldwide. Helminths possess an abundance of complex and unique glycoconjugates that interact with both the innate and adaptive arms of immunity in definitive and intermediate hosts. These glycoconjugates represent a major untapped reservoir of immunomodulatory compounds, which have the potential to treat autoimmune and inflammatory disorders, and antigenic glycans, which could be exploited as vaccines and diagnostics. This review will survey current knowledge of the interactions between helminth glycans and host immunity and highlight the gaps in our understanding which are relevant to advancing therapeutics, vaccine development, and diagnostics. PMID:24009607

An antibacterial vaccination strategy based on a glycoconjugate containing the core lipopolysaccharide tetrasaccharide Hep2Kdo2

NASA Astrophysics Data System (ADS)

Kong, Lingbing; Vijayakrishnan, Balakumar; Kowarik, Michael; Park, Jin; Zakharova, Alexandra N.; Neiwert, Larissa; Faridmoayer, Amirreza; Davis, Benjamin G.

2016-03-01

Certain non-mammalian cell wall sugars are conserved across a variety of pathogenic bacteria. This conservation of structure, combined with their structural differences when compared with mammalian sugars, make them potentially powerful epitopes for immunization. Here, we report the synthesis of a glycoconjugate that displays the so-called ‘inner core’ sugars of Gram-negative bacterial cell walls. We also describe an antibacterial vaccination strategy based on immunization with the glycoconjugate and the subsequent administration of an inhibitor that uncovers the corresponding epitope in pathogenic bacteria. The core tetrasaccharide, Hep2Kdo2, a common motif in bacterial lipopolysaccharides, was synthesized and attached via a chain linker to a diphtheria toxin mutant carrier protein. This glycoconjugate generated titres of antibodies towards the inner core tetrasaccharide of the lipopolysaccharide, which were capable of binding the cell-surface sugars of bacterial pathogenic strains including Neisseria meningitidis, Pseudomonas aeruginosa and Escherichia coli. Exposure of bacterial lipopolysaccharide in in vitro experiments, using an inhibitor of capsular polysaccharide transport, enabled potent bacterial killing with antiserum.

Acute oral safety study of sodium caseinate glycosylated via maillard reaction with galactose in rats.

PubMed

Anadón, Arturo; Martínez, Maria A; Ares, Irma; Castellano, Victor; Martínez-Larrañaga, Maria R; Corzo-Martínez, Marta; Moreno, F Javier; Villamiel, Mar

2014-03-01

In order to potentially use sodium caseinate (SC) glycated with galactose (Gal) in the food industry as a new functional ingredient with proved technological and biological properties, an evaluation of oral acute toxicity has been carried out. An acute safety study with SC-Gal glycoconjugates in the Wistar rat with a single oral gavage dose of 2,000 mg/kg of body weight was conducted. The SC-Gal glycoconjugates were well tolerated; no adverse effects or mortality was observed during the 2-week observation period. No abnormal signs, behavioral changes, body weight changes, or alterations in food and water consumption occurred. After this period, no changes in hematological and serum chemistry parameters, organ weights, or gross pathology or histopathology were detected. It was concluded that SC-Gal glycoconjugates obtained via the Maillard reaction were well tolerated in rats at an acute oral dose of 2,000 mg/kg of body weight. The SC-Gal glycoconjugates have a low order of acute toxicity, and the oral 50 % lethal dose for male and female rats is in excess of 2,000 mg/kg of body weight.

Chemical characterization of the glycated myofibrillar proteins from grass carp (Ctenopharyngodon idella) and their impacts on the human gut microbiota in vitro fermentation.

PubMed

Han, Kaining; Yao, Ye; Dong, Shiyuan; Jin, Sun; Xiao, Hang; Wu, Haohao; Zeng, Mingyong

2017-03-22

In this study, the chemical characterization of glycoconjugates of myofibrillar proteins from grass carp conjugated with glucose via Maillard reaction for up to 24 h of dry-heating was investigated, and their impacts on the microbial community in vitro human fecal fermentation were firstly evaluated by high-throughput sequencing technologies. The glycation greatly increased the furosine levels in glycoconjugates, which reached the maximum level (2.87 ± 0.08 mg per 100 mg protein) for 9 h of heating, and resulted in the structural changes of myofibrillar proteins based on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR) analysis. Size-exclusion chromatography (SEC) analysis of digested glycoconjugates showed that the gradually increased proportion between 1423 Da (bacitracin)-12 588 Da (cytochrome C) with the prolongation of heating time, suggesting that glycation decreased the digestibility of myofibrillar proteins. Furthermore, glycoconjugates with a higher level of Amadori products and lower browning intensity enhanced fecal microbiota diversity based on species-level phylotypes. The production of butyrate in fermentation of digested glycoconjugates was affected by the glycation extent of myofibrillar proteins, and significantly and positively correlated with Mitsuokella, Lachnospiraceae_UCG-004, Sutterella, Salinimicrobium, Fodinibius and Nitriliruptor (p < 0.05), but negatively correlated with Enterococcus, Dorea (p < 0.05), Escherichia-Shigella and Phascolarctobacterium (p < 0.01). Our findings demonstrated that the glycation of myofibrillar proteins could have potentially positive effects to intestinal health.

Desialylation of glycoconjugates on the surface of monocytes activates the extracellular signal-related kinases ERK 1/2 and results in enhanced production of specific cytokines.

PubMed

Stamatos, Nicholas M; Curreli, Sabrina; Zella, Davide; Cross, Alan S

2004-02-01

Modulation of the sialic acid content of cell-surface glycoproteins and glycolipids influences the functional capacity of cells of the immune system. The role of sialidase(s) and the consequent desialylation of cell surface glycoconjugates in the activation of monocytes have not been established. In this study, we show that desialylation of glycoconjugates on the surface of purified monocytes using exogenous neuraminidase (NANase) activated extracellular signal-regulated kinase 1/2 (ERK 1/2), an intermediate in intracellular signaling pathways. Elevated levels of phosphorylated ERK 1/2 were detected in desialylated monocytes after 2 h of NANase treatment, and increased amounts persisted for at least 2 additional hours. Desialylation of cell surface glycoconjugates also led to increased production of interleukin (IL)-6, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta by NANase-treated monocytes that were maintained in culture. Neither increased levels of phosphorylated ERK 1/2 nor enhanced production of cytokines were detected when NANase was heat-inactivated before use, demonstrating the specificity of NANase action. Treatment of monocytes with gram-negative bacterial lipopolysaccharide (LPS) also led to enhanced production of IL-6, MIP-1alpha, and MIP-1beta. The amount of each of these cytokines that was produced was markedly increased when monocytes were desialylated with NANase before exposure to LPS. These results suggest that changes in the sialic acid content of surface glycoconjugates influence the activation of monocytes.

In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation.

PubMed

Kämpf, Michael M; Braun, Martin; Sirena, Dominique; Ihssen, Julian; Thöny-Meyer, Linda; Ren, Qun

2015-01-23

Glycoconjugated vaccines composed of polysaccharide antigens covalently linked to immunogenic carrier proteins have proved to belong to the most effective and safest vaccines for combating bacterial pathogens. The functional transfer of the N-glycosylation machinery from Campylobacter jejuni to the standard prokaryotic host Escherichia coli established a novel bioconjugation methodology termed bacterial glycoengineering. In this study, we report on the production of a new recombinant glycoconjugate vaccine against Shigella flexneri 2a representing the major serotype for global outbreaks of shigellosis. We demonstrate that S. flexneri 2a O-polysaccharides can be transferred to a detoxified variant of Pseudomonas aeruginosa carrier protein exotoxin A (EPA) by the C. jejuni oligosaccharyltransferase PglB, resulting in glycosylated EPA-2a. Moreover, we optimized the in vivo production of this novel vaccine by identification and quantitative analysis of critical process parameters for glycoprotein synthesis. It was found that sequential induction of oligosaccharyltransferase PglB and carrier protein EPA increased the specific productivity of EPA-2a by a factor of 1.6. Furthermore, by the addition of 10 g/L of the monosaccharide N-acetylglucosamine during induction, glycoconjugate vaccine yield was boosted up to 3.1-fold. The optimum concentration of Mg2+ ions for N-glycan transfer was determined to be 10 mM. Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production. The present study is the first attempt to identify stimulating parameters for improved productivity of S. flexneri 2a bioconjugates. Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective in vivo production process for a glycoconjugate vaccine against S. flexneri 2a in E. coli. This study is an important step towards this goal and provides a starting point for further optimization studies.

Solution conformation and flexibility of capsular polysaccharides from Neisseria meningitidis and glycoconjugates with the tetanus toxoid protein

NASA Astrophysics Data System (ADS)

Abdelhameed, Ali Saber; Morris, Gordon A.; Almutairi, Fahad; Adams, Gary G.; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E.

2016-10-01

The structural integrity of meningococcal native, micro-fluidized and activated capsular polysaccharides and their glycoconjugates - in the form most relevant to their potential use as vaccines (dilute solution) - have been investigated with respect to their homogeneity, conformation and flexibility. Sedimentation velocity analysis showed that the polysaccharide size distributions were generally bimodal with some evidence for higher molar mass forms at higher concentration. Weight average molar masses Mw where lower for activated polysaccharides. Conjugation with tetanus toxoid protein however greatly increased the molar mass and polydispersity of the final conjugates. Glycoconjugates had an approximately unimodal log-normal but broad and large molar mass profiles, confirmed by sedimentation equilibrium “SEDFIT MSTAR” analysis. Conformation analysis using HYDFIT (which globally combines sedimentation and viscosity data), “Conformation Zoning” and Wales-van Holde approaches showed a high degree of flexibility - at least as great as the unconjugated polysaccharides, and very different from the tetanus toxoid (TT) protein used for the conjugation. As with the recently published finding for Hib-TT complexes, it is the carbohydrate component that dictates the solution behaviour of these glycoconjugates, although the lower intrinsic viscosities suggest some degree of compaction of the carbohydrate chains around the protein.

Solution conformation and flexibility of capsular polysaccharides from Neisseria meningitidis and glycoconjugates with the tetanus toxoid protein.

PubMed

Abdelhameed, Ali Saber; Morris, Gordon A; Almutairi, Fahad; Adams, Gary G; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E

2016-10-26

The structural integrity of meningococcal native, micro-fluidized and activated capsular polysaccharides and their glycoconjugates - in the form most relevant to their potential use as vaccines (dilute solution) - have been investigated with respect to their homogeneity, conformation and flexibility. Sedimentation velocity analysis showed that the polysaccharide size distributions were generally bimodal with some evidence for higher molar mass forms at higher concentration. Weight average molar masses M w where lower for activated polysaccharides. Conjugation with tetanus toxoid protein however greatly increased the molar mass and polydispersity of the final conjugates. Glycoconjugates had an approximately unimodal log-normal but broad and large molar mass profiles, confirmed by sedimentation equilibrium "SEDFIT MSTAR" analysis. Conformation analysis using HYDFIT (which globally combines sedimentation and viscosity data), "Conformation Zoning" and Wales-van Holde approaches showed a high degree of flexibility - at least as great as the unconjugated polysaccharides, and very different from the tetanus toxoid (TT) protein used for the conjugation. As with the recently published finding for Hib-TT complexes, it is the carbohydrate component that dictates the solution behaviour of these glycoconjugates, although the lower intrinsic viscosities suggest some degree of compaction of the carbohydrate chains around the protein.

Glycoconjugates in New World species of Leishmania: polymorphisms in lipophosphoglycan and glycoinositolphospholipids and interaction with hosts.

PubMed

de Assis, Rafael Ramiro; Ibraim, Izabela Coimbra; Nogueira, Paula Monalisa; Soares, Rodrigo Pedro; Turco, Salvatore J

2012-09-01

Protozoan parasites of the genus Leishmania cause a number of important diseases in humans and undergo a complex life cycle, alternating between a sand fly vector and vertebrate hosts. The parasites have a remarkable capacity to avoid destruction in which surface molecules are determinant for survival. Amongst the many surface molecules of Leishmania, the glycoconjugates are known to play a central role in host-parasite interactions and are the focus of this review. The most abundant and best studied glycoconjugates are the Lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs). This review summarizes the main studies on structure and biological functions of these molecules in New World Leishmania species. LPG and GIPLs are complex molecules that display inter- and intraspecies polymorphisms. They are key elements for survival inside the vector and to modulate the vertebrate immune response during infection. Most of the studies on glycoconjugates focused on Old World Leishmania species. Here, it is reported some of the studies involving New World species and their biological significance on host-parasite interaction. This article is part of a Special Issue entitled Glycoproteomics. Copyright © 2011 Elsevier B.V. All rights reserved.

Differential modulation of macrophage response elicited by glycoinositolphospholipids and lipophosphoglycan from Leishmania (Viannia) shawi.

PubMed

Passero, Luiz F D; Assis, Rafael R; da Silva, Thays N F; Nogueira, Paula M; Macedo, Diego H; Pessoa, Natália L; Campos, Marco A; Laurenti, Márcia D; Soares, Rodrigo P

2015-08-01

In this work, some aspects of the glycobiology of Leishmania shawi were examined, as it is a causative agent of cutaneous leishmaniasis in the New World. Additionally, the interaction of L. shawi's main glycoconjugates [lipophosphoglycan (LPG) and glycoinositolphospholipids (GIPLs)] with macrophages was evaluated in vitro. L. shawi LPG was devoid of side-chains in its repeat units, whereas monosaccharide analysis showed that GIPLs were suggestive of mannose-rich (type I or hybrid). In order to evaluate the biological roles of those molecules, BALB/c resident peritoneal macrophages were incubated with these glycoconjugates for 24h, and the levels of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-12p70 and IL-10, were determined. In general, the GIPLs exhibited a greater proinflammatory role than the LPGs did. However, for the first time, the GIPLs from this species were able to trigger the production of IL-10, an anti-inflammatory cytokine. In conclusion, L. shawi glycoconjugates were able to interact with the innate immune compartment. These data reinforce the role of parasite glycoconjugates during parasite and host cell interactions. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Silibinin phosphodiester glyco-conjugates: Synthesis, redox behaviour and biological investigations.

PubMed

Romanucci, Valeria; Agarwal, Chapla; Agarwal, Rajesh; Pannecouque, Christophe; Iuliano, Mauro; De Tommaso, Gaetano; Caruso, Tonino; Di Fabio, Giovanni; Zarrelli, Armando

2018-04-01

New silibinin phosphodiester glyco-conjugates were synthesized by efficient phosphoramidite chemistry and were fully characterized by 2D-NMR. A wide-ranging study focused on the determination of their pKa and E° values as well as on their radical scavenging activities by different assays (DPPH, ABTS + and HRSA) was conducted. The new glyco-conjugates are more water-soluble than silibinin, and their radical scavenging activities are higher than those of silibinin. The conjugation therefore improves both the water solubilities and antioxidant activities of the flavonolignan moieties. The serum stability was evaluated under physiological conditions, and the glyco-conjugates degraded with half-lives of 40-70 h, making them useful in pro-drug approaches. We started by treating androgen-dependent prostate cancer (PCa) LNCaP cells and then expanded our studies to androgen-independent PCa PC3 and DU145 cells. In most cases, the new derivatives significantly reduced both total and live cell numbers, albeit at different levels. Anti-HIV activities were evaluated and the glucosamine-phosphate silibinin derivative showed higher activity (IC 50  = 73 μM) than silibinin. Copyright © 2018 Elsevier Inc. All rights reserved.

Development of dialyzer with immobilized glycoconjugate polymers for removal of Shiga-toxin.

PubMed

Miyagawa, Atsushi; Watanabe, Miho; Igai, Katsura; Kasuya, Maria Carmelita Z; Natori, Yasuhiro; Nishikawa, Kiyotaka; Hatanaka, Kenichi

2006-06-01

The dialyzer for Shiga-toxin elimination was developed and its performance was established. The dialyzer was prepared by immobilization of multivalent ligands. Glycoconjugate polymers having oligosaccharides and amino groups were synthesized to function as Shiga-toxin adsorbents. The amino group was utilized to immobilize the polymer inside the cellulose hollow fiber of the dialyzer. Cellulose hollow fibers packed in the dialyzer were carboxymethylated under moderate conditions. The glycoconjugate polymers were bound covalently to the hollow fibers of the dialyzer by condensation reaction between the amino group of the polymer and the carboxyl group of the cellulose hollow fiber. Shiga-toxin eliminabilities of the prepared dialyzers were evaluated at various conditions. Even at high concentration of protein such as FCS, the dialyzer showed an excellent performance for Shiga-toxin adsorption.

Invited review: Anti-adhesive properties of bovine oligosaccharides and bovine milk fat globule membrane-associated glycoconjugates against bacterial food enteropathogens.

PubMed

Douëllou, T; Montel, M C; Thevenot Sergentet, D

2017-05-01

The prevalence of the main raw milk and raw milk-derived dairy product enteropathogens (Campylobacter, Shiga toxin-producing Escherichia coli, Listeria, and Salmonella) is higher than the number of epidemiological cases related to ingesting these foodstuffs. Bovine milk oligosaccharides and milk fat globule membrane (MFGM)-linked glycoconjugates interact with foodborne enteropathogens to inhibit their adhesion to intestinal cells and tissues. This review examines the main mechanisms and strategies used by enteropathogens to adhere to their target, details the anti-adhesive properties of MFGM against enteropathogens and enterotoxins, assesses the integrity of bacteria-MFGM complexes during dairy product manufacture and digestion, and discusses the potential for using these macromolecules and glycoconjugates in foods for public health. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Plant as a plenteous reserve of lectin

PubMed Central

Hivrale, AU; Ingale, AG

2013-01-01

Lectins are clusters of glycoproteins of nonimmune foundation that combine specifically and reversibly to carbohydrates, mainly the sugar moiety of glycoconjugates, resulting in cell agglutination and precipitation of glycoconjugates. They are universally distributed in nature, being established in plants, fungi, viruses, bacteria, crustacea, insects, and animals, but leguminacae plants are rich source of lectins. The present review reveals the structure, biological properties, and application of plant lectins. PMID:24084524

A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate.

PubMed

Abdelhameed, Ali Saber; Adams, Gary G; Morris, Gordon A; Almutairi, Fahad M; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E

2016-02-26

Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 10(6) g.mol(-1)) compared to the native (Mw ~ 1.2 × 10(6) g.mol(-1)). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 10(6) g.mol(-1)), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein (slightly extended and hydrodynamically compact structure with an aspect ratio of ~3). This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution.

The glycoconjugate sugar residues of the sessile and motile cells in the thymus of normal and cyclosporin-A-treated rats: lectin histochemistry.

PubMed

Gheri, G; Gheri Bryk, S; Riccardi, R; Sgambati, E; Cirri Borghi, M B

2002-01-01

It is well known that cell surface glycoconjugates play a determinant role in cellular recognition, cell-to-cell adhesion and serve as receptor molecules. T-lymphocytes are in strict contact with the thymic epithelial cells, which control their process of maturation and proliferation. On the other hand the normal maturation of the epithelial cells is believed to be induced by T-lymphocytes. For these reasons we have studied the glycoconjugates saccharidic moieties of the sessile and motile cells in the thymus of normal male albino Wistar rats and their changes following cyclosporin-A treatment, using a battery of seven HRP-lectins. Cytochemical controls were performed for specificity of lectin-sugar reaction. Some sections were pre-treated with neuraminidase prior to staining with HRP-lectins. Our results have demonstrated, in the control rats, a large amount and a variety of terminal and subterminal oligosaccharides within and/or on the epithelial thymic cells and in macrophages. After cyclosporin-A treatment, among the thymic epithelial cells, the subcapsular, paraseptal and perivascular cells showed the loss of some sugar residues, which characterized the same cells in the intact thymus. Some hypotheses are reported on the role played by the glycoconjugate sugar residues in control and cyclosporin-A treated rats.

Investigating the candidacy of the serotype specific rhamnan polysaccharide based glycoconjugates to prevent disease caused by the dental pathogen Streptococcus mutans.

PubMed

St Michael, Frank; Yang, Qingling; Cairns, Chantelle; Vinogradov, Evgeny; Fleming, Perry; Hayes, Alexander C; Aubry, Annie; Cox, Andrew D

2018-02-01

Dental caries remains a major health issue and the Gram-positive bacterium Streptococcus mutans is considered as the major pathogen causing caries. More recently, S. mutans has been recognised as a cause of endocarditis, ulcerative colitis and fatty acid liver disease along with the likelihood of increased cerebral hemorrhage following a stroke if S. mutans is present systemically. We initiated this study to examine the vaccine candidacy of the serotype specific polysaccharides elaborated by S. mutans. We have confirmed the carbohydrate structures for the serotype specific rhamnan containing polysaccharides from serotypes c, f and k. We have prepared glycoconjugate vaccines using the rhamnan containing polymers from serotypes f and k and immunised mice and rabbits. We consistently obtained a robust immune response to the glycoconjugates with cross-reactivity consistent with the structural similarities of the polymers from the different serotypes. We developed an opsonophagocytic assay which illustrated the ability of the post-immune sera to facilitate opsonophagocytic killing of the homologous and heterologous serotypes at titers consistent with the structural homologies. We conclude that glycoconjugates of the rhamnan polymers of S. mutans are a potential vaccine candidate to target dental caries and other sequelae following the escape of S. mutans from the oral cavity.

Visualizing the dental biofilm matrix by means of fluorescence lectin-binding analysis.

PubMed

Tawakoli, Pune N; Neu, Thomas R; Busck, Mette M; Kuhlicke, Ute; Schramm, Andreas; Attin, Thomas; Wiedemeier, Daniel B; Schlafer, Sebastian

2017-01-01

The extracellular matrix is a poorly studied, yet important component of dental biofilms. Fluorescence lectin-binding analysis (FLBA) is a powerful tool to characterize glycoconjugates in the biofilm matrix. This study aimed to systematically investigate the ability of 75 fluorescently labeled lectins to visualize and quantify extracellular glycoconjugates in dental biofilms. Lectin binding was screened on pooled supragingival biofilm samples collected from 76 subjects using confocal microscopy. FLBA was then performed with 10 selected lectins on biofilms grown in situ for 48 h in the absence of sucrose. For five lectins that proved particularly suitable, stained biovolumes were quantified and correlated to the bacterial composition of the biofilms. Additionally, combinations of up to three differently labeled lectins were tested. Of the 10 lectins, five bound particularly well in 48-h-biofilms: Aleuria aurantia (AAL), Calystega sepiem (Calsepa), Lycopersicon esculentum (LEA), Morniga-G (MNA-G) and Helix pomatia (HPA). No significant correlation between the binding of specific lectins and bacterial composition was found. Fluorescently labeled lectins enable the visualization of glycoconjugates in the dental biofilm matrix. The characterization and quantification of glycoconjugates in dental biofilms require a combination of several lectins. For 48-h-biofilms grown in absence of sucrose, AAL, Calsepa, HPA, LEA, and MNA-G are recommendable.

A gold nanoparticle-linked glycoconjugate vaccine against Burkholderia mallei.

PubMed

Gregory, Anthony E; Judy, Barbara M; Qazi, Omar; Blumentritt, Carla A; Brown, Katherine A; Shaw, Andrew M; Torres, Alfredo G; Titball, Richard W

2015-02-01

Burkholderia mallei are Gram-negative bacteria, responsible for the disease glanders. B. mallei has recently been classified as a Tier 1 agent owing to the fact that this bacterial species can be weaponised for aerosol release, has a high mortality rate and demonstrates multi-drug resistance. Furthermore, there is no licensed vaccine available against this pathogen. Lipopolysaccharide (LPS) has previously been identified as playing an important role in generating host protection against Burkholderia infection. In this study, we present gold nanoparticles (AuNPs) functionalised with a glycoconjugate vaccine against glanders. AuNPs were covalently coupled with one of three different protein carriers (TetHc, Hcp1 and FliC) followed by conjugation to LPS purified from a non-virulent clonal relative, B. thailandensis. Glycoconjugated LPS generated significantly higher antibody titres compared with LPS alone. Further, they improved protection against a lethal inhalation challenge of B. mallei in the murine model of infection. Burkholderia mallei is associated with multi-drug resistance, high mortality and potentials for weaponization through aerosol inhalation. The authors of this study present gold nanoparticles (AuNPs) functionalized with a glycoconjugate vaccine against this Gram negative bacterium demonstrating promising results in a murine model even with the aerosolized form of B. Mallei. Copyright © 2015 Elsevier Inc. All rights reserved.

Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and host–pathogen interaction

PubMed Central

Mishra, Arun K; Driessen, Nicole N; Appelmelk, Ben J; Besra, Gurdyal S

2011-01-01

Approximately one third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. This bacterium has an unusual lipid-rich cell wall containing a vast repertoire of antigens, providing a hydrophobic impermeable barrier against chemical drugs, thus representing an attractive target for vaccine and drug development. Apart from the mycolyl–arabinogalactan–peptidoglycan complex, mycobacteria possess several immunomodulatory constituents, notably lipomannan and lipoarabinomannan. The availability of whole-genome sequences of M. tuberculosis and related bacilli over the past decade has led to the identification and functional characterization of various enzymes and the potential drug targets involved in the biosynthesis of these glycoconjugates. Both lipomannan and lipoarabinomannan possess highly variable chemical structures, which interact with different receptors of the immune system during host–pathogen interactions, such as Toll-like receptors-2 and C-type lectins. Recently, the availability of mutants defective in the synthesis of these glycoconjugates in mycobacteria and the closely related bacterium, Corynebacterium glutamicum, has paved the way for host–pathogen interaction studies, as well as, providing attenuated strains of mycobacteria for the development of new vaccine candidates. This review provides a comprehensive account of the structure, biosynthesis and immunomodulatory properties of these important glycoconjugates. PMID:21521247

Use of lectins to in situ visualize glycoconjugates of extracellular polymeric substances in acidophilic archaeal biofilms

PubMed Central

Zhang, R Y; Neu, T R; Bellenberg, S; Kuhlicke, U; Sand, W; Vera, M

2015-01-01

Biofilm formation and the production of extracellular polymeric substances (EPS) by meso- and thermoacidophilic metal-oxidizing archaea on relevant substrates have been studied to a limited extent. In order to investigate glycoconjugates, a major part of the EPS, during biofilm formation/bioleaching by archaea on pyrite, a screening with 75 commercially available lectins by fluorescence lectin-binding analysis (FLBA) has been performed. Three representative archaeal species, Ferroplasma acidiphilum DSM 28986, Sulfolobus metallicus DSM 6482T and a novel isolate Acidianus sp. DSM 29099 were used. In addition, Acidianus sp. DSM 29099 biofilms on elemental sulfur were studied. The results of FLBA indicate (i) 22 lectins bound to archaeal biofilms on pyrite and 21 lectins were binding to Acidianus sp. DSM 29099 biofilms on elemental sulfur; (ii) major binding patterns, e.g. tightly bound EPS and loosely bound EPS, were detected on both substrates; (iii) the three archaeal species produced various EPS glycoconjugates on pyrite surfaces. Additionally, the substratum induced different EPS glycoconjugates and biofilm structures of cells of Acidianus sp. DSM 29099. Our data provide new insights into interactions between acidophilic archaea on relevant surfaces and also indicate that FLBA is a valuable tool for in situ investigations on archaeal biofilms. PMID:25488256

A gold nanoparticle-linked glycoconjugate vaccine against Burkholderia mallei

PubMed Central

Gregory, Anthony E.; Judy, Barbara M.; Qazi, Omar; Blumentritt, Carla A.; Brown, Katherine A.; Shaw, Andrew M.; Torres, Alfredo G.; Titball, Richard W.

2014-01-01

Burkholderia mallei are Gram-negative bacteria, responsible for the disease glanders. B. mallei has recently been classified as a Tier 1 agent owing to the fact that this bacterial species can be weaponised for aerosol release, has a high mortality rate and demonstrates multi-drug resistance. Furthermore, there is no licensed vaccine available against this pathogen. Lipopolysaccharide (LPS) has previously been identified as playing an important role in generating host protection against Burkholderia infection. In this study, we present gold nanoparticles (AuNPs) functionalised with a glycoconjugate vaccine against glanders. AuNPs were covalently coupled with one of three different protein carriers (TetHc, Hcp1 and FliC) followed by conjugation to LPS purified from a non-virulent clonal relative, B. thailandensis. Glycoconjugated LPS generated significantly higher antibody titres and compared with LPS alone. Further, they improved protection against a lethal inhalation challenge of B. mallei in the murine model of infection. PMID:25194998

Electronic Detection of Lectins Using Carbohydrate Functionalized Nanostructures: Graphene versus Carbon Nanotubes

PubMed Central

Chen, Yanan; Vedala, Harindra; Kotchey, Gregg P.; Audfray, Aymeric; Cecioni, Samy; Imberty, Anne; Vidal, Sébastien; Star, Alexander

2012-01-01

Here we investigated the interactions between lectins and carbohydrates using field-effect transistor (FET) devices comprised of chemically converted graphene (CCG) and single-walled carbon nanotubes (SWNTs). Pyrene- and porphyrin-based glycoconjugates were functionalized noncovalently on the surface of CCG-FET and SWNT-FET devices, which were then treated with 2 µM of nonspecific and specific lectins. In particular, three different lectins (PA-IL, PA-IIL and ConA) and three carbohydrate epitopes (galactose, fucose and mannose) were tested. The responses of 36 different devices were compared and rationalized using computer-aided models of carbon nanostructure/glycoconjugate interactions. Glycoconjugates surface coverage in addition to one-dimensional structures of SWNTs resulted in optimal lectin detection. Additionally, lectin titration data of SWNT- and CCG-based biosensors were used to calculate lectin dissociation constants (Kd) and compare them to the values obtained from the isothermal titration microcalorimetry (ITC) technique. PMID:22136380

Chemical glycosylation of cytochrome c improves physical and chemical protein stability.

PubMed

Delgado, Yamixa; Morales-Cruz, Moraima; Hernández-Román, José; Martínez, Yashira; Griebenow, Kai

2014-08-06

Cytochrome c (Cyt c) is an apoptosis-initiating protein when released into the cytoplasm of eukaryotic cells and therefore a possible cancer drug candidate. Although proteins have been increasingly important as pharmaceutical agents, their chemical and physical instability during production, storage, and delivery remains a problem. Chemical glycosylation has been devised as a method to increase protein stability and thus enhance their long-lasting bioavailability. Three different molecular weight glycans (lactose and two dextrans with 1 kD and 10 kD) were chemically coupled to surface exposed Cyt c lysine (Lys) residues using succinimidyl chemistry via amide bonds. Five neo-glycoconjugates were synthesized, Lac4-Cyt-c, Lac9-Cyt-c, Dex5(10kD)-Cyt-c, Dex8(10kD)-Cyt-c, and Dex3(1kD)-Cyt-c. Subsequently, we investigated glycoconjugate structure, activity, and stability. Circular dichroism (CD) spectra demonstrated that Cyt c glycosylation did not cause significant changes to the secondary structure, while high glycosylation levels caused some minor tertiary structure perturbations. Functionality of the Cyt c glycoconjugates was determined by performing cell-free caspase 3 and caspase 9 induction assays and by measuring the peroxidase-like pseudo enzyme activity. The glycoconjugates showed ≥94% residual enzyme activity and 86 ± 3 to 95 ± 1% relative caspase 3 activation compared to non-modified Cyt c. Caspase 9 activation by the glycoconjugates was with 92 ± 7% to 96 ± 4% within the error the same as the caspase 3 activation. There were no major changes in Cyt c activity upon glycosylation. Incubation of Dex3(1 kD)-Cyt c with mercaptoethanol caused significant loss in the tertiary structure and a drop in caspase 3 and 9 activation to only 24 ± 8% and 26 ± 6%, respectively. This demonstrates that tertiary structure intactness of Cyt c was essential for apoptosis induction. Furthermore, glycosylation protected Cyt c from detrimental effects by some stresses (i.e., elevated temperature and humidity) and from proteolytic degradation. In addition, non-modified Cyt c was more susceptible to denaturation by a water-organic solvent interface than its glycoconjugates, important for the formulation in polymers. The results demonstrate that chemical glycosylation is a potentially valuable method to increase Cyt c stability during formulation and storage and potentially during its application after administration.

Binding of heavy metal ions in aggregates of microbial cells, EPS and biogenic iron minerals measured in-situ using metal- and glycoconjugates-specific fluorophores

NASA Astrophysics Data System (ADS)

Hao, Likai; Guo, Yuan; Byrne, James M.; Zeitvogel, Fabian; Schmid, Gregor; Ingino, Pablo; Li, Jianli; Neu, Thomas R.; Swanner, Elizabeth D.; Kappler, Andreas; Obst, Martin

2016-05-01

Aggregates consisting of bacterial cells, extracellular polymeric substances (EPS) and Fe(III) minerals formed by Fe(II)-oxidizing bacteria are common at bulk or microscale chemical interfaces where Fe cycling occurs. The high sorption capacity and binding capacity of cells, EPS, and minerals controls the mobility and fate of heavy metals. However, it remains unclear to which of these component(s) the metals will bind in complex aggregates. To clarify this question, the present study focuses on 3D mapping of heavy metals sorbed to cells, glycoconjugates that comprise the majority of EPS constituents, and Fe(III) mineral aggregates formed by the phototrophic Fe(II)-oxidizing bacteria Rhodobacter ferrooxidans SW2 using confocal laser scanning microscopy (CLSM) in combination with metal- and glycoconjugates-specific fluorophores. The present study evaluated the influence of glycoconjugates, microbial cell surfaces, and (biogenic) Fe(III) minerals, and the availability of ferrous and ferric iron on heavy metal sorption. Analyses in this study provide detailed knowledge on the spatial distribution of metal ions in the aggregates at the sub-μm scale, which is essential to understand the underlying mechanisms of microbe-mineral-metal interactions. The heavy metals (Au3+, Cd2+, Cr3+, CrO42-, Cu2+, Hg2+, Ni2+, Pd2+, tributyltin (TBT) and Zn2+) were found mainly sorbed to cell surfaces, present within the glycoconjugates matrix, and bound to the mineral surfaces, but not incorporated into the biogenic Fe(III) minerals. Statistical analysis revealed that all ten heavy metals tested showed relatively similar sorption behavior that was affected by the presence of sorbed ferrous and ferric iron. Results in this study showed that in addition to the mineral surfaces, both bacterial cell surfaces and the glycoconjugates provided most of sorption sites for heavy metals. Simultaneously, ferrous and ferric iron ions competed with the heavy metals for sorption sites on the organic compounds. In summary, the information obtained by the present approach using a microbial model system provides important information to better understand the interactions between heavy metals and biofilms, and microbially formed Fe(III) minerals and heavy metals in complex natural environments.

Structural analysis and profiling of phenolic secondary metabolites of Mexican lupine species using LC-MS techniques.

PubMed

Wojakowska, Anna; Piasecka, Anna; García-López, Pedro M; Zamora-Natera, Francisco; Krajewski, Paweł; Marczak, Łukasz; Kachlicki, Piotr; Stobiecki, Maciej

2013-08-01

Flavonoid glycoconjugates from roots and leaves of eight North America lupine species (Lupinus elegans, Lupinus exaltatus, Lupinus hintonii, Lupinus mexicanus, Lupinus montanus, Lupinus rotundiflorus, Lupinus stipulatus, Lupinus sp.), three Mediterranean species (Lupinus albus, Lupinus angustifolius, Lupinus luteus) and one species from South America domesticated in Europe (Lupinus mutabilis) were analyzed using two LC/MS systems: low-resolution ion trap instrument and high-resolution quadrupole-time-of-flight spectrometer. As a result of the LC/MS profiling using the CID/MS(n) experiments structures of 175 flavonoid glycoconjugates found in 12 lupine species were identified at three confidence levels according to the Metabolomic Standard Initiative, mainly at level 2 and 3, some of them were classified to the level 1. Among the flavonoid derivatives recognized in the plant extracts were isomeric or isobaric compounds, differing in the degree of hydroxylation of the aglycones and the presence of glycosidic, acyl or alkyl groups in the molecules. The elemental composition of the glycoconjugate molecules was established from the exact m/z values of the protonated/deprotonated molecules ([M+H](+)/[M-H](-)) measured with the accuracy better than 5 ppm. Information concerning structures of the aglycones, the type of sugar moieties (hexose, deoxyhexose or pentose) and, in some cases, their placement on the aglycones as well as the acyl substituents of the flavonoid glycoconjugates was achieved in experiments, in which collision-induced dissociation was applied. Flavonoid aglycones present in the studied O-glycoconjugates were unambiguously identified after the comparison of the pseudo-MS(3) spectra with the spectra registered for the standards. Isomers of flavonoid glycoconjugates, in which one or two sugar moieties were attached to 4'- or 7-hydroxyl groups or directly to the C-6 or C-8 of the aglycones, could be distinguished on the basis of the MS(2) spectra. However, the collision energy applied in the CID experiments had to be optimized for each group of the compounds and there were no universal settings that allowed the acquisition of structural information for all the compounds present in the sample. Information obtained from the flavonoid conjugate profiling was used for the chemotaxonomic comparison of the studied lupine species. A clear-cut discrimination of the Mediterranean and North American lupines was obtained as a result of this analysis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Rebamipide increases the mucin-like glycoprotein production in corneal epithelial cells.

PubMed

Takeji, Yasuhiro; Urashima, Hiroki; Aoki, Akihiro; Shinohara, Hisashi

2012-06-01

Dry eye is a multifactorial disease of tears and the ocular surface due to tear deficiency or excessive tear evaporation. Tear film instability is due to a disturbance in ocular surface mucin leading to a dysfunction of mucin, resulting in dry eye. In this study, we examined the effect of rebamipide, an anti-ulcer agent, on glycoconjugate production, as an indicator of mucin-like glycoprotein in cultured corneal epithelial cells. Further, we investigated the effect of rebamipide on the gene expression of membrane-associated mucins. Confluent cultured human corneal epithelial cells were incubated with rebamipide for 24 h. The glycoconjugate content in the supernatant and the cell extracts was measured by wheat germ agglutinin-enzyme-linked lectin assay combined gel-filtration method. In the experiment on mucin gene expression, cultured human corneal epithelial cells were collected at 0, 3, 6, and 12 h after administration of rebamipide. Real-time quantitative polymerase chain reaction was used to analyze the quantity of MUC1, MUC 4, and MUC16 gene expression. Rebamipide significantly increased the glycoconjugate contents in the supernatant and cell extract. In the mucin gene expression in the cells, rebamipide increased MUC1 and MUC4 gene expression, but did not increase MUC16 gene expression. Rebamipide promoted glycoconjugate, which has a property as a mucin-like glycoprotein, in human corneal epithelial cells. The increased production was mediated by MUC1 and MUC4 gene expression.

Fluorescence Lectin Bar-Coding of Glycoconjugates in the Extracellular Matrix of Biofilm and Bioaggregate Forming Microorganisms.

PubMed

Neu, Thomas R; Kuhlicke, Ute

2017-02-10

Microbial biofilm systems are defined as interface-associated microorganisms embedded into a self-produced matrix. The extracellular matrix represents a continuous challenge in terms of characterization and analysis. The tools applied in more detailed studies comprise extraction/chemical analysis, molecular characterization, and visualisation using various techniques. Imaging by laser microscopy became a standard tool for biofilm analysis, and, in combination with fluorescently labelled lectins, the glycoconjugates of the matrix can be assessed. By employing this approach a wide range of pure culture biofilms from different habitats were examined using the commercially available lectins. From the results, a binary barcode pattern of lectin binding can be generated. Furthermore, the results can be fine-tuned and transferred into a heat map according to signal intensity. The lectin barcode approach is suggested as a useful tool for investigating the biofilm matrix characteristics and dynamics at various levels, e.g. bacterial cell surfaces, adhesive footprints, individual microcolonies, and the gross biofilm or bio-aggregate. Hence fluorescence lectin bar-coding (FLBC) serves as a basis for a subsequent tailor-made fluorescence lectin-binding analysis (FLBA) of a particular biofilm. So far, the lectin approach represents the only tool for in situ characterization of the glycoconjugate makeup in biofilm systems.  Furthermore, lectin staining lends itself to other fluorescence techniques in order to correlate it with cellular biofilm constituents in general and glycoconjugate producers in particular.

Strain Selection for Generation of O-Antigen-Based Glycoconjugate Vaccines against Invasive Nontyphoidal Salmonella Disease

PubMed Central

Saul, Allan; MacLennan, Calman A.; Micoli, Francesca; Rondini, Simona

2015-01-01

Nontyphoidal Salmonellae, principally S. Typhimurium and S. Enteritidis, are a major cause of invasive bloodstream infections in sub-Saharan Africa with no vaccine currently available. Conjugation of lipopolysaccharide O-antigen to a carrier protein constitutes a promising vaccination strategy. Here we describe a rational process to select the most appropriate isolates of Salmonella as source of O-antigen for developing a bivalent glycoconjugate vaccine. We screened a library of 30 S. Typhimurium and 21 S. Enteritidis in order to identify the most suitable strains for large scale O-antigen production and generation of conjugate vaccines. Initial screening was based on growth characteristics, safety profile of the isolates, O-antigen production, and O-antigen characteristics in terms of molecular size, O-acetylation and glucosylation level and position, as determined by phenol sulfuric assay, NMR, HPLC-SEC and HPAEC-PAD. Three animal isolates for each serovar were identified and used to synthesize candidate glycoconjugate vaccines, using CRM197 as carrier protein. The immunogenicity of these conjugates and the functional activity of the induced antibodies was investigated by ELISA, serum bactericidal assay and flow cytometry. S. Typhimurium O-antigen showed high structural diversity, including O-acetylation of rhamnose in a Malawian invasive strain generating a specific immunodominant epitope. S. Typhimurium conjugates provoked an anti-O-antigen response primarily against the O:5 determinant. O-antigen from S. Enteritidis was structurally more homogeneous than from S. Typhimurium, and no idiosyncratic antibody responses were detected for the S. Enteritidis conjugates. Of the three initially selected isolates, two S. Typhimurium (1418 and 2189) and two S. Enteritidis (502 and 618) strains generated glycoconjugates able to induce high specific antibody levels with high breadth of serovar-specific strain coverage, and were selected for use in vaccine production. The strain selection approach described is potentially applicable to the development of glycoconjugate vaccines against other bacterial pathogens. PMID:26445460

Leishmania cell wall as a potent target for antiparasitic drugs. A focus on the glycoconjugates.

PubMed

Cabezas, Yari; Legentil, Laurent; Robert-Gangneux, Florence; Daligault, Franck; Belaz, Sorya; Nugier-Chauvin, Caroline; Tranchimand, Sylvain; Tellier, Charles; Gangneux, Jean-Pierre; Ferrières, Vincent

2015-08-21

Although leishmaniasis has been studied for over a century, the fight against cutaneous, mucocutaneous and visceral forms of the disease remains a hot topic. This review refers to the parasitic cell wall and more particularly to the constitutive glycoconjugates. The structures of the main glycolipids and glycoproteins, which are species-dependent, are described. The focus is on the disturbance of the lipid membrane by existing drugs and possible new ones, in order to develop future therapeutic agents.

Hijacking bacterial glycosylation for the production of glycoconjugates, from vaccines to humanised glycoproteins.

PubMed

Cuccui, Jon; Wren, Brendan

2015-03-01

Glycosylation or the modification of a cellular component with a carbohydrate moiety has been demonstrated in all three domains of life as a basic post-translational process important in a range of biological processes. This review will focus on the latest studies attempting to exploit bacterial N-linked protein glycosylation for glycobiotechnological applications including glycoconjugate vaccine and humanised glycoprotein production. The challenges that remain for these approaches to reach full biotechnological maturity will be discussed. Oligosaccharyltransferase-dependent N-linked glycosylation can be exploited to make glycoconjugate vaccines against bacterial pathogens. Few technical limitations remain, but it is likely that the technologies developed will soon be considered a cost-effective and flexible alternative to current chemical-based methods of vaccine production. Some highlights from current glycoconjugate vaccines developed using this in-vivo production system include a vaccine against Shigella dysenteriae O1 that has passed phase 1 clinical trials, a vaccine against the tier 1 pathogen Francisella tularensis that has shown efficacy in mice and a vaccine against Staphylococcus aureus serotypes 5 and 8. Generation of humanised glycoproteins within bacteria was considered impossible due to the distinct nature of glycan modification in eukaryotes and prokaryotes. We describe the method used to overcome this conundrum to allow engineering of a eukaryotic pentasaccharide core sugar modification within Escherichia coli. This core was assembled by combining the function of the initiating transferase WecA, several Alg genes from Saccharomyces cerevisiae and the oligosaccharyltransferase function of the Campylobacter jejuni PglB. Further exploitation of a cytoplasmic N-linked glycosylation system found in Actinobacillus pleuropneumoniae where the central enzyme is known as N-linking glycosyltransferase has overcome some of the limitations demonstrated by the oligosaccharyltransferase-dependent system. Characterisation of the first bacterial N-linked glycosylation system in the human enteropathogen Campylobacter jejuni has led to substantial biotechnological applications. Alternative methods for glycoconjugate vaccine production have been developed using this N-linked system. Vaccines against both Gram-negative and Gram-positive organisms have been developed, and efficacy testing has thus far demonstrated that the vaccines are safe and that robust immune responses are being detected. These are likely to complement and reduce the cost of current technologies thus opening new avenues for glycoconjugate vaccines. These new markets could potentially include glycoconjugate vaccines tailored specifically for animal vaccination, which has until today thought to be non-viable due to the cost of current in-vitro chemical conjugation methods. Utilisation of N-linked glycosylation to generate humanised glycoproteins is also close to becoming reality. This 'bottom up' assembly mechanism removes the heterogeneity seen in current humanised products. The majority of developments reported in this review exploit a single N-linked glycosylation system from Campylobacter jejuni; however, alternative N-linked glycosylation systems have been discovered which should help to overcome current technical limitations and perhaps more systems remain to be discovered. The likelihood is that further glycosylation systems exist and are waiting to be exploited. © 2014 Royal Pharmaceutical Society.

Lectin histochemistry of enterocytes sugar residues in the gut of the chick embryo and of the newborn.

PubMed

Bryk, S Gheri; Gheri, G

2002-01-01

The glycoconjugates sugar residues content, distribution and changes in the enterocytes of different tracts of the developing intestine of the chick embryo and of 1-day-old chick were investigated, using a battery of seven HRP-conjugated lectins (DBA, SBA, PNA, WGA, ConA, LTA and UEAI). The results of the present research have shown the presence of a large amount of glycoconjugates sugar residues in the enterocytes of duodenal, ileal and colonic anlage, starting from the beginning of the second week of incubation. Differences were detected among the three investigated intestinal segments, as to the time of appearance of the glycoconjugates sugar residues in the enterocytes. The duodenal enterocytes showed the most precocious appearance of lectin-reactive material, followed by the ileal enterocytes and afterwards by colonic enterocytes. The duodenal enterocytes were characterised by the presence of SBA binding sites, which were not detectable in the duodenal enterocytes of the adult animal.

ASGPR-Mediated Uptake of Multivalent Glycoconjugates for Drug Delivery in Hepatocytes.

PubMed

Monestier, Marie; Charbonnier, Peggy; Gateau, Christelle; Cuillel, Martine; Robert, Faustine; Lebrun, Colette; Mintz, Elisabeth; Renaudet, Olivier; Delangle, Pascale

2016-04-01

Liver cells are an essential target for drug delivery in many diseases. The hepatocytes express the asialoglycoprotein receptor (ASGPR), which promotes specific uptake by means of N-acetylgalactosamine (GalNAc) recognition. In this work, we designed two different chemical architectures to treat Wilson's disease by intracellular copper chelation. Two glycoconjugates functionalized with three or four GalNAc units each were shown to enter hepatic cells and chelate copper. Here, we studied two series of compounds derived from these glycoconjugates to find key parameters for the targeting of human hepatocytes. Efficient cellular uptake was demonstrated by flow cytometry using HepG2 human heptic cells that express the human oligomeric ASGPR. Dissociation constants in the nanomolar range showed efficient multivalent interactions with the receptor. Both architectures were therefore concluded to be able to compete with endogeneous asialoglycoproteins and serve as good vehicles for drug delivery in hepatocytes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The influence of conjugation variables on the design and immunogenicity of a glycoconjugate vaccine against Salmonella Typhi

PubMed Central

Arcuri, M.; Di Benedetto, R.; Cunningham, A. F.; Saul, A.; MacLennan, C. A.

2017-01-01

In recent years there have been major efforts to develop glycoconjugate vaccines based on the Vi polysaccharide that will protect against Salmonella enterica Typhi infections, particularly typhoid fever, which remains a major public health concern in low-income countries. The design of glycoconjugate vaccines influences the immune responses they elicit. Here we systematically test the response in mice to Vi glycoconjugates that differ in Vi chain length (full-length and fragmented), carrier protein, conjugation chemistry, saccharide to protein ratio and size. We show that the length of Vi chains, but not the ultimate size of the conjugate, has an impact on the anti-Vi IgG immune response induced. Full-length Vi conjugates, independent of the carrier protein, induce peak IgG responses rapidly after just one immunization, and secondary immunization does not enhance the magnitude of these responses. Fragmented Vi linked to CRM197 and diphtheria toxoid, but not to tetanus toxoid, gives lower anti-Vi antibody responses after the first immunization than full-length Vi conjugates, but antibody titres are similar to those induced by full-length Vi conjugates following a second dose. The chemistry to conjugate Vi to the carrier protein, the linker used, and the saccharide to protein ratio do not significantly alter the response. We conclude that Vi length and carrier protein are the variables that influence the anti-Vi IgG response to immunization the most, while other parameters are of lesser importance. PMID:29287062

Immunobiological Activity of Synthetically Prepared Immunodominant Galactomannosides Structurally Mimicking Aspergillus Galactomannan

PubMed Central

Paulovičová, Ema; Paulovičová, Lucia; Hrubiško, Martin; Krylov, Vadim B.; Argunov, Dmitry A.; Nifantiev, Nikolay E.

2017-01-01

The study is oriented at the in vitro evaluation of the immunobiological activity and efficacy of synthetically prepared isomeric pentasaccharides representing fragments of Aspergillus fumigatus cell-wall galactomannan and containing β-(1→5)-linked tetragalactofuranoside chain attached to O-6 (GM-1) or O-3 (GM-2) of a spacer-armed mannopyranoside residue. These compounds were studied as biotinylated conjugates which both demonstrated immunomodulatory activities on the RAW 264.7 cell line murine macrophages as in vitro innate immunity cell model. Immunobiological studies revealed time- and concentration-dependent efficient immunomodulation. The proliferation of RAW 264.7 macrophages was induced at higher concentration (100 µg/mL) of studied glycoconjugates and longer exposure (48 h), with more pronounced efficacy for GM-1. The increase of proliferation followed the previous increase of IL-2 production. The cytokine profile of the macrophages treated with the glycoconjugates was predominantly pro-inflammatory Th1 type with significant increase of TNFα, IL-6, and IL-12 release for both glycoconjugates. The RAW 264.7 macrophages production of free radicals was not significantly affected by glycoconjugates stimulation. The phagocytic activity of RAW 264.7 cells was reduced following GM-1 treatment and was significantly increased after 24 h stimulation with GM-2, contrary to 48 h stimulation. Moreover, the synthetically prepared galactomannoside derivatives have been evaluated as efficient serodiagnostic antigens recognized by specific Ig isotypes, and significant presence of specific IgM antibodies in serum of patients suffering from vulvovaginitis was observed. PMID:29081774

Application of enzymes, sodium tripolyphosphate and cation exchange resin for the release of extracellular polymeric substances from sewage sludge. Characterization of the extracted polysaccharides/glycoconjugates by a panel of lectins.

PubMed

Wawrzynczyk, J; Szewczyk, E; Norrlöw, O; Dey, E Szwajcer

2007-06-30

The study describes extraction of extracellular polymeric substances (EPS) from sewage sludge by applying enzymes and enzymes combined with sodium tripolyphosphate (STPP). Additionally, a systematic study of two non-enzymatic extraction agents is described. The assessment of the released products is made by colorimetrical methods and polysaccharides/glycoconjugates identification by the interaction with four immobilized lectins. Bio-sludge from Helsingborg (Sweden) and Damhusåen (Denmark) were used as two case studies for testing enzymatic extractability and thereby to make useful prediction of sludge bio-digestibility. From Helsingborg sludge the enzymes extracted about 40% more of EPS than from Damhusåen. The polysaccharides/glycoconjugates in both sludges maintained the same level, and showed substantial different interaction motifs with lectins panel. Damhusåen enzymatic extracted EPS had an enhanced amount of suspended material that was post-hydrolysed by the use of polygalacturonase and lysozyme resulting in pectin like polymers and petiptidoglycans. Petiptidoglycan is a marker from bacterial cell debris. STPP and cation exchange resin (CER) released different quantities of EPS. The CER released polysaccharides/glycoconjugates had higher molecular weight and stronger affinity towards Concanavalin A than the one released by the action of STPP. Independent of the extraction conditions, STPP released elevated amounts of polyvalent cations and humic substances in contrast to the very low amounts of released by CER.

Glycoconjugate Vaccines: The Regulatory Framework.

PubMed

Jones, Christopher

2015-01-01

Most vaccines, including the currently available glycoconjugate vaccines, are administered to healthy infants, to prevent future disease. The safety of a prospective vaccine is a key prerequisite for approval. Undesired side effects would not only have the potential to damage the individual infant but also lead to a loss of confidence in the respective vaccine-or vaccines in general-on a population level. Thus, regulatory requirements, particularly with regard to safety, are extremely rigorous. This chapter highlights regulatory aspects on carbohydrate-based vaccines with an emphasis on analytical approaches to ensure the consistent quality of successive manufacturing lots.

Histochemical analysis of glycoconjugates in the skin of a catfish (arius tenuispinis, day).

PubMed

Al-Banaw, A; Kenngott, R; Al-Hassan, J M; Mehana, N; Sinowatz, F

2010-02-01

A histochemical study using conventional carbohydrate histochemistry (periodic-acid staining including diastase controls, alcian blue staining at pH 1 and 2.5) as well as using a battery of 14 fluorescein isothiocyanate (FITC)-labelled lectins to identify glycoconjugates present in 10 different areas of the skin of a catfish (Arius tenuispinis) was carried out. The lectins used were: mannose-binding lectins (Con A, LCA and PSA), galactose-binding lectins (PNA, RCA), N-acetylgalactosamine-binding lectins (DBA, SBA, SJA and GSL I), N-acetylglucosamine-binding lectins (WGA and WGAs), fucose-binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E, PHA L). Conventional glycoconjugate staining (PAS staining, alcian blue at pH 1 and 2.5) showed that the mucous goblet cells contain a considerable amount of glycoconjugates in all locations of the skin, whereas the other unicellular gland type, the club cells, lacked these glycoconjugates. The glycoproteins found in goblet cells are neutral and therefore stain magenta when subjected to PAS staining. Alcian blue staining indicating acid glycoproteins was distinctly positive at pH 1, but gave only a comparable staining at pH 2.5. The mucus of the goblet cells therefore also contains acid glycoproteins rich in sulphate groups. Using FITC-labelled lectins, the carbohydrate composition of the glycoproteins of goblet cells could be more fully characterized. A distinct staining of the mucus of goblet cells was found with the mannose-binding lectins LCA and PSA; the galactosamine-binding lectins DBA, SBA and GLS I; the glucosamine-binding lectin WGA; and PHA E which stains glycoproteins with complex carbohydrate configurations. No reaction occurred with the fucose-binding lectin UEA and the sialic acid-specific lectin SNA. In addition, the galactose-binding lectins PNA and RCA showed only a weak or completely negative staining of the mucus in the goblet cells. The specificity of the lectin staining could be proved by inhibiting binding of the lectins by competitive inhibition with the corresponding sugars. From these data, we can conclude that the mucus produced by the epidermal goblet cells of A. tenuispinis is rich in mannose, N-acetylgalactosamine and N-acetylglucosamine residues.

Human milk glycoconjugates that inhibit pathogens.

PubMed

Newburg, D S

1999-02-01

Breast-fed infants have lower incidence of diarrhea, respiratory disease, and otitis media. The protection by human milk has long been attributed to the presence of secretory IgA. However, human milk contains large numbers and amounts of complex carbohydrates, including glycoproteins, glycolipids, glycosaminoglycans, mucins, and especially oligosaccharides. The oligosaccharides comprise the third most abundant solid constituent of human milk, and contain a myriad of structures. Complex carbohydrate moieties of glycoconjugates and oligosaccharides are synthesized by the many glycosyltransferases in the mammary gland; those with homology to cell surface glycoconjugate pathogen receptors may inhibit pathogen binding, thereby protecting the nursing infant. Several examples are reviewed: A fucosyloligosaccharide inhibits the diarrheagenic effect of stable toxin of Escherichia coli. A different fucosyloligosaccharide inhibits infection by Campylobacter jejuni. Binding of Streptococcus pneumoniae and of enteropathogenic E. coli to their respective receptors is inhibited by human milk oligosaccharides. The 46-kD glycoprotein, lactadherin, inhibits rotavirus binding and infectivity. Low levels of lactadherin in human milk are associated with a higher incidence of symptomatic rotavirus in breast-fed infants. A mannosylated glycopeptide inhibits binding by enterohemorrhagic E. coli. A glycosaminoglycan inhibits binding of gp120 to CD4, the first step in HIV infection. Human milk mucin inhibits binding by S-fimbriated E. coli. The ganglioside, GM1, reduces diarrhea production by cholera toxin and labile toxin of E. coli. The neutral glycosphingolipid, Gb3, binds to Shigatoxin. Thus, many complex carbohydrates of human milk may be novel antipathogenic agents, and the milk glycoconjugates and oligosaccharides may be a major source of protection for breastfeeding infants.

Resolvin D1 Increases Mucin Secretion in Cultured Rat Conjunctival Goblet Cells via Multiple Signaling Pathways

PubMed Central

Lippestad, Marit; Hodges, Robin R.; Utheim, Tor P.; Serhan, Charles N.; Dartt, Darlene A.

2017-01-01

Purpose Goblet cells in the conjunctiva secrete mucin into the tear film protecting the ocular surface. The proresolution mediator resolvin D1 (RvD1) regulates mucin secretion to maintain homeostasis during physiological conditions and in addition, actively terminates inflammation. We determined the signaling mechanisms used by RvD1 in cultured rat conjunctival goblet cells to increase intracellular [Ca2+] ([Ca2+]i) and induce glycoconjugate secretion. Methods Increase in [Ca2+]i were measured using fura 2/AM and glycoconjugate secretion determined using an enzyme-linked lectin assay with the lectin Ulex Europaeus Agglutinin 1. Signaling pathways activated by RvD1 were studied after goblet cells were pretreated with signaling pathway inhibitors before stimulation with RvD1. The results were compared with results when goblet cells were stimulated with RvD1 alone and percent inhibition calculated. Results The increase in [Ca2+]i stimulated by RvD1 was blocked by inhibitors to phospholipases (PL-) -D, -C, -A2, protein kinase C (PKC), extracellular signal-regulated kinases (ERK)1/2 and Ca2+/calmodulin-dependent kinase (Ca2+/CamK). Glycoconjugate secretion was significantly inhibited by PLD, -C, -A2, ERK1/2 and Ca2+/CamK, but not PKC. Conclusions We conclude that RvD1 increases glycoconjugate secretion from goblet cells via multiple signaling pathways including PLC, PLD, and PLA2, as well as their signaling components ERK1/2 and Ca2+/CamK to preserve the mucous layer and maintain homeostasis by protecting the eye from desiccating stress, allergens, and pathogens. PMID:28892824

Study of chemical stability of lemon oil components in sodium caseinate-lactose glycoconjugate-stabilized oil-in-water emulsions using solid-phase microextraction-gas chromatography.

PubMed

Sabik, Hassan; Achouri, Allaoua; Alfaro, Maria; Pelletier, Marylène; Belanger, Denis; Britten, Michel; Fustier, Patrick

2014-07-25

A headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC/MS) method was developed to quantify lemon oil components and their degradation products in oil-in-water (O/W) emulsions prepared with sodium caseinate-heated-lactose (NaC-T + Lact) glycoconjugates as wall materials at two pH values (3.0 and 6.8). NaC-T + Lact conjugates had a significantly lower solubility at both pHs. Hydrolysis prior to glycation enhanced the solubility of glycoconjugates. Glycation with lactose did not improve the emulsion activity of NaC, while caseinate glycoconjugates showed much stronger antioxidant activity than the NaC-control sample. This might be due to the presence of melanoidins formed between the sugar and amino acid compounds as supported by the increase in browning intensity. Among the SPME-fibres tested, carboxen/polydimethylsiloxane (CAR/PDMS) provided better results in terms of sensitivity and selectivity for oil lemon components and their degradation products. Storage studies of these emulsions demonstrated that glycated NaC-T + Lact showed protection against peroxidation compared to the control. However, acidic pH conditions altered their stability over storage time. The major off-flavor components (α-terpineol and carvone) were inhibited in emulsions stabilized with glycated NaC, particularly at pH 6.8. The use of NaC-T + Lact conjugates showed improved encapsulation efficiency and stability and could be used as potential food ingredient-emulsifiers for stabilising citrus oils against oxidative degradation in food and beverage applications.

Contribution of several volatile phenols and their glycoconjugates to smoke-related sensory properties of red wine.

PubMed

Parker, Mango; Osidacz, Patricia; Baldock, Gayle A; Hayasaka, Yoji; Black, Cory A; Pardon, Kevin H; Jeffery, David W; Geue, Jason P; Herderich, Markus J; Francis, I Leigh

2012-03-14

Guaiacol and 4-methylguaiacol are well-known as contributors to the flavor of wines made from smoke-affected grapes, but there are other volatile phenols commonly found in smoke from forest fires that are also potentially important. The relationships between the concentration of a range of volatile phenols and their glycoconjugates with the sensory characteristics of wines and model wines were investigated. Modeling of the attribute ratings from a sensory descriptive analysis of smoke-affected wines with their chemical composition indicated the concentrations of guaiacol, o-cresol, m-cresol, and p-cresol were related to smoky attributes. The best-estimate odor thresholds of these compounds were determined in red wine, together with the flavor threshold of guaiacol. Guaiacol β-D-glucoside and m-cresol β-D-glucoside in model wine were found to give rise to a smoky/ashy flavor in-mouth, and the respective free volatiles were released. The study indicated that a combination of volatile phenols and their glycosides produces an undesirable smoke flavor in affected wines. The observation of flavor generation from nonvolatile glycoconjugates in-mouth has potentially important implications.

Analytical Scheme Leading to Integrated High-Sensitivity Profiling of Glycosphingolipids Together with N- and O-Glycans from One Sample

NASA Astrophysics Data System (ADS)

Benktander, John D.; Gizaw, Solomon T.; Gaunitz, Stefan; Novotny, Milos V.

2018-05-01

Glycoconjugates are directly or indirectly involved in many biological processes. Due to their complex structures, the structural elucidation of glycans and the exploration of their role in biological systems have been challenging. Glycan pools generated through release from glycoprotein or glycolipid mixtures can often be very complex. For the sake of procedural simplicity, many glycan profiling studies choose to concentrate on a single class of glycoconjugates. In this paper, we demonstrate it feasible to cover glycosphingolipids, N-glycans, and O-glycans isolated from the same sample. Small volumes of human blood serum and ascites fluid as well as small mouse brain tissue samples are sufficient to profile sequentially glycans from all three classes of glycoconjugates and even positively identify some mixture components through MALDI-MS and LC-ESI-MS. The results show that comprehensive glycan profiles can be obtained from the equivalent of 500-μg protein starting material or possibly less. These methodological improvements can help accelerating future glycomic comprehensive studies, especially for precious clinical samples.

The metabolism of galactose in the human gastric mucous membrane.

PubMed

Kopacz-Jodczyk, T; Zwierz, K; Gałasiński, W

1984-12-01

After incubating pieces of human gastric mucous membrane with radioactive galactose, labeled metabolites of glycolysis (FDP,PEP,pyruvate):hexose and hexosamine intermediates in glycoconjugate biosynthesis (gal-1P, UDP-gal,acetylated hexosamines, and their phosphate esters), amino acids (glycine, alanine, and serine), and oxoglutarate as a metabolite of the citric acid cycle were isolated from the acid-soluble fraction. These results suggest that galactose in the human gastric mucous membrane is epimerized to glucose and metabolized in the glycolytic pathway together with oxidation in the citric acid cycle and in the direction of glycoconjugate biosynthesis.

Structural studies of novel glycoconjugates from polymerized allergens (allergoids) and mannans as allergy vaccines.

PubMed

Manzano, Ana I; Javier Cañada, F; Cases, Bárbara; Sirvent, Sofia; Soria, Irene; Palomares, Oscar; Fernández-Caldas, Enrique; Casanovas, Miguel; Jiménez-Barbero, Jesús; Subiza, José L

2016-02-01

Immunotherapy for treating IgE-mediated allergies requires high doses of the corresponding allergen. This may result in undesired side effects and, to avoid them, hypoallergenic allergens (allergoids) polymerized with glutaraldehyde are commonly used. Targeting allergoids to dendritic cells to enhance cell uptake may result in a more effective immunotherapy. Allergoids coupled to yeast mannan, as source of polymannoses, would be suitable for this purpose, since mannose-binding receptors are expressed on these cells. Conventional conjugation procedures of mannan to proteins use oxidized mannan to release reactive aldehydes able to bind to free amino groups in the protein; yet, allergoids lack these latter because their previous treatment with glutaraldehyde. The aim of this study was to obtain allergoids conjugated to mannan by an alternative approach based on just glutaraldehyde treatment, taking advantage of the mannoprotein bound to the polymannose backbone. Allergoid-mannan glycoconjugates were produced in a single step by treating with glutaraldehyde a defined mixture of allergens derived from Phleum pratense grass pollen and native mannan (non-oxidized) from Saccharomyces cerevisae. Analytical and structural studies, including 2D-DOSY and (1)H-(13)C HSQC nuclear magnetic resonance spectra, demonstrated the feasibility of such an approach. The glycoconjugates obtained were polymers of high molecular weight showing a higher stability than the native allergen or the conventional allergoid without mannan. The allergoid-mannan glycoconjugates were hypoallergenic as detected by the IgE reactivity with sera from grass allergic patients, even with lower reactivity than conventional allergoid without mannan. Thus, stable hypoallergenic allergoids conjugated to mannan suitable for using in immunotherapy can be achieved using glutaraldehyde. In contrast to mannan oxidation, the glutaraldehyde approach allows to preserve mannoses with their native geometry, which may be functionally important for its receptor-mediated recognition.

Protection against Streptococcus suis Serotype 2 Infection Using a Capsular Polysaccharide Glycoconjugate Vaccine

PubMed Central

Calzas, Cynthia; Shiao, Tze Chieh; Neubauer, Axel; Kempker, Jennifer; Roy, René; Gottschalk, Marcelo

2016-01-01

Streptococcus suis serotype 2 is an encapsulated bacterium and one of the most important bacterial pathogens in the porcine industry. Despite decades of research for an efficient vaccine, none is currently available. Based on the success achieved with other encapsulated pathogens, a glycoconjugate vaccine strategy was selected to elicit opsonizing anti-capsular polysaccharide (anti-CPS) IgG antibodies. In this work, glycoconjugate prototypes were prepared by coupling S. suis type 2 CPS to tetanus toxoid, and the immunological features of the postconjugation preparations were evaluated in vivo. In mice, experiments evaluating three different adjuvants showed that CpG oligodeoxyribonucleotide (ODN) induces very low levels of anti-CPS IgM antibodies, while the emulsifying adjuvants Stimune and TiterMax Gold both induced high levels of IgGs and IgM. Dose-response trials comparing free CPS with the conjugate vaccine showed that free CPS is nonimmunogenic independently of the dose used, while 25 μg of the conjugate preparation was optimal in inducing high levels of anti-CPS IgGs postboost. With an opsonophagocytosis assay using murine whole blood, sera from immunized mice showed functional activity. Finally, the conjugate vaccine showed immunogenicity and induced protection in a swine challenge model. When conjugated and administered with emulsifying adjuvants, S. suis type 2 CPS is able to induce potent IgM and isotype-switched IgGs in mice and pigs, yielding functional activity in vitro and protection against a lethal challenge in vivo, all features of a T cell-dependent response. This study represents a proof of concept for the potential of glycoconjugate vaccines in veterinary medicine applications against invasive bacterial infections. PMID:27113360

Carbohydrates in Supramolecular Chemistry.

PubMed

Delbianco, Martina; Bharate, Priya; Varela-Aramburu, Silvia; Seeberger, Peter H

2016-02-24

Carbohydrates are involved in a variety of biological processes. The ability of sugars to form a large number of hydrogen bonds has made them important components for supramolecular chemistry. We discuss recent advances in the use of carbohydrates in supramolecular chemistry and reveal that carbohydrates are useful building blocks for the stabilization of complex architectures. Systems are presented according to the scaffold that supports the glyco-conjugate: organic macrocycles, dendrimers, nanomaterials, and polymers are considered. Glyco-conjugates can form host-guest complexes, and can self-assemble by using carbohydrate-carbohydrate interactions and other weak interactions such as π-π interactions. Finally, complex supramolecular architectures based on carbohydrate-protein interactions are discussed.

The oligosaccharidic content of the glycoconjugates of the prepubertal descended and undescended testis: lectin histochemical study.

PubMed

Gheri, Gherardo; Sgambati, Eleonora; Thyrion, Giorgia D Zappoli; Vichi, Debora; Orlandini, Giovanni E

2004-01-01

The saccharidic content of the glycoconjugates has been studied in the descended the undescended testes of a 8 years old boy. For this purpose, a battery of seven HRP-conjugated lectins (SBA, DBA,PNA,WGA,UEAI, LTA and ConA) was used. D-galactose-N-acetyl-D-galactosamine and alpha-L-fucose sugar residues, which were present in the cytoplasm of the Sertoli cells of the normally positioned prepubertal testis, were not detected in the same cells of the undescended testis. The Leydig's cells of the descended testis appeared characterized by N-acetyl-D-glucosamine which was absent in the rare and atrophic Leydig's cells of the cryptorchid testis. Differences in sugar residues distribution between the descended and the undescended testis were also detected in the lamina propria of the seminiferous tubules. Peritubular myoid cells in the undescended testis only reacted with PNA, after neuraminidase digestion, thus revealing the presence of D-galactose (beta1-->3)-N-acetyl-D-galactosamine and sialic acid. In this study a complete distributional map of the sugar residues of the glycoconjugates in the descended and undescended prepubertal testis is reported.

Lectin-binding histochemistry of non-decalcified growth plate cartilage: a postembedment method for light microscopy of epon-embedded tissue.

PubMed

Farnum, C E; Wilsman, N J

1984-06-01

A postembedment method for the localization of lectin-binding glycoconjugates was developed using Epon-embedded growth plate cartilage from Yucatan miniature swine. By testing a variety of etching, blocking, and incubation procedures, a standard protocol was developed for 1 micron thick sections that allowed visualization of both intracellular and extracellular glycoconjugates with affinity for wheat germ agglutinin and concanavalin A. Both fluorescent and peroxidase techniques were used, and comparisons were made between direct methods and indirect methods using the biotin-avidin bridging system. Differential extracellular lectin binding allowed visualization of interterritorial , territorial, and pericellular matrices. Double labeling experiments showed the precision with which intracellular binding could be localized to specific cytoplasmic compartments, with resolution of binding to the Golgi apparatus, endoplasmic reticulum, and nuclear membrane at the light microscopic level. This method allows the localization of both intracellular and extracellular lectin-binding glycoconjugates using fixation and embedment procedures that are compatible with simultaneous ultrastructural analysis. As such it should have applicability both to the morphological analysis of growth plate organization during normal endochondral ossification, as well as to the diagnostic pathology of matrix abnormalities in disease states of growing cartilage.

Distribution of binding sites for the plant lectin Ulex europaeus agglutinin I on primary sensory neurones in seven different mammalian species.

PubMed

Gerke, Michelle B; Plenderleith, Mark B

2002-01-01

There is an increasing body of evidence to suggest that different functional classes of neurones express characteristic cell-surface carbohydrates. Previous studies have shown that the plant lectin Ulex europaeus agglutinin-I (UEA) binds to a population of small to medium diameter primary sensory neurones in rabbits and humans. This suggests that a fucose-containing glycoconjugate may be expressed by nociceptive primary sensory neurones. In order to determine the extent to which this glycoconjugate is expressed by other species, in the current study, we have examined the distribution of UEA-binding sites on primary sensory neurones in seven different mammals. Binding sites for UEA were associated with the plasma membrane and cytoplasmic granules of small to medium dorsal root ganglion cells and their axon terminals in laminae I-III of the grey matter of the spinal cord, in the rabbit, cat and marmoset monkey. However, no binding was observed in either the dorsal root ganglia or spinal cord in the mouse, rat, guinea pig or flying fox. These results indicate an inter-species variation in the expression of cell-surface glycoconjugates on mammalian primary sensory neurones.

Emerging facets of prokaryotic glycosylation

PubMed Central

Schäffer, Christina; Messner, Paul

2017-01-01

Glycosylation of proteins is one of the most prevalent post-translational modifications occurring in nature, with a wide repertoire of biological implications. Pathways for the main types of this modification, the N- and O-glycosylation, can be found in all three domains of life—the Eukarya, Bacteria and Archaea—thereby following common principles, which are valid also for lipopolysaccharides, lipooligosaccharides and glycopolymers. Thus, studies on any glycoconjugate can unravel novel facets of the still incompletely understood fundamentals of protein N- and O-glycosylation. While it is estimated that more than two-thirds of all eukaryotic proteins would be glycosylated, no such estimate is available for prokaryotic glycoproteins, whose understanding is lagging behind, mainly due to the enormous variability of their glycan structures and variations in the underlying glycosylation processes. Combining glycan structural information with bioinformatic, genetic, biochemical and enzymatic data has opened up an avenue for in-depth analyses of glycosylation processes as a basis for glycoengineering endeavours. Here, the common themes of glycosylation are conceptualised for the major classes of prokaryotic (i.e. bacterial and archaeal) glycoconjugates, with a special focus on glycosylated cell-surface proteins. We describe the current knowledge of biosynthesis and importance of these glycoconjugates in selected pathogenic and beneficial microbes. PMID:27566466

Protection of non-human primates against glanders with a gold nanoparticle glycoconjugate vaccine

PubMed Central

Torres, Alfredo G.; Gregory, Anthony E.; Hatcher, Christopher L.; Vinet-Oliphant, Heather; Morici, Lisa A.; Titball, Richard W.; Roy, Chad J.

2014-01-01

The Gram-negative Burkholderia mallei is a zoonotic pathogen and the causative agent of glanders disease. Because the bacteria maintain the potential to be used as a biothreat agent, vaccine strategies are required for human glanders prophylaxis. A rhesus macaque (Macaca mulatta) model of pneumonic (inhalational) glanders was established and the protective properties of a nanoparticle glycoconjugate vaccine composed of B. thailandensis LPS conjugated to FliC was evaluated. An aerosol challenge dose of ~1×104 CFU B. mallei produced mortality in 50% of naïve animals (n = 2/4), 2–3 days post-exposure. Although survival benefit was not observed by vaccination with a glycoconjugate glanders vaccine (p=0.42), serum LPS-specific IgG titres were significantly higher on day 80 in 3 vaccinated animals who survived compared with 3 vaccinated animals who died. Furthermore, B. mallei was isolated from multiple organs of both non-vaccinated survivors, but not from any organs of 3 vaccinated survivors at 30 days post-challenge. Taken together, this is the first time a candidate vaccine has been evaluated in a non-human primate aerosol model of glanders and represents the initial step for consideration in pre-clinical studies. PMID:25533326

Potential targets for next generation antimicrobial glycoconjugate vaccines

PubMed Central

Micoli, Francesca; Costantino, Paolo; Adamo, Roberto

2018-01-01

Abstract Cell surface carbohydrates have been proven optimal targets for vaccine development. Conjugation of polysaccharides to a carrier protein triggers a T-cell-dependent immune response to the glycan moiety. Licensed glycoconjugate vaccines are produced by chemical conjugation of capsular polysaccharides to prevent meningitis caused by meningococcus, pneumococcus and Haemophilus influenzae type b. However, other classes of carbohydrates (O-antigens, exopolysaccharides, wall/teichoic acids) represent attractive targets for developing vaccines. Recent analysis from WHO/CHO underpins alarming concern toward antibiotic-resistant bacteria, such as the so called ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) and additional pathogens such as Clostridium difficile and Group A Streptococcus. Fungal infections are also becoming increasingly invasive for immunocompromised patients or hospitalized individuals. Other emergencies could derive from bacteria which spread during environmental calamities (Vibrio cholerae) or with potential as bioterrorism weapons (Burkholderia pseudomallei and mallei, Francisella tularensis). Vaccination could aid reducing the use of broad-spectrum antibiotics and provide protection by herd immunity also to individuals who are not vaccinated. This review analyzes structural and functional differences of the polysaccharides exposed on the surface of emerging pathogenic bacteria, combined with medical need and technological feasibility of corresponding glycoconjugate vaccines. PMID:29547971

Ultrastructure and Glycoconjugate Pattern of the Foot Epithelium of the Abalone Haliotis tuberculata (Linnaeus, 1758) (Gastropoda, Haliotidae)

PubMed Central

Bravo Portela, I.; Martinez-Zorzano, V. S.; Molist- Perez, I.; Molist García, P.

2012-01-01

The foot epithelium of the gastropod Haliotis tuberculata is studied by light and electron microscopy in order to contribute to the understanding of the anatomy and functional morphology of the mollusks integument. Study of the external surface by scanning electron microscopy reveals that the side foot epithelium is characterized by a microvillus border with a very scant presence of small ciliary tufts, but the sole foot epithelium bears a dense field of long cilia. Ultrastructural examination by transmission electron microscopy of the side epithelial cells shows deeply pigmented cells with high electron-dense granular content which are not observed in the epithelial sole cells. Along the pedal epithelium, seven types of secretory cells are present; furthermore, two types of subepithelial glands are located just in the sole foot. The presence and composition of glycoconjugates in the secretory cells and subepithelial glands are analyzed by conventional and lectin histochemistry. Subepithelial glands contain mainly N-glycoproteins rich in fucose and mannose whereas secretory cells present mostly acidic sulphated glycoconjugates such as glycosaminoglycans and mucins, which are rich in galactose, N-acetyl-galactosamine, and N-acetyl-glucosamine. No sialic acid is present in the foot epithelium. PMID:22645482

Designer vaccines to prevent infections due to group B Streptococcus.

PubMed

Kasper, D L

1995-10-01

Group B streptococci (GBS) are the major cause of serious infections in neonates and an important cause of infection in adults, particularly peripartum women and patients with diabetes mellitus and malignancy. Immunity to GBS in neonates is associated with naturally acquired maternal antibodies to the type-specific capsular polysaccharides of these organisms. IgG class antibodies directed to these polysaccharides are passed transplacentally and protect the child from invasive GBS disease. Phase I and II clinical trials showed that the purified polysaccharides had limited immunogenicity. However, vaccine responders passed functional IgG class antibodies to their children. A glycoconjugate vaccine has been designed so that the type-specific polysaccharides are covalently linked to a carrier protein. This secondary amine linkage is between aldehyde groups created on the eighth carbon of a selected number of periodate-oxidized sialic acid residues of the polysaccharide and epsilon-amino groups on lysine residues of tetanus toxoid. Careful epitope mapping studies had demonstrated that modification by controlled periodate oxidation could be accomplished and that an important conformational epitope on the polysaccharide would be preserved. Preclinical testing of the glycoconjugate vaccines in animal models of GBS disease demonstrated the immunogenicity and protective efficacy of the vaccine-induced antibodies. Phase I clinical testing of the glycoconjugate vaccine is in progress, and the early results appear promising.

Structural characterization of bovine beta-lactoglobulin-galactose/tagatose Maillard complexes by electrophoretic, chromatographic, and spectroscopic methods.

PubMed

Corzo-Martínez, Marta; Moreno, F Javier; Olano, Agustín; Villamiel, Mar

2008-06-11

To investigate the influence of the type of carbonyl group of the sugar on the structural changes of proteins during glycation, an exhaustive structural characterization of glycated beta-lactoglobulin with galactose (aldose) and tagatose (ketose) has been carried out. Conjugates were prepared via Maillard reaction at 40 and 50 degrees C, pH 7, and a w = 0.44. The progress of the Maillard reaction was followed by indirect formation of Amadori and Heyns compounds, advanced glycation end products, and brown polymers. The structural characterization of glycoconjugates was conducted by using a number of analytical techniques such as RP-HPLC, isoelectric focusing, MALDI-ToF, SDS-PAGE, size exclusion chromatography, and spectrofluorimetry (tryptophan fluorescence). In addition, the surface hydrophobicity of the beta-lactoglobulin glycoconjugates was also assessed. The results showed a higher reactivity of galactose than tagatose to form the glycoconjugates, probably due to the higher electrophilicity of the aldehyde group. At 40 degrees C, more aggregation was produced when beta-lactoglobulin was conjugated with tagatose as compared to galactose. However, at 50 degrees C hardly any difference was observed in the aggregation produced by galactose and tagatose. These results afford more insight into the importance of the functional group of the carbohydrate moiety during the formation of protein-carbohydrate conjugates via Maillard reaction.

Evaluation of glycophenotype in breast cancer by quantum dot-lectin histochemistry

PubMed Central

Andrade, Camila G; Cabral Filho, Paulo E; Tenório, Denise PL; Santos, Beate S; Beltrão, Eduardo IC; Fontes, Adriana; Carvalho, Luiz B

2013-01-01

Cell surface glycoconjugates play an important role in differentiation/dedifferentiation processes and lectins are employed to evaluate them by several methodologies. Fluorescent probes are considered a valuable tool because of their ability to provide a particular view, and are more detailed and sensitive in terms of cell structure and molecular content. The aim of this study was to evaluate and compare the expression and distribution of glycoconjugates in normal human breast tissue, and benign (fibroadenoma), and malignantly transformed (invasive ductal carcinoma) breast tissues. For this, we used mercaptosuccinic acid-coated Cadmium Telluride (CdTe) quantum dots (QDs) conjugated with concanavalin A (Con A) or Ulex europaeus agglutinin I (UEA I) lectins to detect α-D-glucose/mannose and L-fucose residues, respectively. The QD-lectin conjugates were evaluated by hemagglutination activity tests and carbohydrate inhibition assays, and were found to remain functional, keeping their fluorescent properties and carbohydrate recognition ability. Fluorescence images showed that different regions of breast tissue expressed particular types of carbohydrates. While the stroma was preferentially and intensely stained by QD-Con A, ductal cells were preferentially labeled by QD-UEA I. These results indicate that QD-lectin conjugates can be used as molecular probes and can help to elucidate the glycoconjugate profile in biological processes. PMID:24324334

Protection of non-human primates against glanders with a gold nanoparticle glycoconjugate vaccine.

PubMed

Torres, Alfredo G; Gregory, Anthony E; Hatcher, Christopher L; Vinet-Oliphant, Heather; Morici, Lisa A; Titball, Richard W; Roy, Chad J

2015-01-29

The Gram-negative Burkholderia mallei is a zoonotic pathogen and the causative agent of glanders disease. Because the bacteria maintain the potential to be used as a biothreat agent, vaccine strategies are required for human glanders prophylaxis. A rhesus macaque (Macaca mulatta) model of pneumonic (inhalational) glanders was established and the protective properties of a nanoparticle glycoconjugate vaccine composed of Burkholderia thailandensis LPS conjugated to FliC was evaluated. An aerosol challenge dose of ∼1×10(4) CFU B. mallei produced mortality in 50% of naïve animals (n=2/4), 2-3 days post-exposure. Although survival benefit was not observed by vaccination with a glycoconjugate glanders vaccine (p=0.42), serum LPS-specific IgG titers were significantly higher on day 80 in 3 vaccinated animals who survived compared with 3 vaccinated animals who died. Furthermore, B. mallei was isolated from multiple organs of both non-vaccinated survivors, but not from any organs of 3 vaccinated survivors at 30 days post-challenge. Taken together, this is the first time a candidate vaccine has been evaluated in a non-human primate aerosol model of glanders and represents the initial step for consideration in pre-clinical studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Consumption of human milk glycoconjugates by infant-associated bifidobacteria: mechanisms and implications

PubMed Central

Garrido, Daniel; Dallas, David C.

2013-01-01

Human milk is a rich source of nutrients and energy, shaped by mammalian evolution to provide all the nutritive requirements of the newborn. In addition, several molecules in breast milk act as bioactive agents, playing an important role in infant protection and guiding a proper development. While major breast milk nutrients such as lactose, lipids and proteins are readily digested and consumed by the infant, other molecules, such as human milk oligosaccharides and glycosylated proteins and lipids, can escape intestinal digestion and transit through the gastrointestinal tract. In this environment, these molecules guide the composition of the developing infant intestinal microbiota by preventing the colonization of enteric pathogens and providing carbon and nitrogen sources for other colonic commensals. Only a few bacteria, in particular Bifidobacterium species, can gain access to the energetic content of milk as it is displayed in the colon, probably contributing to their predominance in the intestinal microbiota in the first year of life. Bifidobacteria deploy exquisite molecular mechanisms to utilize human milk oligosaccharides, and recent evidence indicates that their activities also target other human milk glycoconjugates. Here, we review advances in our understanding of how these microbes have been shaped by breast milk components and the strategies associated with their consumption of milk glycoconjugates. PMID:23460033

Sugar‐coated sperm: Unraveling the functions of the mammalian sperm glycocalyx

PubMed Central

Tecle, Eillen

2015-01-01

SUMMARY Mammalian spermatozoa are coated with a thick glycocalyx that is assembled during sperm development, maturation, and upon contact with seminal fluid. The sperm glycocalyx is critical for sperm survival in the female reproductive tract and is modified during capacitation. The complex interplay among the various glycoconjugates generates numerous signaling motifs that may regulate sperm function and, as a result, fertility. Nascent spermatozoa assemble their own glycans while the cells still possess a functional endoplasmic reticulum and Golgi in the seminiferous tubule, but once spermatogenesis is complete, they lose the capacity to produce glycoconjugates de novo. Sperm glycans continue to be modified, during epididymal transit by extracellular glycosidases and glycosyltransferases. Furthermore, epididymal cells secrete glycoconjugates (glycophosphatidylinositol‐anchored glycoproteins and glycolipids) and glycan‐rich microvesicles that can fuse with the maturing sperm membrane. The sperm glycocalyx mediates numerous functions in the female reproductive tract, including the following: inhibition of premature capacitation; passage through the cervical mucus; protection from innate and adaptive female immunity; formation of the sperm reservoir; and masking sperm proteins involved in fertilization. The immense diversity in sperm‐associated glycans within and between species forms a remarkable challenge to our understanding of essential sperm glycan functions. Mol. Reprod. Dev. 82: 635–650, 2015. © 2015 The Authors. Molecular Reproduction and Development published by Wiley Periodicals, Inc. PMID:26061344

ANALYSIS OF GLYCANS DERIVED FROM GLYCOCONJUGATES BY CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY

PubMed Central

Mechref, Yehia

2012-01-01

The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful mass spectrometric (MS) and tandem MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high order tandem MS (MSn). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC) and gas chromatography (GC). However, capillary electrophoresis (CE) and microfluidics capillary electrophoresis (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to mass spectrometry. PMID:22180203

Determination of glycation sites by tandem mass spectrometry in a synthetic lactose-bovine serum albumin conjugate, a vaccine model prepared by dialkyl squarate chemistry

PubMed Central

Jahouh, Farid; Hou, Shu-jie; Kováč, Pavol; Banoub, Joseph H.

2012-01-01

RATIONALE Neoglycoconjugate vaccines synthesized by the squaric acid spacer method allow single point attachment of the carbohydrate antigen to the protein carrier. However, the localization of the carbohydrate antigen sites of conjugation on the protein carrier has been an elusive task difficult to achieve. METHOD Covalent attachment of the lactose antigen to the bovine serum albumin (BSA) was prepared by the squaric acid method using a hapten:BSA ratio of 20:1. Different reaction times were used during the conjugation reaction and two different lactose-BSA glycoconjugate vaccines were obtained. The carbohydrate antigen hapten:BSA ratios of these lactose-BSA glycoconjugate vaccines were determined by MALDI-TOF/RTOF-MS and the glycation sites in the neoglycoconjugates were determined using nano-LC/ESI-QqTOF-MS/MS analysis of the trypsin and GluC V8 digests of the conjugates. RESULTS We have identified a total of 15 glycation sites located on the BSA lysine residues for the neoglycoconjugate vaccine formed with a hapten:BSA ratio of 5.1:1, However, the tryptic and GluC V8 digests of the hapten-BSA glycoconjugate with a hapten:BSA ratio of 19.0:1 allowed identification of 30 glycation sites located on the BSA. These last results seem to indicate that this conjugation results in formation of various glycoforms. CONCLUSIONS It was observed that the number of identified glycation sites increased when the hapten:BSA ratio of glycoconjugate formation increased, and that the location of the glycation sites appears to be mainly on the outer surface of the BSA carrier molecule which is in line with the assumption that the sterically more accessible lysine residues, namely those located on the outer surface of the BSA, would be conjugated preferentially. PMID:22368054

Investigation on Sugar-Protein Connectivity in Salmonella O-Antigen Glycoconjugate Vaccines.

PubMed

De Benedetto, Gianluigi; Salvini, Laura; Gotta, Stefano; Cescutti, Paola; Micoli, Francesca

2018-05-16

Invasive nontyphoidal Salmonella disease, for which licensed vaccines are not available, is a leading cause of bloodstream infections in Africa. The O-antigen portion of lipopolysaccharide is a good target for protective immunity. Covalent conjugation of the O-antigen to a carrier protein increases its immunogenicity and O-antigen based glycoconjugate vaccines are currently under investigation at the preclinical stage. We developed a conjugation chemistry for linking O-antigen to CRM 197 carrier protein, through sequential insertion of adipic acid dihydrazide (ADH) and adipic acid bis( N-hydroxysuccinimide) ester (SIDEA) as linkers, without impacting O-antigen chain epitopes. Here the resulting sugar-protein connectivity has been investigated in detail. The core portion of the lipopolysaccharide was used as a model molecule to prepare CRM 197 conjugates, making structural investigations easier. The first step of reductive amination with ADH involves the terminal 3-deoxy-d- manno-oct-2-ulosonic acid (KDO) residue of the core region. The second reaction step resulted not to be selective, as SIDEA reacted with both ADH and pyrophosphorylethanolamine (PPEtN) of the core region, independently from the pH at which the reaction was performed. Peptide mapping analysis of the deglycosylated core-CRM 197 conjugates confirmed that lysine residues of CRM 197 were linked to SIDEA not only through KDO-ADH but also through PPEtN. This analysis also confirmed that the conjugation chemistry is random on the protein, involving a large number of lysine residues, particularly the surface exposed ones. The method for core-CRM 197 characterization was successfully extended to O-antigen-CRM 197 conjugate, confirming the results obtained with the core. This study not only allowed full characterization of OAg-CRM 197 conjugates, but can be applied to optimize synthesis and characterization of other OAg-based glycoconjugate vaccines. Analytical methods to investigate saccharide-protein connectivity are also of fundamental importance to study the relationship between glycoconjugate structure and immune response induced.

Targeted delivery of antigen to hamster nasal lymphoid tissue with M-cell-directed lectins.

PubMed Central

Giannasca, P J; Boden, J A; Monath, T P

1997-01-01

The nasal cavity of a rodent is lined by an epithelium organized into distinct regional domains responsible for specific physiological functions. Aggregates of nasal lymphoid tissue (NALT) located at the base of the nasal cavity are believed to be sites of induction of mucosal immune responses to airborne antigens. The epithelium overlying NALT contains M cells which are specialized for the transcytosis of immunogens, as demonstrated in other mucosal tissues. We hypothesized that NALT M cells are characterized by distinct glycoconjugate receptors which influence antigen uptake and immune responses to transcytosed antigens. To identify glycoconjugates that may distinguish NALT M cells from other cells of the respiratory epithelium (RE), we performed lectin histochemistry on sections of the hamster nasal cavity with a panel of lectins. Many classes of glycoconjugates were found on epithelial cells in this region. While most lectins bound to sites on both the RE and M cells, probes capable of recognizing alpha-linked galactose were found to label the follicle-associated epithelium (FAE) almost exclusively. By morphological criteria, the FAE contains >90% M cells. To determine if apical glycoconjugates on M cells were accessible from the nasal cavity, an M-cell-selective lectin and a control lectin in parallel were administered intranasally to hamsters. The M-cell-selective lectin was found to specifically target the FAE, while the control lectin did not. Lectin bound to M cells in vivo was efficiently endocytosed, consistent with the role of M cells in antigen transport. Intranasal immunization with lectin-test antigen conjugates without adjuvant stimulated induction of specific serum immunoglobulin G, whereas antigen alone or admixed with lectin did not. The selective recognition of NALT M cells by a lectin in vivo provides a model for microbial adhesin-host cell receptor interactions on M cells and the targeted delivery of immunogens to NALT following intranasal administration. PMID:9317039

Glycan analysis of Fonsecaea monophora from clinical and environmental origins reveals different structural profile and human antigenic response

PubMed Central

Burjack, Juliana R.; Santana-Filho, Arquimedes P.; Ruthes, Andrea C.; Riter, Daniel S.; Vicente, Vania A.; Alvarenga, Larissa M.; Sassaki, Guilherme L.

2014-01-01

Dematiaceous fungi constitute a large and heterogeneous group, characterized by having a dark pigment, the dihydroxynaftalen melanin—DHN, inside their cell walls. In nature they are found mainly as soil microbiota or decomposing organic matter, and are spread in tropical and subtropical regions. The fungus Fonsecaea monophora causes chromoblastomycosis in humans, and possesses essential mechanisms that may enhance pathogenicity, proliferation and dissemination inside the host. Glycoconjugates confer important properties to these pathogenic microorganisms. In this work, structural characterization of glycan structures present in two different strains of F. monophora MMHC82 and FE5p4, from clinical and environmental origins, respectively, was performed. Each one were grown on Minimal Medium (MM) and Czapeck-Dox (CD) medium, and the water soluble cell wall glycoconjugates and exopolysaccharides (EPS) were evaluated by NMR, methylation and principal component analysis (PCA). By combining the methylation and 2D NMR analyses, it was possible to visualize the glycosidic profiles of the complex carbohydrate mixtures. Significant differences were observed in β-D-Galf-(1→5) and (1→6) linkages, α- and β-D-Glcp-(1→3), (1→4), and (1→6) units, as well as in α-D-Manp. PCA from 1H-NMR data showed that MMHC82 from CD medium showed a higher variation in the cell wall carbohydrates, mainly related to O-2 substituted β-D-Galf (δ 106.0/5.23 and δ 105.3/5.23) units. In order to investigate the antigenic response of the glycoconjugates, these were screened against serum from chromoblastomycosis patients. The antigen which contained the cell wall of MMHC82 grown in MM had β-D-Manp units that promoted higher antigenic response. The distribution of these fungal species in nature and the knowledge of how cell wall polysaccharides and glycoconjugates structure vary, may contribute to the better understanding and the elucidation of the pathology caused by this fungus. PMID:25401093

Synthesis and Spectroscopic Evaluation of Two Novel Glycosylated Zinc(II)-Phthalocyanines.

PubMed

Bächle, Felix; Hanack, Michael; Ziegler, Thomas

2015-10-09

In continuation of our work on glycoconjugated phthalocyanines, two new water soluble, non-ionic zinc(II) phthalocyanines have been prepared and fully characterized by means of ¹H-NMR, 13C-NMR, MALDI-TOF, ESI-TOF, UV-Vis spectroscopy, emission spectroscopy and fluorescence lifetime measurements. The carbohydrate-containing phthalonitrile precursors were synthesized through a copper-catalyzed azide-alkyne cycloaddition (CuAAC). The 2-methoxyethoxymethyl protecting group (MEM) was used to protect the carbohydrate moieties. It resisted the harsh basic cyclotetramerization conditions and could be easily cleaved under mild acidic conditions. The glycoconjugated zinc(II) phthalocyanines described here have molar extinction coefficents εmax>10⁵ m(-1) cm(-1) and absorption maxima λ>680 nm, which make them attractive photosensitizers for photo-dynamic therapy.

Protective effect of vilva juice on glycoconjugate levels in experimentally induced constipation in rats.

PubMed

Padmini, R; Sabitha, K E; Devi, C S Shyamala

2004-10-01

Efficacy of vilva, a polyherbal formulation was evaluated in morphine induced constipated rats. Vilva juice, at a dose of 1.5 ml/100 g body wt was given orally for a period of 7 days. Morphine sulfate was injected to induce constipation on 8th day, 45 min before the experiments. Protein bound glycoconjungates were estimated in intestinal tissue. Altered levels of glycoconjugates were maintained at near normalcy when pretreated with vilva juice in morphine induced rats. Histological changes were observed in the colon tissue. The damage to crypts of Liberkunn in constipated rats were found to be reduced in vilva pretreated rats. Vilva, thus, offered significant protection against morphine induced constipation by way of augmenting mucus secretion.

Antibacterials: A sweet vaccine

NASA Astrophysics Data System (ADS)

Bundle, David

2016-03-01

Vaccination with a synthetic glycoconjugate, in combination with the administration of an inhibitor that blocks capsular polysaccharide synthesis in bacteria, could offer an alternative route to combat bacterial infections.

Boronlectin/Polyelectrolyte Ensembles as Artificial Tongue: Design, Construction, and Application for Discriminative Sensing of Complex Glycoconjugates from Panax ginseng.

PubMed

Zhang, Xiao-Tai; Wang, Shu; Xing, Guo-Wen

2017-02-01

Ginsenoside is a large family of triterpenoid saponins from Panax ginseng, which possesses various important biological functions. Due to the very similar structures of these complex glycoconjugates, it is crucial to develop a powerful analytic method to identify ginsenosides qualitatively or quantitatively. We herein report an eight-channel fluorescent sensor array as artificial tongue to achieve the discriminative sensing of ginsenosides. The fluorescent cross-responsive array was constructed by four boronlectins bearing flexible boronic acid moieties (FBAs) with multiple reactive sites and two linear poly(phenylene-ethynylene) (PPEs). An "on-off-on" response pattern was afforded on the basis of superquenching of fluorescent indicator PPEs and an analyte-induced allosteric indicator displacement (AID) process. Most importantly, it was found that the canonical distribution of ginsenoside data points analyzed by linear discriminant analysis (LDA) was highly correlated with the inherent molecular structures of the analytes, and the absence of overlaps among the five point groups reflected the effectiveness of the sensor array in the discrimination process. Almost all of the unknown ginsenoside samples at different concentrations were correctly identified on the basis of the established mathematical model. Our current work provided a general and constructive method to improve the quality assessment and control of ginseng and its extracts, which are useful and helpful for further discriminating other complex glycoconjugate families.

Hypothalamic digoxin, hemispheric chemical dominance, and sarcoidosis.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2003-11-01

The isoprenoid pathway produces three key metabolites--endogenous digoxin, dolichol, and ubiquinone. This was assessed in patients with systemic sarcoidosis. All l5 patients with sarcoidosis were right-handed/left hemispheric dominant by the dichotic listening test. The pathway was also studied in normal right hemispheric, left hemispheric, and bihemispheric dominant individuals for comparison to find out the role of hemispheric dominance in the pathogenesis of sarcoidosis. In patients with sarcoidosis there was elevated digoxin synthesis, increased dolichol, and glycoconjugate levels, and low ubiquinone and elevated free radical levels. There was also an increase in tryptophan catabolites and a reduction in tyrosine catabolites. There was an increase in cholesterol:phospholipid ratio and a reduction in glycoconjugate level of RBC membrane in these patients. The neurotransmitter/digoxin-mediated increased intra cellular calcium induced immune activation, ubiquinone deficiency-related mitochondrial dysfunction/free radical generation, and increased dolichol-related altered glycoconjugate metabolism/endogenous self-glycoprotein antigen generation are crucial to the pathogenesis of sarcoidosis. The biochemical patterns obtained in sarcoidosis are similar to those obtained in left-handed/right hemispheric chemically dominant individuals by the dichotic listening test. But all the patients with sarcoidosis were right-handed/left hemispheric dominant by the dichotic listening test. Hemispheric chemical dominance has no correlation with handedness or the dichotic listening test. Sarcoidosis occurs in right hemispheric chemically dominant individuals and is a reflection of altered brain function.

Hypothalamic digoxin, hemispheric chemical dominance, and mesenteric artery occlusion.

PubMed

Kurup, Ravi Kumar; Kurup, Paramesware Achutha

2003-12-01

The role of the isoprenoid pathway in vascular thrombosis, especially mesenteric artery occlusion and its relation to hemispheric dominance, was assessed in this study. The following parameters were measured in patients with mesenteric artery occlusion and individuals with right hemispheric, left hemispheric, and bihemispheric dominance: (1) plasma HMG CoA reductase, digoxin, dolichol, ubiquinone, and magnesium levels; (2) tryptophan/tyrosine catabolic patterns; (3) free radical metabolism; (4) glycoconjugate metabolism; and (5) membrane composition. In patients with mesenteric artery occlusion there was elevated digoxin synthesis, increased dolichol and glycoconjugate levels, low ubiquinone, and elevated free radical levels. The RBC membrane Na(+)-K+ ATPase activity and serum magnesium were decreased. There was also an increase in tryptophan catabolites and reduction in tyrosine catabolites in the serum. There was an increase in cholesterol:phospholipid ratio and a reduction in glycoconjugate level of RBC membrane in these patients. The biochemical patterns obtained in mesenteric artery occlusion is similar to those obtained in left-handed/right hemispheric dominant individuals by the dichotic listening test. But all the patients with mesenteric artery occlusion were right-handed/left hemispheric dominant by the dichotic listening test. Hemispheric chemical dominance has no correlation with handedness or the dichotic listening test. Mesenteric artery occlusion occurs in right hemispheric chemically dominant individuals and is a reflection of altered brain function. Hemispheric chemical dominance may thus control the risk for developing vascular thrombosis in individuals.

Improving Analytical Characterization of Glycoconjugate Vaccines through Combined High-Resolution MS and NMR: Application to Neisseria meningitidis Serogroup B Oligosaccharide-Peptide Glycoconjugates.

PubMed

Yu, Huifeng; An, Yanming; Battistel, Marcos D; Cipollo, John F; Freedberg, Darón I

2018-04-17

Conjugate vaccines are highly heterogeneous in terms of glycosylation sites and linked oligosaccharide length. Therefore, the characterization of conjugate vaccines' glycosylation state is challenging. However, improved product characterization can lead to enhancements in product control and product quality. Here, we present a synergistic combination of high-resolution mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) for the analysis of glycoconjugates. We use the power of this strategy to characterize model polysaccharide conjugates and to demonstrate a detailed level of glycoproteomic analysis. These are first steps on model compounds that will help untangle the details of complex product characterization in conjugate vaccines. Ultimately, this strategy can be applied to enhance the characterization of polysaccharide conjugate vaccines. In this study, we lay the groundwork for the analysis of conjugate vaccines. To begin this effort, oligosaccharide-peptide conjugates were synthesized by periodate oxidation of an oligosaccharide of a defined length, α,2-8 sialic acid trimer, followed by a reductive amination, and linking the trimer to an immunogenic peptide from tetanus toxoid. Combined mass spectrometry and nuclear magnetic resonance were used to monitor each reaction and conjugation products. Complete NMR peak assignment and detailed MS information on oxidized oligosialic acid and conjugates are reported. These studies provide a deeper understanding of the conjugation chemistry process and products, which can lead to a better controlled production process.

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: an update for 2009-2010.

PubMed

Harvey, David J

2015-01-01

This review is the sixth update of the original article published in 1999 on the application of MALDI mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2010. General aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, arrays and fragmentation are covered in the first part of the review and applications to various structural typed constitutes the remainder. The main groups of compound that are discussed in this section are oligo and polysaccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals. Many of these applications are presented in tabular form. Also discussed are medical and industrial applications of the technique, studies of enzyme reactions and applications to chemical synthesis. © 2014 Wiley Periodicals, Inc.

Colonize, evade, flourish

PubMed Central

Rubin, Erica J; Trent, M Stephen

2013-01-01

Helicobacter pylori is an adapted gastric pathogen that colonizes the human stomach, causing severe gastritis and gastric cancer. A hallmark of infection is the ability of this organism to evade detection by the human immune system. H. pylori has evolved a number of features to achieve this, many of which involve glyco-conjugates including the lipopolysaccharide, peptidoglycan layer, glycoproteins, and glucosylated cholesterol. These major bacterial components possess unique features from those of other gram-negative organisms, including differences in structure, assembly, and modification. These defining characteristics of H. pylori glycobiology help the pathogen establish a long-lived infection by providing camouflage, modulating the host immune response, and promoting virulence mechanisms. In this way, glyco-conjugates are essential for H. pylori pathogenicity and survival, allowing it to carve out a niche in the formidable environment of the human stomach. PMID:23859890

Distinct human α(1,3)-fucosyltransferases drive Lewis-X/sialyl Lewis-X assembly in human cells.

PubMed

Mondal, Nandini; Dykstra, Brad; Lee, Jungmin; Ashline, David J; Reinhold, Vernon N; Rossi, Derrick J; Sackstein, Robert

2018-05-11

In humans, six α(1,3)-fucosyltransferases (α(1,3)-FTs: FT3/FT4/FT5/FT6/FT7/FT9) reportedly fucosylate terminal lactosaminyl glycans yielding Lewis-X (Le X ; CD15) and/or sialyl Lewis-X (sLe X ; CD15s), structures that play key functions in cell migration, development, and immunity. Prior studies analyzing α(1,3)-FT specificities utilized either purified and/or recombinant enzymes to modify synthetic substrates under nonphysiological reaction conditions or molecular biology approaches wherein α(1,3)-FTs were expressed in mammalian cell lines, notably excluding investigations using primary human cells. Accordingly, although significant insights into α(1,3)-FT catalytic properties have been obtained, uncertainty persists regarding their human Le X /sLe X biosynthetic range across various glycoconjugates. Here, we undertook a comprehensive evaluation of the lactosaminyl product specificities of intracellularly expressed α(1,3)-FTs using a clinically relevant primary human cell type, mesenchymal stem cells. Cells were transfected with modified mRNA encoding each human α(1,3)-FT, and the resultant α(1,3)-fucosylated lactosaminyl glycoconjugates were analyzed using a combination of flow cytometry and MS. The data show that biosynthesis of sLe X is driven by FTs-3, -5, -6, and -7, with FT6 and FT7 having highest potency. FT4 and FT9 dominantly biosynthesize Le X , and, among all FTs, FT6 holds a unique capacity in creating sLe X and Le X determinants across protein and lipid glycoconjugates. Surprisingly, FT4 does not generate sLe X on glycolipids, and neither FT4, FT6, nor FT9 synthesizes the internally fucosylated sialyllactosamine VIM-2 (CD65s). These results unveil the relevant human lactosaminyl glycans created by human α(1,3)-FTs, providing novel insights on how these isoenzymes stereoselectively shape biosynthesis of vital glycoconjugates, thereby biochemically programming human cell migration and tuning human immunologic and developmental processes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Determination of free polysaccharide in Vi glycoconjugate vaccine against typhoid fever.

PubMed

Giannelli, C; Cappelletti, E; Di Benedetto, R; Pippi, F; Arcuri, M; Di Cioccio, V; Martin, L B; Saul, A; Micoli, F

2017-05-30

Glycoconjugate vaccines based on the Vi capsular polysaccharide directed against Salmonella enterica serovar Typhi are licensed or in development against typhoid fever, an important cause of morbidity and mortality in developing countries. Quantification of free polysaccharide in conjugate vaccines is an important quality control for release, to monitor vaccine stability and to ensure appropriate immune response. However, we found that existing separation methods based on size are not appropriate as free Vi non-specifically binds to unconjugated and conjugated protein. We developed a method based on free Vi separation by Capto Adhere resin and quantification by HPAEC-PAD. The method has been tested for conjugates of Vi derived from Citrobacter freundii with different carrier proteins such as CRM 197 , Tetanus Toxoid and Diphtheria Toxoid. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Hypothalamic digoxin, hemispheric chemical dominance, and interstitial lung disease.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2003-10-01

The isoprenoid pathway produces three key metabolites--endogenous digoxin, dolichol, and ubiquinone. This was assessed in patients with idiopathic pulmonary fibrosis and in individuals of differing hemispheric dominance to find out the role of hemispheric dominance in the pathogenesis of idiopathic pulmonary fibrosis. All 15 cases of interstitial lung disease were right-handed/left hemispheric dominant by the dichotic listening test. The isoprenoidal metabolites--digoxin, dolichol, and ubiquinone, RBC membrane Na(+)-K+ ATPase activity, serum magnesium, tyrosine/tryptophan catabolic patterns, free radical metabolism, glycoconjugate metabolism, and RBC membrane composition--were assessed in idiopathic pulmonary fibrosis as well as in individuals with differing hemispheric dominance. In patients with idiopathic pulmonary fibrosis there was elevated digoxin synthesis, increased dolichol and glycoconjugate levels, and low ubiquinone and elevated free radical levels. There was also an increase in tryptophan catabolites and a reduction in tyrosine catabolites. There was an increase in cholesterol phospholipid ratio and a reduction in glycoconjugate level of RBC membrane in patients with idiopathic pulmonary fibrosis. Isoprenoid pathway dysfunction con tributes to the pathogenesis of idiopathic pulmonary fibrosis. The biochemical patterns obtained in interstitial lung disease are similar to those obtained in left-handed/right hemispheric chemically dominant individuals by the dichotic listening test. However, all the patients with interstitial lung disease were right-handed/left hemispheric dominant by the dichotic listening test. Hemispheric chemical dominance has no correlation with handedness or the dichotic listening test. Interstitial lung disease occurs in right hemispheric chemically dominant individuals and is a reflection of altered brain function.

Design, Synthesis of Novel Platinum(II) Glycoconjugates, and Evaluation of Their Antitumor Effects.

PubMed

Han, Jianbin; Gao, Xiangqian; Liu, Ran; Yang, Jinna; Zhang, Menghua; Mi, Yi; Shi, Ying; Gao, Qingzhi

2016-06-01

A new series of sugar-conjugated (trans-R, R-cyclohexane-1, 2-diamine)-2-halo-malonato-platinum(II) complexes were designed and synthesized to target tumor-specific glucose transporters (GLUTs). The water solubility of the sugar-conjugated platinum (II) complexes was greatly improved by average of 570-fold, 33-fold, and 94-fold, respectively, compared to cisplatin (1.0 mg/mL), carboplatin (17.1 mg/mL), and the newest generation of clinical drug oxaliplatin (6.0 mg/mL). Despite the high water solubility, the platinum(II) glycoconjugates exhibited a notable increase in cytotoxicity by a margin of 1.5- to 6.0-fold in six different human cancer cell lines with respect to oxaliplatin. The potential GLUT1 transportability of the complexes was investigated through a molecular docking study and was confirmed with GLUT1 inhibitor-mediated cytotoxicity dependency evaluation. The results showed that the sugar-conjugated platinum(II) complexes can be recognized by the glucose recognition binding site of GLUT1 and their cell killing effect depends highly on the GLUT1 inhibitor, quercetin. The research presenting a prospective concept for targeted therapy anticancer drug design, and with the analysis of the synthesis, water solubility, antitumor activity, and the transportability of the platinum(II) glycoconjugates, this study provides fundamental data supporting the inherent potential of these designed conjugates as lead compounds for GLUT-mediated tumor targeting. © 2016 John Wiley & Sons A/S.

Complete genome sequence of Campylobacter jejuni strain 12567 a livestock-associated clade representative

USDA-ARS?s Scientific Manuscript database

We report the complete genome sequence of the Campylobacter jejuni strain 12567, a member of a C. jejuni livestock-associated clade that expresses glycoconjugates linked to improved gastrointestinal tract persistence....

Milk Glycans and Their Interaction with the Infant-Gut Microbiota

PubMed Central

Kirmiz, Nina; Robinson, Randall C.; Shah, Ishita M.; Barile, Daniela; Mills, David A.

2018-01-01

Human milk is a unique and complex fluid that provides infant nutrition and delivers an array of bioactive molecules that serve various functions. Glycans, abundant in milk, can be found as free oligosaccharides or as glycoconjugates. Milk glycans are increasingly linked to beneficial outcomes in neonates through protection from pathogens and modulation of the immune system. Indeed, these glycans influence the development of the infant and the infant-gut microbiota. Bifidobacterium species commonly are enriched in breastfed infants and are among a limited group of bacteria that readily consume human milk oligosaccharides (HMOs) and milk glycoconjugates. Given the importance of bifidobacteria in infant health, numerous studies have examined the molecular mechanisms they employ to consume HMOs and milk glycans, thus providing insight into this unique enrichment and shedding light on a range of translational opportunities to benefit at-risk infants. PMID:29580136

Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

PubMed Central

Jahouh, Farid; Hou, Shu-jie; Kováč, Pavol; Banoub, Joseph H.

2012-01-01

We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate–protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate–spacer–carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate. PMID:22012665

A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium*

PubMed Central

Kodali, Srinivas; Vinogradov, Evgeny; Lin, Fiona; Khoury, Nancy; Hao, Li; Pavliak, Vilo; Jones, C. Hal; Laverde, Diana; Huebner, Johannes; Jansen, Kathrin U.; Anderson, Annaliesa S.; Donald, Robert G. K.

2015-01-01

The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic acid and legionaminic acid, respectively. A minor high molecular weight polysaccharide component was recognized as the fructose homopolymer levan, and a glucosylated lipoteichoic acid (LTA) was identified in a micellar fraction. The polysaccharides were conjugated to the CRM197 carrier protein, and the resulting glycoconjugates were used to immunize rabbits. Rabbit immune sera were evaluated for their ability to kill Tx16 in opsonophagocytic assays and in a mouse passive protection infection model. Although antibodies raised against levan failed to mediate opsonophagocytic killing, the other glycoconjugates induced effective opsonic antibodies, with the altruronic acid-containing polysaccharide antisera showing the greatest opsonophagocytic assay activity. Antibodies directed against either novel heteroglycan or the LTA reduced bacterial load in mouse liver or kidney tissue. To assess antigen prevalence, we screened a diverse collection of blood isolates (n = 101) with antibodies to the polysaccharides. LTA was detected on the surface of 80% of the strains, and antigens recognized by antibodies to the two major heteroglycans were co-expressed on 63% of these clinical isolates. Collectively, these results represent the first steps toward identifying components of a glycoconjugate vaccine to prevent E. faecium infection. PMID:26109072

Structures and application of oligosaccharides in human milk.

PubMed

Kobata, Akira

2010-01-01

Comparative study of the oligosaccharide profiles of individual human milk revealed the presence of three different patterns. Four oligosaccharides containing the Fucalpha1-2Gal group were missing in the milk of non-secretor, and three oligosaccharides containing the Fucalpha1-4GlcNAc group were missing in the milk of Lewis negative individuals. Disappearance of some major oligosaccharides in these samples led to the finding of five novel minor oligosaccharides, which were hidden under the missing oligosaccharides. Following these studies, structures of many novel milk oligosaccharides were elucidated. At least 13 core oligosaccharides were found in these oligosaccharides. By adding alpha-fucosyl residues and sialic acid residues to these core oligosaccharides, more than one hundred oligosaccharides were formed. All these oligosaccharides contain lactose at their reducing termini. This evidence, together with the deletion phenomena found in the milk oligosaccharides of non-secretor and Lewis negative individuals, suggested that the oligosaccharides are formed from lactose by the concerted action of glycosyltransferases, which are responsible for elongation and branching of the Galbeta1-4GlcNAc group in the sugar chains of glycoconjugates on the surface of epithelial cells. Therefore, oligosaccharides in human milk could include many structures, starting from the Galbeta1-4GlcNAc group in the sugar chains of various glycoconjugates. Many lines of evidence recently indicated that virulent enteric bacteria and viruses start their infection by binding to particular sugar chains of glycoconjugates on the target cell surfaces. Therefore, milk oligosaccharides could be useful for developing drugs, which inhibit the infection of bacteria and viruses.

Draft Genome Sequence of Enterococcus hirae Strain INF E1 Isolated from Cultured Milk.

PubMed

Porcellato, Davide; Ostlie, Hilde M; Skeie, Siv B

2014-07-17

Here, we present the draft genome of Enterococcus hirae INF E1, found as a contaminant in cultured milk and studied for its ability to metabolize milk fat globule membrane glycoconjugates. Copyright © 2014 Porcellato et al.

Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas.

PubMed

Willemer, S; Köhler, H; Naumann, R; Kern, H F; Adler, G

1990-01-01

Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): L-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis- to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.

Hypothalamic digoxin and hemispheric chemical dominance: relation to speech and language dysfunction.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2003-06-01

The isoprenoid pathway produces three key metabolites--endogenous digoxin, dolichol, and ubiquinone. Since endogenous digoxin can regulate neurotransmitter transport and dolichols can modulate glycoconjugate synthesis important in synaptic connectivity, the pathway was assessed in patients with dyslexia, delayed recovery from global aphasia consequent to a dominant hemispheric thrombotic infarct, and developmental delay of speech milestone. The pathway was also studied in right hemispheric, left hemispheric, and bihemispheric dominance to find out the role of hemispheric dominance in the pathogenesis of speech disorders. The plasma/serum--activity of HMG CoA reductase, magnesium, digoxin, dolichol, ubiquinone--and tryptophan/tyrosine catabolic patterns, as well as RBC (Na+)-K+ ATPase activity, were measured in the above mentioned groups. The glycoconjugate metabolism and membrane composition was also studied. The study showed that in dyslexia, developmental delay of speech milestone, and delayed recovery from global aphasia there was an upregulated isoprenoidal pathway with increased digoxin and dolichol levels. The membrane (Na+)-K+ ATPase activity, serum magnesium and ubiquinone levels were low. The tryptophan catabolites were increased and the tyrosine catabolites including dopamine decreased in the serum contributing to a speech dysfunction. There was an increase in carbohydrate residues of glycoproteins, glycosaminoglycans, and glycolipids levels as well as an increased activity of GAG degrading enzymes and glyco hydrolases in the serum. The cholesterol:phospholipid ratio of RBC membrane increased and membrane glycoconjugates showed a decrease. All of these could contribute to altered synaptic inactivity in these disorders. The patterns correlated with those obtained in right hemispheric chemical dominance. Right hemispheric chemical dominance may play a role in the genesis of these disorders. Hemispheric chemical dominance has no correlation with handedness or the dichotic listening test.

Hypothalamic digoxin, hemispheric chemical dominance and sarcoidosis.

PubMed

Ravi Kumar, A; Kurup, Parameswara Achutha

2004-06-01

The isoprenoid pathway produces three key metabolites: endogenous digoxin (membrane sodium-potassium ATPase inhibitor, immunomodulator and regulator of neurotransmitter/amino acid transport), dolichol (regulates N-glycosylation of proteins) and ubiquinone (free radical scavenger). The role of the isoprenoid pathway in the pathogenesis of sarcoidosis in relation to hemispheric dominance was studied. The isoprenoid pathway-related cascade was assessed in patients with systemic sarcoidosis with pulmonary involvement. The pathway was also assessed in patients with right hemispheric, left hemispheric and bihemispheric dominance for comparison to find out the role of hemispheric dominance in the pathogenesis of sarcoidosis. In patients with sarcoidosis there was elevated digoxin synthesis, increased dolichol and glycoconjugate levels and low ubiquinone and elevated free radical levels. There was also an increase in tryptophan catabolites and a reduction in tyrosine catabolites. There was an increase in the cholesterol:phospholipid ratio and a reduction in the glycoconjugate level of red blood cell (RBC) membrane in this group of patients. The same biochemical patterns were obtained in individuals with right hemispheric dominance. In individuals with left hemispheric dominance the patterns were reversed. Endogenous digoxin, by activating the calcineurin signal transduction pathway of T cells, can contribute to immune activation in sarcoidosis. An altered glycoconjugate metabolism can lead to the generation of endogenous self-glycoprotein antigens in the lung as well as other tissues. Increased free radical generation can also lead to immune activation. The role of a dysfunctional isoprenoid pathway and endogenous digoxin in the pathogenesis of sarcoidosis in relation to right hemispheric chemical dominance is discussed. All the patients with sarcoidosis were right-handed/left hemispheric dominant according to the dichotic listening test, but their biochemical patterns were suggestive of right hemispheric chemical dominance. Hemispheric chemical dominance has no correlation with handedness or the dichotic listening test.

Overexpression of O-polysaccharide chain length regulators in Gram-negative bacteria using the Wzx-/Wzy-dependent pathway enhances production of defined modal length O-polysaccharide polymers for use as haptens in glycoconjugate vaccines.

PubMed

Hegerle, N; Bose, J; Ramachandran, G; Galen, J E; Levine, M M; Simon, R; Tennant, S M

2018-03-30

O-polysaccharide (OPS) molecules are protective antigens for several bacterial pathogens, and have broad utility as components of glycoconjugate vaccines. Variability in the OPS chain length is one obstacle towards further development of these vaccines. Introduction of sizing steps during purification of OPS molecules of suboptimal or of mixed lengths introduces additional costs and complexity while decreasing the final yield. The overall goal of this study was to demonstrate the utility of engineering Gram-negative bacteria to produce homogenous O-polysaccharide populations that can be used as the basis of carbohydrate vaccines by overexpressing O-polysaccharide chain length regulators of the Wzx-/Wzy-dependent pathway. The O-polysaccharide chain length regulators wzzB and fepE from Salmonella Typhimurium I77 and wzz2 from Pseudomonas aeruginosa PAO1 were cloned and expressed in the homologous organism or in other Gram-negative bacteria. Overexpression of these Wzz proteins in the homologous organism significantly increased the proportion of long or very long chain O-polysaccharides. The same observation was made when wzzB was overexpressed in Salmonella Paratyphi A and Shigella flexneri, and wzz2 was overexpressed in two other strains of P. aeruginosa. Overexpression of Wzz proteins in Gram-negative bacteria using the Wzx/Wzy-dependant pathway for lipopolysaccharide synthesis provides a genetic method to increase the production of an O-polysaccharide population of a defined size. The methods presented herein represent a cost-effective and improved strategy for isolating preferred OPS vaccine haptens, and could facilitate the further use of O-polysaccharides in glycoconjugate vaccine development. © 2018 The Society for Applied Microbiology.

The effect of phenol composition on the sensory profile of smoke affected wines.

PubMed

Kelly, David; Zerihun, Ayalsew

2015-05-26

Vineyards exposed to wildfire generated smoke can produce wines with elevated levels of lignin derived phenols that have acrid, metallic and smoky aromas and flavour attributes. While a large number of phenols are present in smoke affected wines, the effect of smoke vegetation source on the sensory descriptors has not been reported. Here we report on a descriptive sensory analysis of wines made from grapes exposed to different vegetation sources of smoke to examine: (1) the effect vegetation source has on wine sensory attribute ratings and; (2) associations between volatile and glycoconjugated phenol composition and sensory attributes. Sensory attribute ratings were determined by a trained sensory panel and phenol concentrations determined by gas chromatography-mass spectroscopy. Analysis of variance, principal component analysis and partial least squares regressions were used to evaluate the interrelationships between the phenol composition and sensory attributes. The results showed that vegetation source of smoke significantly affected sensory attribute intensity, especially the taste descriptors. Differences in aroma and taste from smoke exposure were not limited to an elevation in a range of detractive descriptors but also a masking of positive fruit descriptors. Sensory differences due to vegetation type were driven by phenol composition and concentration. In particular, the glycoconjugates of 4-hydroxy-3-methoxybenzaldehyde (vanillin), 1-(4-hydroxy-3-methoxyphenyl)ethanone (acetovanillone), 4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde) and 1-(4-hydroxy-3,5-dimethoxyphenyl)ethanone (acetosyringone) concentrations were influential in separating the vegetation sources of smoke. It is concluded that the detractive aroma attributes of smoke affected wine, especially of smoke and ash, were associated with volatile phenols while the detractive flavour descriptors were correlated with glycoconjugated phenols.

Effects of ozone on the cholinergic secretory responsiveness of ferret tracheal glands

DOE Office of Scientific and Technical Information (OSTI.GOV)

McBride, R.K.; Oberdoerster, G.; Marin, M.G.

1991-06-01

Oxidant air pollutants exacerbate several pulmonary diseases. Inhalation of ozone has been shown to induce airway smooth muscle hyperresponsiveness. Oxidant injury could also affect airway secretory mechanisms. The authors postulated that oxidant exposure would alter the glycoconjugate secretory function of airway submucosal glands. To test this hypothesis they examined the effects of in vivo ozone exposure on the in vitro secretory responsiveness of ferret tracheal glands. Ferrets were exposed to 1 ppm ozone, 24 hr/day for 3 or 7 days. Following exposure, glandular explants, denuded of surface epithelial cells, were prepared and incubated in medium containing 3H-glucosamine for 18 hr.more » Basal secretion of labeled glycoconjugates was significantly increased 31% following 3 days of ozone exposure (P less than or equal to 0.05) and remained elevated 11% after 7 days of exposure compared to the air-exposed group. After 3 or 7 days of exposure to ozone, tracheal gland responsiveness to carbachol was increased as indicated by significantly lower EC50 values (log molar concentration) of -6.43 {plus minus} 0.04 (n = 6) and -6.50 {plus minus} 0.11 (n = 5), respectively; compared to -6.20 {plus minus} 0.08 (n = 6) for the air-exposed group. There was no difference in carbachol EC50 values for air and 7-day ozone-exposed animals treated with dexamethasone. Dexamethasone did not attenuate the ozone-induced increase in basal secretion. Tracheal gland responsiveness to {alpha}- or {beta}-adrenergic agonists was not changed by oxidant exposure. These experiments suggest that oxidant injury not only increases basal secretion of respiratory glycoconjugates but also increases tracheal gland sensitivity to a cholinergic agonist.« less

Differential Impact of LPG-and PG-Deficient Leishmania major Mutants on the Immune Response of Human Dendritic Cells

PubMed Central

Jayakumar, Asha; Hickerson, Suzanne; Mostrom, Janet; Turco, Salvatore J.; Beverley, Stephen M.; McDowell, Mary Ann

2015-01-01

Background Leishmania major infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction. Methodology/Principal Findings Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-), or generally deficient for all PGs, (FV1 lpg2-). Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription. Conclusions/Significance These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12. PMID:26630499

Regional differences in lectin binding patterns of vestibular hair cells

NASA Technical Reports Server (NTRS)

Baird, Richard A.; Schuff, N. R.; Bancroft, J.

1994-01-01

Surface glycoconjugates of hair cells and supporting cells in the vestibular endorgans of the bullfrog were identified using biotinylated lectins with different carbohydrate specificities. Lectin binding in hair cells was consistent with the presence of glucose and mannose (CON A), galactose (RCA-I), N-acetylgalactosamine (VVA), but not fucose (UEA-I) residues. Hair cells in the bullfrog sacculus, unlike those in the utriculus and semicircular canals, did not stain for N-acetylglucosamine (WGA) or N-acetylgalactosamine (VVA). By contrast, WGA and, to a lesser extent, VVA, differentially stained utricular and semicircular canal hair cells, labeling hair cells located in peripheral, but not central, regions. In mammals, WGA uniformly labeled Type 1 hair cells while labeling, as in the bullfrog, Type 2 hair cells only in peripheral regions. These regional variations were retained after enzymatic digestion. We conclude that vestibular hair cells differ in their surface glycoconjugates and that differences in lectin binding patterns can be used to identify hair cell types and to infer the epithelial origin of isolated vestibular hair cells.

Glycoconjugates as Mediators of Nitric Oxide Production upon Exposure to Bacterial Spores by Macrophages

NASA Astrophysics Data System (ADS)

Lahiani, Mohamed; Soderberg, Lee; Tarasenko, Olga

2011-06-01

Phagocytes generate nitric oxide (NO) in large quantities to combat bacteria. The spore-producing Gram-positive organisms of Bacillus cereus family are causative agents from mild to a life threatening infection in humans and domestic animals. Our group have shown that glycoconjugates (GCs) activate macrophages and enhance killing of Bacillus spores. In this investigation, we will explore the effect of different GCs structures on NO production. The objective of this study is to study effects of GCs 2, 4, 6, 8, 10 on NO release upon exposure to B. cereus and Bacillus anthracis spores by macrophages. Our results demonstrated that GCs activated macrophages and increased NO production using studied GCs ligands compared to macrophage only (p<0.001). GC2 and GC8 were able to further increase NO production in macrophages compared to the B. anthracis spores treated macrophages (p<0.001). Our finding suggests that GCs could be used as potential mediators of NO production in macrophages to fight B. anthracis and other pathogens.

Studies on the liver sequestration of lymphocytes bearing membrane-associated galactose-terminal glycoconjugates: reversal with agents that effectively compete for the asialoglycoprotein receptor.

PubMed

Samlowski, W E; Spangrude, G J; Daynes, R A

1984-10-15

The removal of "effete" glycoproteins from the circulation represents a proposed physiologic role for the hepatocyte asialoglycoprotein receptor. Our experiments support the hypothesis that this receptor may also be directly involved in the removal from the circulation of cells bearing asialoglycoconjugates. We report that the enhanced liver localization of neuraminidase-treated lymphocytes can be competitively inhibited by the coinjection of asialofetuin (ASF). Fetuin itself was without effect. Competitive inhibition of the liver receptor allowed normal localization to lymphoid tissues of the enzyme-treated lymphocytes, a condition which persisted as long as free ASF was present in the circulation. Our studies support the concept that cell surface carbohydrates play an important role in the tissue distribution of circulating lymphocytes. The process of thymocyte maturation, bone marrow transplantation, and the adoptive immunotherapy with continuous T-cell lines represent conditions where recirculation potential may be influenced by the presence of galactose terminal glycoconjugates.

Isolation and Purification of Glycoconjugates from Complex Biological Sources by Recycling HPLC

PubMed Central

Alley, William R.; Mann, Benjamin F.; Hruska, Vlastimil; Novotny, Milos V.

2013-01-01

Among of the most urgent needs of the glycobiology community is to generate libraries of pure carbohydrate standards. While many oligosaccharides have recently been synthesized, some glycans of biomedical importance are still missing in existing collections, or are available in only limited amounts. To address this need, we demonstrate the use of the relatively unexplored technique of recycling high-performance liquid chromatography (R-HPLC) to isolate and purify glycoconjugates from several natural sources. We were able to routinely achieve purities greater than 98%. In several cases, we were able to obtain isomerically pure substances, particularly for glycans with different positional isomerism. These purified substances can then be used in different analytical applications, for example, as standards for mass spectrometry (MS) and capillary-based separations. Moreover, using a bifunctional aromatic amine, the same derivatization agent can be used to enable UV detection of oligosaccharides during their purification and link the isolated molecules to functionalized surfaces and potentially create glycan arrays. PMID:24070405

Isolation and purification of glycoconjugates from complex biological sources by recycling high-performance liquid chromatography.

PubMed

Alley, William R; Mann, Benjamin F; Hruska, Vlastimil; Novotny, Milos V

2013-11-05

Among of the most urgent needs of the glycobiology community is to generate libraries of pure carbohydrate standards. While many oligosaccharides have recently been synthesized, some glycans of biomedical importance are still missing in existing collections or are available in only limited amounts. To address this need, we demonstrate the use of the relatively unexplored technique of recycling high-performance liquid chromatography (R-HPLC) to isolate and purify glycoconjugates from several natural sources. We were able to routinely achieve purities greater than 98%. In several cases, we were able to obtain isomerically pure substances, particularly for glycans with different positional isomerism. These purified substances can then be used in different analytical applications, for example, as standards for mass spectrometry (MS) and capillary-based separations. Moreover, using a bifunctional aromatic amine, the same derivatization agent can be used to enable UV detection of oligosaccharides during their purification and link the isolated molecules to functionalized surfaces and potentially create glycan arrays.

HPLC-MS analysis of pheromone glucoconjugates in oral secretions of male Anastrepha Fruit Flies

USDA-ARS?s Scientific Manuscript database

Using high performance liquid chromatography combined with ESi, APCI, and PBEI mass spectroscopy, novel terpenoid glycoconjugates were identified in oral secretions of several Anastrepha fly species; these findings suggest that non-volatile pheromone signals are used in their lek mating strategies. ...

Stereochemical Control in Carbohydrate Chemistry

ERIC Educational Resources Information Center

Batchelor, Rhys; Northcote, Peter T.; Harvey, Joanne E.; Dangerfield, Emma M.; Stocker, Bridget L.

2008-01-01

Carbohydrates, in the form of glycoconjugates, have recently been shown to control a wide range of cellular processes. Accordingly, students interested in the study of organic chemistry and biomedical sciences should be exposed to carbohydrate chemistry. To this end, we have developed a sequence of experiments that leads the student from the…

Histochemical study of lectin binding sites in fourth and fifth instar gypsy moth larval midgut epithelium

Treesearch

Algimantas P. Valaitis

2011-01-01

There is evidence that the gypsy moth, Lymantria dispar, midgut epithelial brush border membrane has membrane-bound glycoconjugates, such as BTR-270 and aminopeptidase N (APN), which function as high affinity binding sites (receptors) for the insecticidal proteins produced by Bacillus thuringiensis (Bt). As gypsy...

Lectin staining of epithelia lining the uterovaginal junction and sperm-storage tubules in chicken hens

USDA-ARS?s Scientific Manuscript database

In most mammals sperm are subject to a transient storage period in the caudal region of the oviduct during which they undergo cellular and molecular modifications associated with capacitation. During this storage period sperm bind to a terminal carbohydrate moiety associated with a glycoconjugate o...

Computational investigation of enthalpy-entropy compensation in complexation of glycoconjugated bile salts with β-cyclodextrin and analogs.

PubMed

Tidemand, Kasper D; Schönbeck, Christian; Holm, René; Westh, Peter; Peters, Günther H

2014-09-18

The inclusion complexes of glycoconjugated bile salts with β-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrins (HP-β-CD) in aqueous solution were investigated by molecular dynamics simulations to provide a molecular explanation of the experimentally observed destabilizing effect of the HP substituents. Good agreement with experimental data was found with respect to penetration depths of CDs. An increased degree of HP substitution (DS) resulted in an increased probability of blocking the cavity opening, thereby hindering the bile salt from entering CD. Further, the residence time of water molecules in the cavity increased with the DS. Release of water from the cavity resulted in a positive enthalpy change, which correlates qualitatively with the experimentally determined increase in complexation enthalpy and contributes to the enthalpy-entropy compensation. The positive change in complexation entropy with DS was not able to compensate for this unfavorable change in enthalpy induced by the HP substituents, resulting in a destabilizing effect. This was found to originate from fixation of the HP substituents and decreased free rotation of the bile salts within the CD cavities.

Regional differences in lectin binding patterns of vestibular hair cells

NASA Technical Reports Server (NTRS)

Baird, R. A.; Schuff, N. R.; Bancroft, J.

1993-01-01

Surface glycoconjugates of hair cells and supporting cells in the vestibular endorgans of the bullfrog were identified using biotinylated lectins with different carbohydrate specificities. Lectin binding in hair cells was consistent with the presence of glucose and mannose (CON A), galactose (RCA-I), N-acetylglucosamine (WGA), N-acetylgalactosamine (VVA), but not fucose (UEA-I) residues. Hair cells in the bullfrog sacculus, unlike those in the utriculus and semicircular canals, did not strain for N-acetylglucosamine (WGA) or N-acetylgalactosamine (VVA). By contrast, WGA and, to a lesser extent, VVA, differentially stained utricular and semicircular canal hair cells, labeling hair cells located in peripheral, but not central, regions. In mammals, WGA uniformly labeled Type I hair cells while labeling, as in the bullfrog, Type II hair cells only in peripheral regions. These regional variations were retained after enzymatic digestion. We conclude that vestibular hair cells differ in their surface glycoconjugates and that differences in lectin binding patterns can be used to identify hair cell types and to infer the epithelial origin of isolated vestibular hair cells.

Mannose glycoconjugates functionalized at positions 1 and 6. Binding analysis to DC-SIGN using biosensors.

PubMed

Reina, José J; Maldonado, Olivia S; Tabarani, Georges; Fieschi, Franck; Rojo, Javier

2007-01-01

The design of glycoconjugates to allow the generation of multivalent ligands capable of interacting with the receptor DC-SIGN is a topic of high interest due to the role played by this lectin in pathogen infections. Mannose, a ligand of this lectin, could be conjugated at two different positions, 1 and 6, not implicated in the binding process. We have prepared mannose conjugates at these two positions with a long spacer to allow their attachment to a biosensor chip surface. Analysis of the interaction between these surfaces and the tetravalent extracellular domain (ECD) of DC-SIGN by SPR biosensor has demonstrated that both positions are available for this conjugation without affecting the protein binding process. These results emphasize the possibility to conjugate mannose at position 6, allowing the incorporation of hydrophobic groups at the anomeric position to interact with hydrophobic residues in the carbohydrate recognition domain of DC-SIGN, increasing binding affinities. This fact is relevant for the future design of new ligands and the corresponding multivalent systems for DC-SIGN.

Gastrin-releasing peptide stimulates glycoconjugate release from feline trachea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lundgren, J.D.; Baraniuk, J.N.; Ostrowski, N.L.

1990-02-01

The effect of gastrin-releasing peptide (GRP) on respiratory glycoconjugate (RGC) secretion was investigated in a feline tracheal organ culture model. RGC secretion was stimulated by GRP in a dose-dependent fashion at concentrations from 10(-8) to 10(-5) M (range 15-38% increase above control) with a peak effect within 0.5-1 h of incubation. GRP-(14-27), the receptor binding portion of GRP, and the related molecule, bombesin, also stimulated RGC secretion by approximately 20% above control. Acetyl-GRP-(20-27) stimulated RGC release by 10%, whereas GRP-(1-16) was inactive. Autoradiographic studies with 125I-GRP revealed that specific binding was restricted to the submucosal glands and the surface epithelium.more » A specific radioimmunoassay showed the content of GRP in feline trachea after extraction with ethanol-acetic acid to be 156 +/- 91 fmol/g wet wt. Indirect immunohistochemistry indicated that ganglion cells located just outside the cartilage contained GRP-immunoreactive materials. GRP is a novel mucus secretagogue that may participate in regulating airway mucosal gland secretion.« less

Immunologic memory response induced by a meningococcal serogroup C conjugate vaccine using the P64k recombinant protein as carrier.

PubMed

Guirola, María; Urquiza, Dioslaida; Alvarez, Anabel; Cannan-Haden, Leonardo; Caballero, Evelin; Guillén, Gerardo

2006-03-01

In this study, we used an adoptive lymphocyte transfer experiment to evaluate the ability of the P64k recombinant protein to recruit T-helper activity and induce immunologic memory response to the polysaccharide moiety in a meningococcal serogroup C conjugate vaccine. Adoptive transfer of splenocytes from mice immunized with the glycoconjugate conferred antipolysaccharide immunologic memory to naive recipient mice. The observed anamnestic immune response was characterized by more rapid kinetics, isotype switching from IgM to IgG and higher antipolysaccharide antibody titers compared with those reached in groups transferred with splenocytes from plain polysaccharide or phosphate-immunized mice. The memory response generated was also long lasting. Sera from mice transferred with cells from conjugate-immunized mice were the only protective in the infant rat passive protection assay, and also showed higher bactericidal titers. We demonstrated that priming the mice immune system with the glycoconjugate using the P64k protein as carrier induced a memory response to the polysaccharide, promoting a switch of the T-cell-independent response to a T-cell dependent one.

Sialyldisaccharide conformations: a molecular dynamics perspective

NASA Astrophysics Data System (ADS)

Selvin, Jeyasigamani F. A.; Priyadarzini, Thanu R. K.; Veluraja, Kasinadar

2012-04-01

Sialyldisaccharides are significant terminal components of glycoconjugates and their negative charge and conformation are extensively utilized in molecular recognition processes. The conformation and flexibility of four biologically important sialyldisaccharides [Neu5Acα(2-3)Gal, Neu5Acα(2-6)Gal, Neu5Acα(2-8)Neu5Ac and Neu5Acα(2-9)Neu5Ac] are studied using Molecular Dynamics simulations of 20 ns duration to deduce the conformational preferences of the sialyldisaccharides and the interactions which stabilize the conformations. This study clearly describes the possible conformational models of sialyldisaccharides deduced from 20 ns Molecular Dynamics simulations and our results confirm the role of water in the structural stabilization of sialyldisaccharides. An extensive analysis on the sialyldisaccharide structures available in PDB also confirms the conformational regions found by experiments are detected in MD simulations of 20 ns duration. The three dimensional structural coordinates for all the MD derived sialyldisaccharide conformations are deposited in the 3DSDSCAR database and these conformational models will be useful for glycobiologists and biotechnologists to understand the biological functions of sialic acid containing glycoconjugates.

Divergent and convergent synthesis of GalNAc-conjugated dendrimers using dual orthogonal ligations.

PubMed

Thomas, Baptiste; Pifferi, Carlo; Daskhan, Gour Chand; Fiore, Michele; Berthet, Nathalie; Renaudet, Olivier

2015-12-21

The synthesis of glycodendrimers remains a challenging task. In this paper we propose a protocol based on both oxime ligation (OL) to combine cyclopeptide repeating units as the dendritic core and the copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC) to conjugate peripheral α and β propargylated GalNAc. By contrast with the oxime-based iterative protocol reported in our group, our current strategy can be used in both divergent and convergent routes with similar efficiency and the resulting hexadecavalent glycodendrimers can be easily characterized compared to oxime-linked analogues. A series of glycoconjugates displaying four or sixteen copies of both α and β GalNAc have been prepared and their ability to inhibit the adhesion of the soybean agglutinin (SBA) lectin to polymeric-GalNAc immobilized on microtiter plates has been evaluated. As was anticipated, the higher inhibitory effect (IC50 = 0.46 μM) was measured with the structure displaying αGalNAc with the higher valency (compound 13), which demonstrates that the binding properties of these glycoconjugates are strongly dependent on the orientation and distribution of the GalNAc units.

Gastrin-releasing peptide in human nasal mucosa.

PubMed

Baraniuk, J N; Lundgren, J D; Goff, J; Peden, D; Merida, M; Shelhamer, J; Kaliner, M

1990-04-01

Gastrin-releasing peptide (GRP), the 27 amino acid mammalian form of bombesin, was studied in human inferior turbinate nasal mucosa. The GRP content of the mucosa measured by radioimmunoassay was 0.60 +/- 0.25 pmol/g tissue (n = 9 patients; mean +/- SEM). GRP-immunoreactive nerves detected by the immunogold method of indirect immunohistochemistry were found predominantly in small muscular arteries, arterioles, venous sinusoids, and between submucosal gland acini. 125I-GRP binding sites determined by autoradiography were exclusively and specifically localized to nasal epithelium and submucosal glands. There was no binding to vessels. The effects of GRP on submucosal gland product release were studied in short-term explant culture. GRP (10 microM) significantly stimulated the release of the serous cell-specific product lactoferrin, and [3H]glucosamine-labeled glycoconjugates which are products of epithelial goblet cells and submucosal gland cells. These observations indicate that GRP released from nerve fibers probably acts on glandular GRP receptors to induce glycoconjugate release from submucosal glands and epithelium and lactoferrin release from serous cells, but that GRP would probably not affect vascular permeability.

Trimodal Control of Ion-Transport Activity on Cyclo-oligo-(1→6)-β-D-glucosamine-Based Artificial Ion-Transport Systems.

PubMed

Roy, Arundhati; Saha, Tanmoy; Gening, Marina L; Titov, Denis V; Gerbst, Alexey G; Tsvetkov, Yury E; Nifantiev, Nikolay E; Talukdar, Pinaki

2015-11-23

Cyclo-oligo-(1→6)-β-D-glucosamines functionalized with hydrophobic tails are reported as a new class of transmembrane ion-transport system. These macrocycles with hydrophilic cavities were introduced as an alternative to cyclodextrins, which are supramolecular systems with hydrophobic cavities. The transport activities of these glycoconjugates were manipulated by altering the oligomericity of the macrocycles, as well as the length and number of attached tails. Hydrophobic tails of 3 different sizes were synthesized and coupled with each glucosamine scaffold through the amide linkage to obtain 18 derivatives. The ion-transport activity increased from di- to tetrameric glucosamine macrocycles, but decreased further when flexible pentameric glucosamine was introduced. The ion-transport activity also increased with increasing length of attached linkers. For a fixed length of linkers, the transport activity decreased when the number of such tails was reduced. All glycoconjugates displayed a uniform anion-selectivity sequence: Cl(-) >Br(-) >I(-) . From theoretical studies, hydrogen bonding between the macrocycle backbone and the anion bridged through water molecules was observed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid assays for lectin toxicity and binding changes that reflect altered glycosylation in mammalian cells.

PubMed

Stanley, Pamela; Sundaram, Subha

2014-06-03

Glycosylation engineering is used to generate glycoproteins, glycolipids, or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans with truncated glycans missing the sugar transferred by that glycosyltransferase, as well as those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain-of-function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large-volume cultures of mammalian cells may also generate spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan-binding assay. Based on these tests, glycosylation changes expressed by a cell can be revealed, and glycosylation mutants classified into phenotypic groups that may reflect a loss-of-function or gain-of-function mutation in a specific gene involved in glycan synthesis. Copyright © 2014 John Wiley & Sons, Inc.

Development of a glycoconjugate vaccine to prevent invasive Salmonella Typhimurium infections in sub-Saharan Africa

PubMed Central

Baliban, Scott M.; Yang, Mingjun; Ramachandran, Girish; Curtis, Brittany; Shridhar, Surekha; Laufer, Rachel S.; Wang, Jin Y.; Van Druff, John; Higginson, Ellen E.; Hegerle, Nicolas; Varney, Kristen M.; Galen, James E.; Tennant, Sharon M.; Lees, Andrew; MacKerell, Alexander D.; Levine, Myron M.; Simon, Raphael

2017-01-01

Invasive infections associated with non-typhoidal Salmonella (NTS) serovars Enteritidis (SE), Typhimurium (STm) and monophasic variant 1,4,[5],12:i:- are a major health problem in infants and young children in sub-Saharan Africa, and currently, there are no approved human NTS vaccines. NTS O-polysaccharides and flagellin proteins are protective antigens in animal models of invasive NTS infection. Conjugates of SE core and O-polysaccharide (COPS) chemically linked to SE flagellin have enhanced the anti-COPS immune response and protected mice against fatal challenge with a Malian SE blood isolate. We report herein the development of a STm glycoconjugate vaccine comprised of STm COPS conjugated to the homologous serovar phase 1 flagellin protein (FliC) with assessment of the role of COPS O-acetyls for functional immunity. Sun-type COPS conjugates linked through the polysaccharide reducing end to FliC were more immunogenic and protective in mice challenged with a Malian STm blood isolate than multipoint lattice conjugates (>95% vaccine efficacy [VE] versus 30–43% VE). Immunization with de-O-acetylated STm-COPS conjugated to CRM197 provided significant but reduced protection against STm challenge compared to mice immunized with native STm-COPS:CRM197 (63–74% VE versus 100% VE). Although OPS O-acetyls were highly immunogenic, post-vaccination sera that contained various O-acetyl epitope-specific antibody profiles displayed similar in vitro bactericidal activity when equivalent titers of anti-COPS IgG were assayed. In-silico molecular modeling further indicated that STm OPS forms a single dominant conformation, irrespective of O-acetylation, in which O-acetyls extend outward and are highly solvent exposed. These preclinical results establish important quality attributes for an STm vaccine that could be co-formulated with an SE-COPS:FliC glycoconjugate as a bivalent NTS vaccine for use in sub-Saharan Africa. PMID:28388624

Carrier priming with CRM 197 or diphtheria toxoid has a different impact on the immunogenicity of the respective glycoconjugates: biophysical and immunochemical interpretation.

PubMed

Pecetta, S; Lo Surdo, P; Tontini, M; Proietti, D; Zambonelli, C; Bottomley, M J; Biagini, M; Berti, F; Costantino, P; Romano, M R

2015-01-03

Glycoconjugate vaccines play an enormous role in preventing infectious diseases. The main carrier proteins used in commercial conjugate vaccines are the non-toxic mutant of diphtheria toxin (CRM197), diphtheria toxoid (DT) and tetanus toxoid (TT). Modern childhood routine vaccination schedules include the administration of several vaccines simultaneously or in close sequence, increasing the concern that the repeated exposure to conjugates based on these carrier proteins might interfere with the anti-polysaccharide response. Extending previous observations we show here that priming mice with CRM197 or DT does not suppress the response to the carbohydrate moiety of CRM197 meningococcal serogroup A (MenA) conjugates, while priming with DT can suppress the response to DT-MenA conjugates. To explain these findings we made use of biophysical and immunochemical techniques applied mainly to MenA conjugates. Differential scanning calorimetry and circular dichroism data revealed that the CRM197 structure was altered by the chemical conjugation, while DT and the formaldehyde-treated form of CRM197 were less impacted, depending on the degree of glycosylation. Investigating the binding and avidity properties of IgGs induced in mice by non-conjugated carriers, we found that CRM197 induced low levels of anti-carrier antibodies, with decreased avidity for its MenA conjugates and poor binding to DT and respective MenA conjugates. In contrast, DT induced high antibody titers able to bind with comparable avidity both the protein and its conjugates but showing very low avidity for CRM197 and related conjugates. The low intrinsic immunogenicity of CRM197 as compared to DT, the structural modifications induced by glycoconjugation and detoxification processes, resulting in conformational changes in CRM197 and DT epitopes with consequent alteration of the antibody recognition and avidity, might explain the different behavior of CRM197 and DT in a carrier priming context. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hypothalamic digoxin, hemispheric chemical dominance, and inflammatory bowel disease.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2003-09-01

The isoprenoid pathway produces three key metabolites--endogenous digoxin, dolichol, and ubiquinone. It was considered pertinent to assess the pathway in inflammatory bowel disease (ulcerative colitis and regional ileitis). Since endogenous digoxin can regulate neurotransmitter transport, the pathway and the related cascade were also assessed in individuals with differing hemispheric dominance to find out the role of hemispheric dominance in its pathogenesis. All the patients with inflammatory bowel disease were right-handed/left hemispheric dominant by the dichotic listening test. The following parameters were measured in patients with inflammatory bowel disease and in individuals with differing hemispheric dominance: (1) plasma HMG CoA reductase, digoxin, dolichol, ubiquinone, and magnesium levels; (2) tryptophan/tyrosine catabolic patterns; (3) free-radical metabolism; (4) glycoconjugate metabolism; and (5) membrane composition and RBC membrane Na+-K+ ATPase activity. Statistical analysis was done by ANOVA. In patients with inflammatory bowel disease there was elevated digoxin synthesis, increased dolichol and glycoconjugate levels, and low ubiquinone and elevated free radical levels. There was also an increase in tryptophan catabolites and a reduction in tyrosine catabolites. There was an increase in cholesterol:phospholipid ratio and a reduction in glycoconjugate level of RBC membrane in these groups of patients. Inflammatory bowel disease is associated with an upregulated isoprenoid pathway and elevated digoxin secretion from the hypothalamus. This can contribute to immune activation, defective glycoprotein bowel antigen presentation, and autoimmunity and a schizophreniform psychosis important in its pathogenesis. The biochemical patterns obtained in inflammatory bowel disease is similar to those obtained in left-handed/right hemispheric dominant individuals by the dichotic listening test. But all the patients with peptic ulcer disease were right-handed/left hemispheric dominant by the dichotic listening test. Hemispheric chemical dominance has no correlation with handedness or the dichotic listening test. Inflammatory bowel disease occurs in right hemispheric chemically dominant individuals and is a reflection of altered brain function.

Hypothalamic digoxin and hemispheric chemical dominance--relation to the pathogenesis of senile osteoporosis, degenerative osteoarthritis, and spondylosis.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2003-03-01

The isoprenoid pathway produces three key metabolites: i) digoxin (a membrane sodium-potassium ATPase inhibitor which can regulate intracellular calcium/magnesium ratios), ii) dolichol (which regulates N-glycosylation of proteins), and iii) ubiquinone (a free radical scavenger), all of which are important in bone and joint metabolism. The pathway was assessed in senile osteoporosis, spondylosis, and osteoarthritis. Digoxin could possibly play a role in the genesis of cerebral dominance because it can regulate multiple neurotransmitter systems. The pathway was also assessed in individuals of differing hemispheric dominance for comparison and to find out the role of cerebral dominance in the pathogenesis of these diseases. The plasma/serum-activity of HMG CoA reductase, magnesium, digoxin, dolichol, ubiquinone, and tryptophan/tyrosine catabolic patterns, as well as RBC Na(+)-K+ ATPase activity, were measured in the above mentioned groups. The glycoconjugate metabolism, free radical metabolism, and membrane composition were also studied. The pathway was upregulated with increased digoxin synthesis in patients with spondylosis and osteoarthritis. In this group of patients, the glycoconjugate levels and dolichol levels were increased and lysosomal stability reduced. The ubiquinone levels were low and free radicals increased in spondylosis and osteoarthritis. On the other hand, in senile osteoporosis, the isoprenoid pathway was downregulated and digoxin synthesis reduced. The glycoconjugate and dolichol levels were low and lysosomal stability increased. The ubiquinone levels were increased and free radical production increased in senile osteoporosis. The significance of these changes in the pathogenesis of osteoarthritis, spondylosis, and osteoporosis is discussed. The hyperdigoxinemic state is seen in osteoarthritis and spondylosis and in right hemispheric dominance. The hypodigoxinemic state is seen in left hemispheric dominance and senile osteoporosis. Hemispheric dominance plays a crucial role in deciding the predisposition to bone and joint diseases. Right hemispheric chemical dominance predisposes to spondylosis and osteoarthritis. Left hemispheric chemical dominance predisposes to osteoporosis.

Changes of glycoconjugate expression in nasal respiratory mucosa of rats exposed to welding fumes.

PubMed

Jeong, Gil Nam; Jo, Un Bock; Yu, Il Je

2007-09-01

To investigate the effects of welding fumes on the glycoconjugates in nasal respiratory mucosa, male Sprague-Dawley rats were exposed to manual metal arc stainless steel (MMA-SS) welding fumes at a concentration of 56-76 mg/m(3) total suspended particulate for 2 h/day in an inhalation chamber for 90 days. During the exposure period, the experimental animals were sacrificed after 2 h and 15, 30, 60, and 90 days of exposure; then sections were examined using lectin histochemistry. Some remarkable changes, such as destroyed cilia, desquamation and mucification of epithelial cells, and destruction of nasal septal glands, were seen in the welding fume-exposed groups. Specific changes in the lectin binding patterns were also observed in the welding fume-exposed rats. The Ricinus communis agglutinin-I (RCA-I) staining of the cilia and columnar cells increased slightly when compared with the unexposed rats. The RCA-I and Ulex europaeus agglutinin-I (UEA-I) staining of the goblet cells also increased as the exposure continued. The mucigenous epithelial cells reacted with Bandeiraea simplicifolia lectin-I (BSL-I), RCA-I, and succinylated wheat germ agglutinin A (sWGA) after 15 days of exposure, which was not visible in the control group. The dorsal septal glands exhibited an affinity with peanut agglutinin (PNA), BSL-I, and RCA-I, which was also not visible in the control group. The affinity for Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), PNA, sWGA, BSL-I, and UEA-I in the ventral septal glands of the welding fume-exposed groups tended to increase, whereas the concanavalin A (Con A) reactivity in the dorsal septal glands decreased slightly. In conclusion, it was assumed that the changes in the glycoconjugate residues in the nasal respiratory mucosa of the welding fume-exposed rats represented important components of defense mechanisms against the toxicants in the welding fumes.

Development of a glycoconjugate vaccine to prevent invasive Salmonella Typhimurium infections in sub-Saharan Africa.

PubMed

Baliban, Scott M; Yang, Mingjun; Ramachandran, Girish; Curtis, Brittany; Shridhar, Surekha; Laufer, Rachel S; Wang, Jin Y; Van Druff, John; Higginson, Ellen E; Hegerle, Nicolas; Varney, Kristen M; Galen, James E; Tennant, Sharon M; Lees, Andrew; MacKerell, Alexander D; Levine, Myron M; Simon, Raphael

2017-04-01

Invasive infections associated with non-typhoidal Salmonella (NTS) serovars Enteritidis (SE), Typhimurium (STm) and monophasic variant 1,4,[5],12:i:- are a major health problem in infants and young children in sub-Saharan Africa, and currently, there are no approved human NTS vaccines. NTS O-polysaccharides and flagellin proteins are protective antigens in animal models of invasive NTS infection. Conjugates of SE core and O-polysaccharide (COPS) chemically linked to SE flagellin have enhanced the anti-COPS immune response and protected mice against fatal challenge with a Malian SE blood isolate. We report herein the development of a STm glycoconjugate vaccine comprised of STm COPS conjugated to the homologous serovar phase 1 flagellin protein (FliC) with assessment of the role of COPS O-acetyls for functional immunity. Sun-type COPS conjugates linked through the polysaccharide reducing end to FliC were more immunogenic and protective in mice challenged with a Malian STm blood isolate than multipoint lattice conjugates (>95% vaccine efficacy [VE] versus 30-43% VE). Immunization with de-O-acetylated STm-COPS conjugated to CRM197 provided significant but reduced protection against STm challenge compared to mice immunized with native STm-COPS:CRM197 (63-74% VE versus 100% VE). Although OPS O-acetyls were highly immunogenic, post-vaccination sera that contained various O-acetyl epitope-specific antibody profiles displayed similar in vitro bactericidal activity when equivalent titers of anti-COPS IgG were assayed. In-silico molecular modeling further indicated that STm OPS forms a single dominant conformation, irrespective of O-acetylation, in which O-acetyls extend outward and are highly solvent exposed. These preclinical results establish important quality attributes for an STm vaccine that could be co-formulated with an SE-COPS:FliC glycoconjugate as a bivalent NTS vaccine for use in sub-Saharan Africa.

TYCTWD Programs Strive to Make Science Educational and Fun | Poster

Cancer.gov

By Carolynne Keenan, Contributing Writer Joseph Barchi, Jr, Ph.D., calls teaching “the noblest and most important profession.” So it makes sense that Barchi, senior scientist and head of the Glycoconjugate and NMR Section, Chemical Biology Laboratory, Center for Cancer Research, NCI at Frederick, would encourage his lab to offer a fun, educational program at Take Your Child to

Binding Affinity of Glycoconjugates to BACILLUS Spores and Toxins

NASA Astrophysics Data System (ADS)

Rasol, Aveen; Eassa, Souzan; Tarasenko, Olga

2010-04-01

Early recognition of Bacillus cereus group species is important since they can cause food-borne illnesses and deadly diseases in humans. Glycoconjugates (GCs) are carbohydrates covalently linked to non-sugar moieties including lipids, proteins or other entities. GCs are involved in recognition and signaling processes intrinsic to biochemical functions in cells. They also stimulate cell-cell adhesion and subsequent recognition and activation of receptors. We have demonstrated that GCs are involved in Bacillus cereus spore recognition. In the present study, we have investigated whether GCs possess the ability to bind and recognize B. cereus spores and Bacillus anthracis recombinant single toxins (sTX) and complex toxins (cTX). The affinity of GCs to spores + sTX and spores + cTX toxins was studied in the binding essay. Our results demonstrated that GC9 and GC10 were able to selectively bind to B. cereus spores and B. anthracis toxins. Different binding affinities for GCs were found toward Bacillus cereus spores + sTX and spores + cTX. Dilution of GCs does not impede the recognition and binding. Developed method provides a tool for simultaneous recognition and targeting of spores, bacteria toxins, and/or other entities.

Fungal lectins: a growing family.

PubMed

Kobayashi, Yuka; Kawagishi, Hirokazu

2014-01-01

Fungi are members of a large group of eukaryotic organisms that include yeasts and molds, as well as the most familiar member, mushrooms. Fungal lectins with unique specificity and structures have been discovered. In general, fungal lectins are classified into specific families based on their amino acid sequences and three-dimensional structures. In this chapter, we provide an overview of the approximately 80 types of mushroom and fungal lectins that have been isolated and studied to date. In particular, we have focused on ten fungal lectins (Agaricus bisporus, Agrocybe cylindracea, Aleuria aurantia, Aspergillus oryzae, Clitocybe nebularis, Marasmius oreades, Psathyrella velutina, Rhizopus stolonifer, Pholiota squarrosa, Polyporus squamosus), many of which are commercially available and their properties, sugar-binding specificities, structural grouping into families, and applications for biological research being described. The sialic acid-specific lectins (Agrocybe cylindracea and Polyporus squamosus) and fucose-specific lectins (Aleuria aurantia, Aspergillus oryzae, Rhizopus stolonifer, and Pholiota squarrosa) each showed potential for use in identifying sialic acid glycoconjugates and fucose glycoconjugates. Although not much is currently known about fungal lectins compared to animal and plant lectins, the knowledge accumulated thus far shows great promise for several applications in the fields of taxonomy, biomedicine, and molecular and cellular biology.

Immunization with the conjugate vaccine Vi-CRM₁₉₇ against Salmonella typhi induces Vi-specific mucosal and systemic immune responses in mice.

PubMed

Fiorino, Fabio; Ciabattini, Annalisa; Rondini, Simona; Pozzi, Gianni; Martin, Laura B; Medaglini, Donata

2012-09-21

Typhoid fever is a public health problem, especially among young children in developing countries. To address this need, a glycoconjugate vaccine Vi-CRM₁₉₇, composed of the polysaccharide antigen Vi covalently conjugated to the non-toxic mutant of diphtheria toxin CRM₁₉₇, is under development. Here, we assessed the antibody and cellular responses, both local and systemic, following subcutaneous injection of Vi-CRM₁₉₇. The glycoconjugate elicited Vi-specific serum IgG titers significantly higher than unconjugated Vi, with prevalence of IgG1 that persisted for at least 60 days after immunization. Vi-specific IgG, but not IgA, were present in intestinal washes. Lymphocytes proliferation after restimulation with Vi-CRM₁₉₇ was observed in spleen and mesenteric lymph nodes. These data confirm the immunogenicity of Vi-CRM₁₉₇ and demonstrate that the vaccine-specific antibody and cellular immune responses are present also in the intestinal tract, thus strengthening the suitability of Vi-CRM₁₉₇ as a promising candidate vaccine against Salmonella Typhi. Copyright © 2012 Elsevier Ltd. All rights reserved.

SV2 frustrating exocytosis at the semi-diffusor synapse.

PubMed

Vautrin, Jean

2009-04-01

Presynaptic exocytosis is the mechanism commonly believed to release transmitters by diffusion through a pore opening during vesicular membrane fusion with the plasmalemma, but evidence suggesting that exocytosis and transmitter release are two separate steps of synaptic transmission is accumulating. Vesicular glycoconjugates such as Synaptic Vesicle Protein 2 (SV2) proteoglycans and gangliosides retain transmitters in a nondiffusible form and are transported to the synaptic cleft where they contribute forming a dense synaptomatrix. Transmitters are permanently present in synaptic clefts and readily releasable transmitter is easily accessible from the outer side of the presynaptic membrane suggesting that synaptomatrix glycoconjugates prevent immediate release after PKC-dependent exocytosis. The calcium sensor synaptotagmin is also present at the presynaptic plasma membrane and binds SV2 suggesting a direct coupling between the calcium transient and transmitter release from the synaptomatrix. A quantitative coupling of the cytosolic calcic transient to transmitter release from the synaptomatrix explains better complexity and plasticity of miniature postsynaptic signals hitherto difficult to account for in exocytic terms. This alternative representation of synaptic transmission in which the same components of the synaptomatrix support adhesion and signaling functions may cast new lights on synaptic diseases such as Alzheimer's disease. Copyright 2008 Wiley-Liss, Inc.

The Pneumococcal Alpha-Glycerophosphate Oxidase Enhances Nasopharyngeal Colonization through Binding to Host Glycoconjugates.

PubMed

Mahdi, Layla K; Higgins, Melanie A; Day, Christopher J; Tiralongo, Joe; Hartley-Tassell, Lauren E; Jennings, Michael P; Gordon, David L; Paton, Adrienne W; Paton, James C; Ogunniyi, Abiodun D

2017-04-01

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen, causing a broad spectrum of diseases including otitis media, pneumonia, bacteraemia and meningitis. Here we examined the role of a potential pneumococcal meningitis vaccine antigen, alpha-glycerophosphate oxidase (SpGlpO), in nasopharyngeal colonization. We found that serotype 4 and serotype 6A strains deficient in SpGlpO have significantly reduced capacity to colonize the nasopharynx of mice, and were significantly defective in adherence to human nasopharyngeal carcinoma cells in vitro. We also demonstrate that intranasal immunization with recombinant SpGlpO significantly protects mice against subsequent nasal colonization by wild type serotype 4 and serotype 6A strains. Furthermore, we show that SpGlpO binds strongly to lacto/neolacto/ganglio host glycan structures containing the GlcNAcβ1-3Galβ disaccharide, suggesting that SpGlpO enhances colonization of the nasopharynx through its binding to host glycoconjugates. We propose that SpGlpO is a promising vaccine candidate against pneumococcal carriage, and warrants inclusion in a multi-component protein vaccine formulation that can provide robust, serotype-independent protection against all forms of pneumococcal disease. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

TYCTWD Programs Strive to Make Science Educational and Fun | Poster

Cancer.gov

By Carolynne Keenan, Contributing Writer Joseph Barchi, Jr, Ph.D., calls teaching “the noblest and most important profession.” So it makes sense that Barchi, senior scientist and head of the Glycoconjugate and NMR Section, Chemical Biology Laboratory, Center for Cancer Research, NCI at Frederick, would encourage his lab to offer a fun, educational program at Take Your Child to Work Day (TYCTWD).

Hypothalamic digoxin, hemispheric chemical dominance, and chronic bronchitis emphysema.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2003-09-01

The isoprenoid pathway produces three key metabolites--endogenous digoxin (membrane sodium-potassium ATPase inhibitor, immunomodulator, and regulator of neurotransmitter/amino acid transport), dolichol (regulates N-glycosylation of proteins), and ubiquinone (free radical scavenger). This was assessed in patients with chronic bronchitis emphysema. The pathway was also assessed in patients with right hemispheric, left hemispheric, and bihemispheric dominance to find the role of hemispheric dominance in the pathogenesis of chronic bronchitis emphysema. All the 15 patients with chronic bronchitis emphysema were right-handed/left hemispheric dominant by the dichotic listening test. In patients with chronic bronchitis emphysema there was elevated digoxin synthesis, increased dolichol, and glycoconjugate levels, and low ubiquinone and elevated free radical levels. There was also an increase in tryptophan catabolites and a reduction in tyrosine catabolites. There was an increase in cholesterol:phospholipid ratio and a reduction in glycoconjugate levels of RBC membrane in patients with chronic bronchitis emphysema. The same biochemical patterns were obtained in individuals with right hemispheric dominance. Endogenous digoxin by activating the calcineurin signal transduction pathway of T-cell can contribute to immune activation in chronic bronchitis emphysema. Increased free radical generation can also lead to immune activation. Endogenous synthesis of nicotine can contribute to the pathogenesis of the disease. Altered glycoconjugate metabolism and membranogenesis can lead to defective lysosomal stability contributing to the disease process by increased release of lysosomal proteases. The role of an endogenous digoxin and hemispheric dominance in the pathogenesis of chronic bronchitis emphysema and in the regulation of lung structure/function is discussed. The biochemical patterns obtained in chronic bronchitis emphysema is similar to those obtained in left-handed/right hemispheric chemically dominant individuals by the dichotic listening test. But all the patients with chronic bronchitis emphysema were right-handed/left hemispheric dominant by the dichotic listening test. Hemispheric chemical dominance has no correlation with handedness or the dichotic listening test. Chronic bronchitis emphysema occurs in right hemispheric chemically dominant individuals and is a reflection of altered brain function. Hemispheric chemical dominance can play a role in the regulation of lung function and structure.

Anti-Group B Streptococcus Glycan-Conjugate Vaccines Using Pilus Protein GBS80 As Carrier and Antigen: Comparing Lysine and Tyrosine-directed Conjugation.

PubMed

Nilo, Alberto; Morelli, Laura; Passalacqua, Irene; Brogioni, Barbara; Allan, Martin; Carboni, Filippo; Pezzicoli, Alfredo; Zerbini, Francesca; Maione, Domenico; Fabbrini, Monica; Romano, Maria Rosaria; Hu, Qi-Ying; Margarit, Immaculada; Berti, Francesco; Adamo, Roberto

2015-07-17

Gram-positive Streptococcus agalactiae or group B Streptococcus (GBS) is a leading cause of invasive infections in pregnant women, newborns, and elderly people. Vaccination of pregnant women represents the best strategy for prevention of neonatal disease, and GBS polysaccharide-based conjugate vaccines are currently under clinical testing. The potential of GBS pilus proteins selected by genome-based reverse vaccinology as protective antigens for anti-streptococcal vaccines has also been demonstrated. Dressing pilus proteins with surface glycan antigens could be an attractive approach to extend vaccine coverage. We have recently developed an efficient method for tyrosine-directed ligation of large glycans to proteins via copper-free azide-alkyne [3 + 2] cycloaddition. This method enables targeting of predetermined sites of the protein, ensuring that protein epitopes are preserved prior to glycan coupling and a higher consistency in glycoconjugate batches. Herein, we compared conjugates of the GBS type II polysaccharide (PSII) and the GBS80 pilus protein obtained by classic lysine random conjugation and by the recently developed tyrosine-directed ligation. PSII conjugated to CRM197, a carrier protein used for vaccines in the market, was used as a control. We found that the constructs made from PSII and GBS80 were able to elicit murine antibodies recognizing individually the glycan and protein epitopes on the bacterial surface. The generated antibodies were efficacious in mediating opsonophagocytic killing of strains expressing exclusively PSII or GBS80 proteins. The two glycoconjugates were also effective in protecting newborn mice against GBS infection following vaccination of the dams. Altogether, these results demonstrated that polysaccharide-conjugated GBS80 pilus protein functions as a carrier comparably to CRM197, while maintaining its properties of protective protein antigen. Glycoconjugation and reverse vaccinology can, therefore, be combined to design vaccines with broad coverage. This approach opens a path to a new generation of vaccines. Tyrosine-ligation allows creation of more homogeneous vaccines, correlation of the immune response to defined connectivity points, and fine-tuning of the conjugation site in glycan-protein conjugates.

Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

Treesearch

Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

2001-01-01

BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

Preparation of water-soluble glycoconjugated poly(acrylamide) for NMR analyses of carbohydrate-carbohydrate interactions

NASA Astrophysics Data System (ADS)

Xuan, Trinh Anh; Trung, Phan Nghia; Dinh, Bui Long; Yamaguchi, Takumi; Kato, Koichi

2014-05-01

Oligosaccharide chains of glycoconjugates are important biopolymers not only as carriers of information in cell-cell interactions but also as markers of cellular differentiation, aging, and malignant alteration. Molecular interactions where carbohydrates are involved are usually considered as weak interactions, so the study and evaluation of these interactions is still in its infancy. The evidences and studies of carbohydrate-carbohydrate interactions (CCI) will be confirming the importance of this mechanism for specific cell adhesion and communication. Their development will go hand in hand with the development of new and more sensitive techniques to study weak interactions. Recently, synthetic glycopolymers with functions similar to those of such natural carbohydrates and with specific pendant saccharide moieties were used as a solution for enhancement CCI when forming polyvalent interactions. Carbohydrates are ubiquitous components of cell wall membranes and occur as glycolipids, glycoproteins, proteoglycans, and capsular polysaccharides. As such they can participate in forefront intramolecular and intracellular events. Apart from their recognized roles in the physicochemical properties of glycolipids and glycoproteins. In this study, we designed trisaccharide monomers for free radical polymerization. Subsequently, the trisaccharide unit for chemical conjugation was synthesized from galactosamine in good yield. For further NMR analyses of CCI, glycopolymers composed of these sugar derivatives will be provided.

Glycoconjugate sugar residues in the chick embryo developing lung: a lectin histochemical study.

PubMed

Gheri, G; Sgambati, E; Bryk, S G

2000-03-01

A lectin histochemical study was performed to investigate the distribution and changes of the oligosaccharidic component of the glycoconjugates in the lung of chick embryos, of 1-day-old chick, and of the adult animal. For this purpose, a battery of seven horseradish peroxidase-conjugated lectins (PNA, SBA, DBA, WGA, Con A, LTA, and UEA I) were employed. During the first phase of parabronchi and atria formation, D-galactose-(beta1-->3)-N-acetyl-D-galactosamine, beta-N-acetyl-D-galactosamine, D-glucosamine, alpha-D-mannose, and sialic acid, present at the level of the surface and of cytoplasmic granules of the lining epithelial cells, seem to play a role in regulating morphogenetic phenomena. In the subsequent phases, the parabronchial lumen and the atrial cavities were characterized by the presence of lectin-reactive material rich in terminal D-galactose-(beta1-->3)-N-acetyl-D-galactosamine, beta-N-acetyl-D-galactosamine, D-glucosamine and alpha-D-mannose. From day 18 onwards and immediately after hatching, the free border of the cells lining the air capillaries was characterized by the presence of beta-N-acetyl-D-galactosamine and alpha-D-mannose. The appearance of these sugar residues was concomitant with the beginning of respiratory activity. Copyright 2000 Wiley-Liss, Inc.

Hypothalamic-mediated model for systemic lupus erythematosis: relation to hemispheric chemical dominance.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2003-11-01

The isoprenoid pathway including endogenous digoxin was assessed in systemic lupus erythematosis (SLE). All the patients with SLE were right-handed/left hemispheric dominant by the dichotic listening test. This was also studied for comparison in patients with right hemispheric and left hemispheric dominance. The isoprenoid pathway was upregulated with increased digoxin synthesis in patients with SLE and in those with right hemispheric dominance. In this group of patients (i) the tryptophan catabolites were increased and the tyrosine catabolites reduced, (ii) the dolichol and glycoconjugate levels were elevated, (iii) lysosomal stability was reduced, (iv) ubiquinone levels were low and free radical levels increased, and (v) the membrane cholesterol:phospholipid ratios were increased and membrane glycoconjugates reduced. On the other hand, in patients with left hemispheric dominance the reverse patterns were obtained. The biochemical patterns obtained in SLE is similar to those obtained in left-handed/right hemispheric chemically dominant individuals. But all the patients with SLE were right-handed/left hemispheric dominant by the dichotic listening test. Hemispheric chemical dominance has no correlation with handedness or the dichotic listening test. SLE occurs in right hemispheric chemically dominant individuals, and is a reflection of altered brain function. The role of the isoprenoid pathway in the pathogenesis of SLE and its relation to hemispheric dominance is discussed.

Glycoconjugates Effects: do Gender and Ethnicity Influence Exposure of Pathogen by Peripheral Mononuclear Cells ?

NASA Astrophysics Data System (ADS)

Lahiani, Mohamed; Tarasenko, Olga

2010-04-01

Members of the Bacillus cereus group demonstrate different pathological effects. B. cereus is a spore-forming, gram positive bacterium responsible for most foodborne illnesses. It was shown that susceptibility to infection and response to vaccines or treatments can be attributed to specific immunogenetic factors including gender and ethnicity. Glycoconjugate polymers (GCs) are potentially important in pharmaceutical and biomedical research. Our group has shown that GCs activate murine macrophages and promote killing of Bacillus cereus spores during phagocytosis. We hypothesized that the GCs effects are independent from gender and race. The goal of the present study was two-folds: A) determine whether GCs influence on human PMNC exposure of B. cereus spores and B) analyze whether gender and ethnicity influence of the effect of GCs. GCs were studied during exposure and post-exposure conditions. Phagocytosis was performed during exposure of PMNC to Bacillus spores. Post-exposure analysis involved cytotoxicity, cell viability and activation, and colonies forming unit. GC1 and GC3 enhance Bacillus spore killing. GC1 proved more effective than GC3 in spore killing while activating PMNC. Results demonstrate GCs effect were independent from ethnicity or gender. Findings of this research demonstrated that GC can be used as ligands to stimulate PMNC and kill B. cereus spores.

Differential Lectin Binding Patterns Identify Distinct Heart Regions in Giant Danio (Devario aequipinnatus) and Zebrafish (Danio rerio) Hearts

PubMed Central

Manalo, Trina; May, Adam; Quinn, Joshua; Lafontant, Dominique S.; Shifatu, Olubusola; He, Wei; Gonzalez-Rosa, Juan M.; Burns, Geoffrey C.; Burns, Caroline E.; Burns, Alan R.; Lafontant, Pascal J.

2016-01-01

Lectins are carbohydrate-binding proteins commonly used as biochemical and histochemical tools to study glycoconjugate (glycoproteins, glycolipids) expression patterns in cells, tissues, including mammalian hearts. However, lectins have received little attention in zebrafish (Danio rerio) and giant danio (Devario aequipinnatus) heart studies. Here, we sought to determine the binding patterns of six commonly used lectins—wheat germ agglutinin (WGA), Ulex europaeus agglutinin, Bandeiraea simplicifolia lectin (BS lectin), concanavalin A (Con A), Ricinus communis agglutinin I (RCA I), and Lycopersicon esculentum agglutinin (tomato lectin)—in these hearts. Con A showed broad staining in the myocardium. WGA stained cardiac myocyte borders, with binding markedly stronger in the compact heart and bulbus. BS lectin, which stained giant danio coronaries, was used to measure vascular reconstruction during regeneration. However, BS lectin reacted poorly in zebrafish. RCA I stained the compact heart of both fish. Tomato lectin stained the giant danio, and while low reactivity was seen in the zebrafish ventricle, staining was observed in their transitional cardiac myocytes. In addition, we observed unique staining patterns in the developing zebrafish heart. Lectins’ ability to reveal differential glycoconjugate expression in giant danio and zebrafish hearts suggests they can serve as simple but important tools in studies of developing, adult, and regenerating fish hearts. PMID:27680670

Single-molecule analysis of the major glycopolymers of pathogenic and non-pathogenic yeast cells

NASA Astrophysics Data System (ADS)

El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Alsteens, David; Sarazin, Aurore; Jouault, Thierry; Dufrêne, Yves F.

2013-05-01

Most microbes are coated with carbohydrates that show remarkable structural variability and play a crucial role in mediating microbial-host interactions. Understanding the functions of cell wall glycoconjugates requires detailed knowledge of their molecular organization, diversity and heterogeneity. Here we use atomic force microscopy (AFM) with tips bearing specific probes (lectins, antibodies) to analyze the major glycopolymers of pathogenic and non-pathogenic yeast cells at molecular resolution. We show that non-ubiquitous β-1,2-mannans are largely exposed on the surface of native cells from pathogenic Candida albicans and C. glabrata, the former species displaying the highest glycopolymer density and extensions. We also find that chitin, a major component of the inner layer of the yeast cell wall, is much more abundant in C. albicans. These differences in molecular properties, further supported by flow cytometry measurements, may play an important role in strengthening cell wall mechanics and immune interactions. This study demonstrates that single-molecule AFM, combined with immunological and fluorescence methods, is a powerful platform in fungal glycobiology for probing the density, distribution and extension of specific cell wall glycoconjugates. In nanomedicine, we anticipate that this new form of AFM-based nanoglycobiology will contribute to the development of sugar-based drugs, immunotherapeutics, vaccines and diagnostics.

Specific storage of glycoconjugates with terminal α-galactosyl moieties in the exocrine pancreas of Fabry disease patients with blood group B.

PubMed

Rybová, Jitka; Kuchar, Ladislav; Hulková, Helena; Asfaw, Befekadu; Dobrovolný, Robert; Sikora, Jakub; Havlícek, Vladimír; Škultéty, Ludovít; Ledvinová, Jana

2018-06-01

Blood group B glycosphingolipids (B-GSLs) are substrates of the lysosomal alpha-galactosidase A (AGAL). Similar to its major substrate-globotriaosylceramide (Gb3Cer)-B-GSLs are not degraded and accumulate in the cells of patients affected by an inherited defect of AGAL activity (Fabry disease-FD).The pancreas is a secretory organ known to have high biosynthesis of blood group GSLs. Herein, we provide a comprehensive overview of the biochemical and structural abnormalities in pancreatic tissue from two male FD patients with blood group B. In both patients, we found major accumulation of a variety of complex B-GSLs carrying predominantly hexa- and hepta-saccharide structures. The subcellular pathology was dominated by deposits containing B-glycoconjugates and autofluorescent ceroid. The contribution of Gb3Cer to the storage was minor. This abnormal storage pattern was specific for the pancreatic acinar epithelial cells. Other pancreatic cell types including those of islets of Langerhans were affected much less or not at all.Altogether, we provide evidence for a key role of B-antigens in the biochemical and morphological pathology of the exocrine pancreas in FD patients with blood group B. We believe that our findings will trigger further studies aimed at assessing the potential pancreatic dysfunction in this disease.

ABC Transporters Involved in Export of Cell Surface Glycoconjugates

PubMed Central

Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

2010-01-01

Summary: Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles. PMID:20805402

Quantitation and histochemical localization of galectin-1 and galectin-1-reactive glycoconjugates in fetal development of bovine organs.

PubMed

Kaltner, H; Lips, K S; Reuter, G; Lippert, S; Sinowatz, F; Gabius, H J

1997-10-01

The display of cellular oligosaccharide chains is known to undergo marked developmental changes, as monitored histochemically with plant lectins. In conjunction with endogenous lectins respective ligand structures may have a functional role during fetal development. The assumption of a recognitive, functionally productive interplay prompts the study of the expression of a tissue lectin and of lectin-reactive glycoconjugates concomitantly. Focusing on common beta-galactosides as constituents of oligosaccharide chains and the predominant member of the family of galectins in mammals, namely galectin-1, the question therefore is addressed as to whether expression of lectin and lectin-reactive glycoconjugates exhibits alterations, assessed in three morphologically defined fetal stages and in adult bovine organs. Using a sandwich ELISA, the level of the rather ubiquitous galectin-1 is mostly increased in adult organs relative to respective fetal stages, except for the case of kidney. This developmental course is seen rather seldom, when the amounts of lectin-reactive glycoproteins or glycolipids are quantitated in solid-phase assays after tissue homogenization. Western blotting, combined with probing by labeled galectin-1, discloses primarily quantitative changes in the reactivity of individual glycoproteins. Performing the same assays on extract aliquots with a plant agglutinin, namely the galactoside-binding mistletoe lectin, whose fine specificity is different from galectin-1, its reduced extent of binding in solid-phase assays and the disparate profile of lectin-reactive glycoproteins reveal a non-uniform developmental alteration within the group of structural variants of beta-galactosides. Although sample preparation can affect ligand preservation and/or presentation and thus restricts the comparability of biochemical and histochemical results, especially for soluble reactants, the histochemical studies on frozen and paraffin-embedded sections of bovine heart, kidney and liver demonstrate that the localization of the galectin and of lectin-reactive epitopes can show a similar distribution, as seen in liver and heart, with organ-typical quantitative changes of a rather similar staining profile (heart, kidney) or notable changes in the spatial distribution (liver) in the course of development. This report emphasizes the potential value of combined monitoring of the lectin and its potential in vivo ligands to contribute to eventually unravel organ-related function(s) of a tissue lectin.

Meningococcal vaccine development--from glycoconjugates against MenACWY to proteins against MenB--potential for broad protection against meningococcal disease.

PubMed

Dull, Peter M; McIntosh, E David

2012-05-30

Novartis Vaccines has a long-standing research and development interest in the prevention of invasive meningococcal disease. From the initial licensure of the monovalent meningococcal C glycoconjugate vaccine, Menjugate(®), in response to the emergence of a virulent serogroup C ST-11 strain in the United Kingdom to the more recent development and licensure of a quadrivalent meningococcal ACWY glycoconjugate vaccine, Menveo(®), Novartis has a continuing commitment to the development of more effective tools for the control of meningococcal disease. Menveo is now licensed for use in adolescents and adults in over 50 countries and results from phase III studies have shown the vaccine to be well-tolerated and highly immunogenic in infants with vaccination beginning from 2 months of age. The 'holy grail' of meningococcal disease control is a broadly protective vaccine against serogroup B (MenB), preferably a vaccine that protects all age groups including infants. As the serogroup B capsule is poorly immunogenic, efforts over the past 40 years have focused on identifying conserved proteins expressed on the bacterial surface that elicit bactericidal antibodies. Novartis has approached this problem utilizing genomic tools to identify proteins meeting these criteria in a process now known as 'reverse vaccinology'[1]. This process has resulted in a novel multicomponent MenB vaccine (4CMenB) that consists of four major immunogenic components (three subcapsular MenB protein antigens plus outer membrane vesicles (OMVs) which themselves provide multiple subcapsular antigens, the immunodominant one being PorA). These all induce bactericidal antibodies against the antigens that are important in determining the survival, function, and virulence of the meningococci. Phase II studies of 4CMenB have been completed and have demonstrated that the vaccine is highly immunogenic against reference meningococcal strains selected to support licensure. Post-vaccination sera from clinical studies have also been tested against a diverse panel of serogroup B strains to support the development of the Meningococcal Antigen Typing System (MATS), a tool used to predict vaccine strain coverage [2] This overview is intended to give a broad summary of the key clinical data derived from the Menveo and 4CMenB clinical development programs. Copyright © 2012. Published by Elsevier Ltd.

Three-Dimensional Scaffold from Decellularized Human Gingiva for Cell Cultures: Glycoconjugates and Cell Behavior

PubMed Central

Naderi, Somayeh; Khayat Zadeh, Jina; Mahdavi Shahri, Nasser; Nejad Shahrokh Abady, Khadijeh; Cheravi, Mojtaba; Baharara, Javad; Banihashem Rad, Seyed Ali; Bahrami, Ahmad Reza

2013-01-01

Objective: We studied both the presence of some carbohydrate compounds in a threedimensional (3D) matrix harvested from human gingiva and the cell behavior in this matrix. Materials and Methods: In this experimental research, in order to prepare 3D scaffolds, human palatal gingival biopsies were harvested and physically decellularized by freezethawing and sodium dodecyl sulfate (SDS). The scaffolds were placed within the rings of blastema tissues obtained from a pinna rabbit, in vitro. We evaluated the presence of glycoconjugatesand cellular behavior according to histological, histochemical and spectrophotometry techniques at one, two and three weeks after culture. One-way analysis of variance (ANOVA)comparedthe groups. Results: Extracellular matrix (ECM) remained after decellularization of tissue with 1% SDS. Glycoconjugate contents decreased meaningfully at a higher SDS concentration (p<0.0001). After culture of the ECM scaffold with blastema, we observed increased staining of alcian blue, periodic acid-Schiff (PAS) and toluidine blue in the scaffold and a number of other migrant cells which was caused by cell penetrationinto the scaffold. Spectrophotometry results showed an increase in glycosaminoglycans (GAGs) of the decellularized scaffolds at three weeks after culture. Conclusion: The present study has shown that a scaffold generated from palatal gingival tissue ECM is a suitable substrate for blastema cell migration and activity.This scaffold maypotentially be useful as a biological scaffold in tissue engineering applications. PMID:23862119

Terminal alkenes as versatile chemical reporter groups for metabolic oligosaccharide engineering.

PubMed

Späte, Anne-Katrin; Schart, Verena F; Schöllkopf, Sophie; Niederwieser, Andrea; Wittmann, Valentin

2014-12-08

The Diels-Alder reaction with inverse electron demand (DAinv reaction) of 1,2,4,5-tetrazines with electron rich or strained alkenes was proven to be a bioorthogonal ligation reaction that proceeds fast and with high yields. An important application of the DAinv reaction is metabolic oligosaccharide engineering (MOE) which allows the visualization of glycoconjugates in living cells. In this approach, a sugar derivative bearing a chemical reporter group is metabolically incorporated into cellular glycoconjugates and subsequently derivatized with a probe by means of a bioorthogonal ligation reaction. Here, we investigated a series of new mannosamine and glucosamine derivatives with carbamate-linked side chains of varying length terminated by alkene groups and their suitability for labeling cell-surface glycans. Kinetic investigations showed that the reactivity of the alkenes in DAinv reactions increases with growing chain length. When applied to MOE, one of the compounds, peracetylated N-butenyloxycarbonylmannosamine, was especially well suited for labeling cell-surface glycans. Obviously, the length of its side chain represents the optimal balance between incorporation efficiency and speed of the labeling reaction. Sialidase treatment of the cells before the bioorthogonal labeling reaction showed that this sugar derivative is attached to the glycans in form of the corresponding sialic acid derivative and not epimerized to another hexosamine derivative to a considerable extent. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Apicomplexa-specific glucosamine-6-phosphate N-acetyltransferase gene family encodes a key enzyme for glycoconjugate synthesis with potential as therapeutic target.

PubMed

Cova, Marta; López-Gutiérrez, Borja; Artigas-Jerónimo, Sara; González-Díaz, Aida; Bandini, Giulia; Maere, Steven; Carretero-Paulet, Lorenzo; Izquierdo, Luis

2018-03-05

Apicomplexa form a phylum of obligate parasitic protozoa of great clinical and veterinary importance. These parasites synthesize glycoconjugates for their survival and infectivity, but the enzymatic steps required to generate the glycosylation precursors are not completely characterized. In particular, glucosamine-phosphate N-acetyltransferase (GNA1) activity, needed to produce the essential UDP-N-acetylglucosamine (UDP-GlcNAc) donor, has not been identified in any Apicomplexa. We scanned the genomes of Plasmodium falciparum and representatives from six additional main lineages of the phylum for proteins containing the Gcn5-related N-acetyltransferase (GNAT) domain. One family of GNAT-domain containing proteins, composed by a P. falciparum sequence and its six apicomplexan orthologs, rescued the growth of a yeast temperature-sensitive GNA1 mutant. Heterologous expression and in vitro assays confirmed the GNA1 enzymatic activity in all lineages. Sequence, phylogenetic and synteny analyses suggest an independent origin of the Apicomplexa-specific GNA1 family, parallel to the evolution of a different GNA1 family in other eukaryotes. The inability to disrupt an otherwise modifiable gene target suggests that the enzyme is essential for P. falciparum growth. The relevance of UDP-GlcNAc for parasite viability, together with the independent evolution and unique sequence features of Apicomplexa GNA1, highlights the potential of this enzyme as a selective therapeutic target against apicomplexans.

Lectin histochemistry as a tool to identify apoptotic cells in the seminiferous epithelium of Syrian hamster (Mesocricetus auratus) subjected to short photoperiod.

PubMed

Seco-Rovira, V; Beltrán-Frutos, E; Ferrer, C; Sánchez-Huertas, M M; Madrid, J F; Saez, F J; Pastor, L M

2013-12-01

Lectins have been widely used to study the pattern of cellular glycoconjugates in numerous species. In the process of cellular apoptosis, it has been observed that changes occur in the membrane sugar sequences of these apoptotic cells. The aim of our work was to identify which lectins, out of an extensive battery of the same (PNA, SBA, HPA, LTA, Con-A, UEA-I, WGA, DBA, MAA, GNA, AAA, SNA), show affinity for germinal cells in apoptosis, at what stage of cell death they do so and in which germinal cell types they can be detected. For this, we studied testis sections during testicular regression in Syrian hamster (Mesocricetus auratus) subjected to short photoperiod. Several lectins showed an affinity for the glycoconjugate residues of germ cells in apoptosis: Gal β1,3-GalNAcα1, α-d-mannose, N-acetylgalactosamine and l-fucose. Furthermore, lectin specificity was observed for some specific germinal cells and in certain stages of apoptosis. It was also observed that one of these lectins (PNA) showed affinity for Sertoli cells undergoing apoptosis. Therefore, we conclude that the use of lectin histochemistry could be a very useful tool for studying apoptosis in the seminiferous epithelium because of the specificity shown towards germinal cells in pathological or experimentally induced epithelial depletion models. © 2013 Blackwell Verlag GmbH.

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse.

PubMed

Schulte, B A; Spicer, S S

1983-12-01

Mouse salivary glands and pancreases were stained with a battery of ten horseradish peroxidase-conjugated lectins. Lectin staining revealed striking differences in the structure of oligosaccharides of stored intracellular secretory glycoproteins and glycoconjugates associated with the surface of epithelial cells lining excretory ducts. The percentage of acinar cells containing terminal alpha-N-acetylgalactosamine residues varied greatly in submandibular glands of 30 male mice, but all submandibular acinar cells contained oligosaccharides with terminal sialic acid and penultimate beta-galactose residues. The last named dimer was abundant in secretory glycoprotein of all mucous acinar cells in murine sublingual glands and an additional 20-50% of these cells in all glands contained terminal N-acetylglucosamine residues. In contrast, terminal alpha-N-acetylgalactosamine was abundant in sublingual serous demilune secretions. Serous acinar cells in the exorbital lacrimal gland, posterior lingual gland, parotid gland and pancreas exhibited a staining pattern unique to each organ. In contrast, the apical cytoplasm and surface of striated duct epithelial cells in the submandibular, sublingual, parotid and exorbital lacrimal gland stained similarly. A comparison of staining with conjugated lectins reported biochemically to have very similar carbohydrate binding specificity has revealed some remarkable differences in their reactivity, suggesting different binding specificity for the same terminal sugars having different glycosidic linkages or with different penultimate sugar residues.

Hypothalamic digoxin and hemispheric chemical dominance in relation to the pathogenesis of bronchial asthma.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2003-08-01

The isoprenoid pathway produces three key metabolites--digoxin (membrane sodium-potassium ATPase inhibitor and regulator of neurotransmitter transport), dolichol (regulator of N-glycosylation of proteins), and ubiquinone (free radical scavenger). The isoprenoid pathway was assessed in patients with bronchial asthma. The pathway was also assessed in patients with right hemispheric, left hemispheric, and bihemispheric dominance to find out the role of hemispheric dominance in the pathogenesis of bronchial asthma. The pathway was upregulated with increase in digoxin synthesis in bronchial asthma. There was an increase in tryptophan catabolites and a reduction in tyrosine catabolites in patients with bronchial asthma. The ubiquinone levels were low and lipid peroxidation increased in these patients. There was increase in dolichol and glycoconjugate levels and reduction in lysosomal stability in these patients. The cholesterol:phospholipid ratio was increased and glycoconjugate levels were reduced in the membranes of these patients. The patterns noticed in bronchial asthma were similar to those in patients with right hemispheric chemical dominance. Bronchial asthma occurs in right hemispheric chemically dominant individuals. Ninety percent of the patients with bronchial asthma were right-handed and left hemispheric dominant by the dichotic listening test. But their biochemical patterns were similar to those obtained in right hemispheric chemical dominance. Hemispheric chemical dominance is a different entity and has no correlation with handedness or the dichotic listening test.

N-glycolyl groups of nonhuman chondroitin sulfates survive in ancient fossils.

PubMed

Bergfeld, Anne K; Lawrence, Roger; Diaz, Sandra L; Pearce, Oliver M T; Ghaderi, Darius; Gagneux, Pascal; Leakey, Meave G; Varki, Ajit

2017-09-26

Biosynthesis of the common mammalian sialic acid N -glycolylneuraminic acid (Neu5Gc) was lost during human evolution due to inactivation of the CMAH gene, possibly expediting divergence of the Homo lineage, due to a partial fertility barrier. Neu5Gc catabolism generates N -glycolylhexosamines, which are potential precursors for glycoconjugate biosynthesis. We carried out metabolic labeling experiments and studies of mice with human-like Neu5Gc deficiency to show that Neu5Gc degradation is the metabolic source of UDP-GlcNGc and UDP-GalNGc and the latter allows an unexpectedly selective incorporation of N -glycolyl groups into chondroitin sulfate (CS) over other potential glycoconjugate products. Partially N -glycolylated-CS was chemically synthesized as a standard for mass spectrometry to confirm its natural occurrence. Much lower amounts of GalNGc in human CS can apparently be derived from Neu5Gc-containing foods, a finding confirmed by feeding Neu5Gc-rich chow to human-like Neu5Gc-deficient mice. Unlike the case with Neu5Gc, N -glycolyl-CS was also stable enough to be detectable in animal fossils as old as 4 My. This work opens the door for investigating the biological and immunological significance of this glycosaminoglycan modification and for an "ancient glycans" approach to dating of Neu5Gc loss during the evolution of Homo .

Aminooxylated Carbohydrates: Synthesis and Applications.

PubMed

Pifferi, Carlo; Daskhan, Gour Chand; Fiore, Michele; Shiao, Tze Chieh; Roy, René; Renaudet, Olivier

2017-08-09

Among other classes of biomolecules, carbohydrates and glycoconjugates are widely involved in numerous biological functions. In addition to addressing the related synthetic challenges, glycochemists have invested intense efforts in providing access to structures that can be used to study, activate, or inhibit these biological processes. Over the past few decades, aminooxylated carbohydrates have been found to be key building blocks for achieving these goals. This review provides the first in-depth overview covering several aspects related to the syntheses and applications of aminooxylated carbohydrates. After a brief introduction to oxime bonds and their relative stabilities compared to related C═N functions, synthetic aspects of oxime ligation and methodologies for introducing the aminooxy functionality onto both glycofuranosyls and glycopyranosyls are described. The subsequent section focuses on biological applications involving aminooxylated carbohydrates as components for the construcion of diverse architectures. Mimetics of natural structures represent useful tools for better understanding the features that drive carbohydrate-receptor interaction, their biological output and they also represent interesting structures with improved stability and tunable properties. In the next section, multivalent structures such as glycoclusters and glycodendrimers obtained through oxime ligation are described in terms of synthetic design and their biological applications such as immunomodulators. The second-to-last section discusses miscellaneous applications of oxime-based glycoconjugates, such as enantioselective catalysis and glycosylated oligonucleotides, and conclusions and perspectives are provided in the last section.

Functional Mapping of the Lectin Activity Site on the β-Prism Domain of Vibrio cholerae Cytolysin

PubMed Central

Rai, Anand Kumar; Paul, Karan; Chattopadhyay, Kausik

2013-01-01

Vibrio cholerae cytolysin (VCC) is a prominent member in the family of β-barrel pore-forming toxins. It induces lysis of target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. VCC also exhibits prominent lectin-like activity in interacting with β1-galactosyl-terminated glycoconjugates. Apart from the cytolysin domain, VCC harbors two lectin-like domains: the β-Trefoil and the β-Prism domains; however, precise contribution of these domains in the lectin property of VCC is not known. Also, role(s) of these lectin-like domains in the mode of action of VCC remain obscure. In the present study, we show that the β-Prism domain of VCC acts as the structural scaffold to determine the lectin activity of the protein toward β1-galactosyl-terminated glycoconjugates. Toward exploring the physiological implication of the β-Prism domain, we demonstrate that the presence of the β-Prism domain-mediated lectin activity is crucial for an efficient interaction of the toxin toward the target cells. Our results also suggest that such lectin activity may act to regulate the oligomerization ability of the membrane-bound VCC toxin. Based on the data presented here, and also consistent with the existing structural information, we propose a novel mechanism of regulation imposed by the β-Prism domain's lectin activity, implicated in the process of membrane pore formation by VCC. PMID:23209283

Restricted N-glycan conformational space in the PDB and its implication in glycan structure modeling.

PubMed

Jo, Sunhwan; Lee, Hui Sun; Skolnick, Jeffrey; Im, Wonpil

2013-01-01

Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan's crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures.

Canatoxin, a toxic protein from jack beans (Canavalia ensiformis), is a variant form of urease (EC 3.5.1.5): biological effects of urease independent of its ureolytic activity.

PubMed Central

Follmer, C; Barcellos, G B; Zingali, R B; Machado, O L; Alves, E W; Barja-Fidalgo, C; Guimarães, J A; Carlini, C R

2001-01-01

Canatoxin is a toxic protein from Canavalia ensiformis seeds, lethal to mice (LD(50)=2 mg/kg) and insects. Further characterization of canatoxin showed that its main native form (184 kDa) is a non-covalently linked dimer of a 95 kDa polypeptide containing zinc and nickel. Partial sequencing of internal peptides indicated homology with urease (EC 3.5.1.5) from the same seed. Canatoxin has approx. 30% of urease's activity for urea, and K(m) of 2-7 mM. The proteins differ in their affinities for metal ions and were separated by affinity chromatography on a Zn(2+) matrix. Similar to canatoxin, urease activates blood platelets and interacts with glycoconjugates. In contrast with canatoxin, no lethality was seen in mice injected with urease (10 mg/kg). Pretreatment with p-hydroxymercuribenzoate irreversibly abolished the ureolytic activity of both proteins. On the other hand, p-hydroxymercuribenzoate-treated canatoxin was still lethal to mice, and both treated proteins were fully active in promoting platelet aggregation and binding to glycoconjugates. Taken together, our data indicate that canatoxin is a variant form of urease. Moreover, we show for the first time that these proteins display several biological effects that are unrelated to their enzymic activity for urea. PMID:11696010

Restricted N-glycan Conformational Space in the PDB and Its Implication in Glycan Structure Modeling

PubMed Central

Jo, Sunhwan; Lee, Hui Sun; Skolnick, Jeffrey; Im, Wonpil

2013-01-01

Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan's crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures. PMID:23516343

Interaction study between synthetic glycoconjugate ligands and endocytic receptors using flow cytometry.

PubMed

Yura, Hirofumi; Ishihara, Masayuki; Kanatani, Yasuhiro; Takase, Bonpei; Hattori, Hidemi; Suzuki, Shinya; Kawakami, Mitsuyuki; Matsui, Takemi

2006-04-01

Flow cytometric analysis of synthetic galactosyl polymers, asialofetuin and LDL derivatives labeled with FITC (Fluorescein Isothiocyanate) was carried out to determine the phenotypes of endocytic receptors, such as asialoglycoprotein (ASPG) and the LDL receptor, on various types of cells. When FITC-labeled galactosyl polystyrene (GalCPS), being a synthetic ligand of ASPG, was applied to rat hepatocytes and human cancer cells (Hep G2 and Chang Liver), surface fluorescence intensities varied according to receptor expression on the cells. The fluorescence intensity originates from the calcium-dependent binding of the FITC-labeled GalCPS. Although unaltered by pre-treatment with glucosyl polystyrene (GluCPS), fetuin and LDL, the fluorescence intensity was suppressed by pre-treatment with (non-labeled) GalCPS and asialofetuin. Flow cytometry allowed us to demonstrate that the calcium-dependent binding of FITC-labeled LDL (prepared from rabbits) upon the addition of 17alpha-ethinyl estradiol enhances LDL receptor expression, and the expression is suppressed upon the addition of a monoclonal antibody to the LDL receptor. The binding efficiency based on the combination of FITC-labeled ligands suggests a possible application for the classification of cell types and conditions corresponding to endocytic receptor expression without the need for immuno-active antibodies or radiolabeled substances. Furthermore, the synthetic glycoconjugate (GalCPS) is shown to be a sensitive and useful marker for classification based on cell phenotype using flow cytometry.

Expedient Route To Access Rare Deoxy Amino l-Sugar Building Blocks for the Assembly of Bacterial Glycoconjugates.

PubMed

Sanapala, Someswara Rao; Kulkarni, Suvarn S

2016-04-13

Bacterial glycoproteins and oligosaccharides contain several rare deoxy amino l-sugars which are virtually absent in the human cells. This structural difference between the bacterial and host cell surface glycans can be exploited for the development of carbohydrate based vaccines and target specific drugs. However, the unusual deoxy amino l-sugars present in the bacterial glycoconjugates are not available from natural sources. Thus, procurement of orthogonally protected rare l-sugar building blocks through efficient chemical synthesis is a crucial step toward the synthesis of structurally well-defined and homogeneous complex glycans. Herein, we report a general and expedient methodology to access a variety of unusual deoxy amino l-sugars starting from readily available l-rhamnose and l-fucose via highly regioselective, one-pot double serial and double parallel displacements of the corresponding 2,4-bistriflates using azide and nitrite anions as nucleophiles. Alternatively, regioselective monotriflation at O2, O3, and O4 of l-rhamnose/l-fucose allowed selective inversions at respective positions leading to diverse rare sugars. The orthogonally protected deoxy amino l-sugar building blocks could be stereoselectively assembled to obtain biologically relevant bacterial O-glycans, as exemplified by the first total synthesis of the amino linker-attached, conjugation-ready tetrasaccharide of O-PS of Yersinia enterocolitica O:50 strain 3229 and the trisaccharide of Pseudomonas chlororaphis subsp. aureofaciens strain M71.

Fasciola hepatica glycoconjugates immuneregulate dendritic cells through the Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin inducing T cell anergy

PubMed Central

Rodríguez, Ernesto; Kalay, Hakan; Noya, Verónica; Brossard, Natalie; Giacomini, Cecilia; van Kooyk, Yvette; García-Vallejo, Juan J.; Freire, Teresa

2017-01-01

Dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) expressed on a variety of DCs, is a C-type lectin receptor that recognizes glycans on a diverse range of pathogens, including parasites. The interaction of DC-SIGN with pathogens triggers specific signaling events that modulate DC-maturation and activity and regulate T-cell activation by DCs. In this work we evaluate whether F. hepatica glycans can immune modulate DCs via DC-SIGN. We demonstrate that DC-SIGN interacts with F. hepatica glycoconjugates through mannose and fucose residues. We also show that mannose is present in high-mannose structures, hybrid and trimannosyl N-glycans with terminal GlcNAc. Furthermore, we demonstrate that F. hepatica glycans induce DC-SIGN triggering leading to a strong production of TLR-induced IL-10 and IL-27p28. In addition, parasite glycans induced regulatory DCs via DC-SIGN that decrease allogeneic T cell proliferation, via the induction of anergic/regulatory T cells, highlighting the role of DC-SIGN in the regulation of innate and adaptive immune responses by F. hepatica. Our data confirm the immunomodulatory properties of DC-SIGN triggered by pathogen-derived glycans and contribute to the identification of immunomodulatory glyans of helminths that might eventually be useful for the design of vaccines against fasciolosis. PMID:28436457

Fasciola hepatica glycoconjugates immuneregulate dendritic cells through the Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin inducing T cell anergy.

PubMed

Rodríguez, Ernesto; Kalay, Hakan; Noya, Verónica; Brossard, Natalie; Giacomini, Cecilia; van Kooyk, Yvette; García-Vallejo, Juan J; Freire, Teresa

2017-04-24

Dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) expressed on a variety of DCs, is a C-type lectin receptor that recognizes glycans on a diverse range of pathogens, including parasites. The interaction of DC-SIGN with pathogens triggers specific signaling events that modulate DC-maturation and activity and regulate T-cell activation by DCs. In this work we evaluate whether F. hepatica glycans can immune modulate DCs via DC-SIGN. We demonstrate that DC-SIGN interacts with F. hepatica glycoconjugates through mannose and fucose residues. We also show that mannose is present in high-mannose structures, hybrid and trimannosyl N-glycans with terminal GlcNAc. Furthermore, we demonstrate that F. hepatica glycans induce DC-SIGN triggering leading to a strong production of TLR-induced IL-10 and IL-27p28. In addition, parasite glycans induced regulatory DCs via DC-SIGN that decrease allogeneic T cell proliferation, via the induction of anergic/regulatory T cells, highlighting the role of DC-SIGN in the regulation of innate and adaptive immune responses by F. hepatica. Our data confirm the immunomodulatory properties of DC-SIGN triggered by pathogen-derived glycans and contribute to the identification of immunomodulatory glyans of helminths that might eventually be useful for the design of vaccines against fasciolosis.

Total cellular glycomics allows characterizing cells and streamlining the discovery process for cellular biomarkers.

PubMed

Fujitani, Naoki; Furukawa, Jun-ichi; Araki, Kayo; Fujioka, Tsuyoshi; Takegawa, Yasuhiro; Piao, Jinhua; Nishioka, Taiki; Tamura, Tomohiro; Nikaido, Toshio; Ito, Makoto; Nakamura, Yukio; Shinohara, Yasuro

2013-02-05

Although many of the frequently used pluripotency biomarkers are glycoconjugates, a glycoconjugate-based exploration of novel cellular biomarkers has proven difficult due to technical difficulties. This study reports a unique approach for the systematic overview of all major classes of oligosaccharides in the cellular glycome. The proposed method enabled mass spectrometry-based structurally intensive analyses, both qualitatively and quantitatively, of cellular N- and O-linked glycans derived from glycoproteins, glycosaminoglycans, and glycosphingolipids, as well as free oligosaccharides of human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs), and various human cells derived from normal and carcinoma cells. Cellular total glycomes were found to be highly cell specific, demonstrating their utility as unique cellular descriptors. Structures of glycans of all classes specifically observed in hESCs and hiPSCs tended to be immature in general, suggesting the presence of stem cell-specific glycosylation spectra. The current analysis revealed the high similarity of the total cellular glycome between hESCs and hiPSCs, although it was suggested that hESCs are more homogeneous than hiPSCs from a glycomic standpoint. Notably, this study enabled a priori identification of known pluripotency biomarkers such as SSEA-3, -4, and -5 and Tra-1-60/81, as well as a panel of glycans specifically expressed by hESCs and hiPSCs.

Molecular cloning, characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01.

PubMed

Chae, J P; Valeriano, V D; Kim, G-B; Kang, D-K

2013-01-01

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01. The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l(-1) isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel-nitrilotriacetic acid (Ni(2+) -NTA) agarose column and their activities characterized. BSH A hydrolysed tauro-conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco-conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety. BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate-binding sites, these remain functional through motif conservation. This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco-conjugated or tauro-conjugated bile salts. Future structural homology studies and site-directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes. © 2012 The Society for Applied Microbiology.

Bacillus thuringiensis Cry1A toxin-binding glycoconjugates present on the brush border membrane and in the peritrophic membrane of the Douglas-fir tussock moth are peritrophins

Treesearch

Algimantas P. Valaitis; John D. Podgwaite

2013-01-01

Bacillus thuringiensis (Bt) Cry1A toxin-binding sites in the Douglas fir tussock moth (DFTM) larval gut were localized using immunofluorescence microscopy. Cry1Aa, Cry1Ab and Cry1Ac all bound strongly to the DFTM peritrophic membrane (PM); weaker binding of the Cry1A toxins was observed along the apical brush border of the midgut epithelium....

Joint Workshop on Aspects of Halophilism Held in Jerusalem, March 23-28, 1986. Program/Abstracts.

DTIC Science & Technology

1986-04-30

different proteins. The sulfate residues are not reduced, but rather incorporated into glycoconjugates as sulfuric acid esters ( 1 ). In vivo studies with...References: 1 . Gupta, R. and C. R. Woese. 1980. Unusual modification patterns in the transfer ribonuclel acids of arohaebacteria. Curr. 4icrobiol. 4:245-249...of large amounts of rain floods in the winter of 1979- 1 )60, diluting the upper water layers by up to 1 ( 1 %. The bacerial bloom consisted for 80% of

Development of approaches to a third-generation carbohydrate-conjugate vaccine against Streptococcus pneumoniae: the search for optimal oligosaccharide ligands

NASA Astrophysics Data System (ADS)

Gening, M. L.; Kurbatova, E. A.; Tsvetkov, Yu E.; Nifantiev, N. E.

2015-11-01

The review addresses the application of synthetic oligosaccharides related to fragments of capsular polysaccharides from different serotypes of the bacterium Streptococcus pneumoniae for the design of third-generation pneumococcal conjugate vaccines. Special focus is given to characteristic features of the chemical structures of oligosaccharides required for the induction of the protective immune response when using synthetic glycoconjugate vaccines based on oligosaccharide ligands and carrier proteins. The bibliography includes 101 references.

β-1,2-Mannosyltransferases 1 and 3 Participate in Yeast and Hyphae O- and N-Linked Mannosylation and Alter Candida albicans Fitness During Infection.

PubMed

Courjol, Flavie; Jouault, Thierry; Mille, Céline; Hall, Rebecca; Maes, Emmanuel; Sendid, Boualem; Mallet, Jean Maurice; Guerardel, Yann; Gow, Neil A R; Poulain, Daniel; Fradin, Chantal

2015-09-01

β-1,2-mannosylation of Candida albicans glycoconjugates has been investigated through the identification of enzymes involved in the addition of β-1,2-oligomannosides (β-Mans) to phosphopeptidomannan and phospholipomannan. β-1,2-oligomannosides are supposed to have virulence properties that they confer to these glycoconjugates. In a previous study, we showed that cell wall mannoproteins (CWMPs) harbor β-Mans in their O-mannosides; therefore, we analyzed their biosynthesis and impact on virulence. In this study, we demonstrate that O-mannans are heterogeneous and that α-mannosylated O-mannosides, which are biosynthesized by Mnt1 and Mnt2 α-1,2-mannosyltransferases, can be modified with β-Mans but only at the nonreducing end of α-1,2-mannotriose. β-1,2-mannosylation of this O-mannotriose depends on growth conditions, and it involves 2 β-1,2-mannosyltransferases, Bmt1 and Bmt3. These Bmts are essential for β-1,2-mannosylation of CWMPs and expression of β-Mans on germ tubes. A bmt1Δ mutant and a mutant expressing no β-Mans unexpectedly disseminated more in BALB/c mice, whereas they had neither attenuated nor enhanced virulence in C57BL/6 mice. In galectin (Gal)3 knockout mice, the reference strain was more virulent than in C57BL/6 mice, suggesting that the β-Mans innate receptor Gal3 is involved in C. albicans fitness during infection.

Rational chemical design of the carbohydrate in a glycoconjugate vaccine enhances IgM-to-IgG switching.

PubMed

Guttormsen, Hilde-Kari; Paoletti, Lawrence C; Mansfield, Keith G; Jachymek, Wojcieck; Jennings, Harold J; Kasper, Dennis L

2008-04-15

Many pathogens are sheltered from host immunity by surface polysaccharides that would be ideal as vaccines except that they are too similar to host antigens to be immunogenic. The production of functional IgG is a desirable response to vaccines; because IgG is the only isotype that crosses the placenta, it is of particular importance in maternal vaccines against neonatal disease due to group B Streptococcus (GBS). Clinical studies found a substantially lower proportion of IgG-relative to IgM-among antibodies elicited by conjugates prepared with purified GBS type V capsular polysaccharide (CPS) than among those evoked by CPSs of other GBS serotypes. The epitope specificity of IgG elicited in humans by a conjugate prepared with type V CPS is for chemically desialylated type V CPS (dV CPS). We studied desialylation as a mechanism for enhancing the ability of type V CPS to induce IgM-to-IgG switching. Desialylation did not affect the structural conformation of type V CPS. Rhesus macaques, whose isotype responses to GBS conjugates match those of humans, produced functionally active IgG in response to a dV CPS-tetanus toxoid conjugate (dV-TT), and 98% of neonatal mice born to dams vaccinated with dV-TT survived lethal challenge with viable GBS. Targeted chemical engineering of a carbohydrate to create a molecule less like host self may be a rational approach for improving other glycoconjugates.

Correlation of serum biomarkers (TSA & LSA) and epithelial dysplasia in early diagnosis of oral precancer and oral cancer.

PubMed

Sawhney, Hemant; Kumar, C Anand

Oral cancer is currently the most frequent cause of cancer-related deaths, which is usually preceded by oral pre-cancerous lesions and conditions. Altered glycosylation of glycoconjugates, such as sialic acid, fucose, etc. are amongst the important molecular changes that accompany malignant transformation. The purpose of our study was to evaluate usefulness of serum Total Sialic Acid (TSA) and serum Lipid-Bound Sialic Acid (LSA) as markers of oral precancerous lesions and histopathologically correlating them with grades of epithelial dysplasia. Blood samples were collected from 50 patients with oral precancer (Leukoplakia & OSMF), 25 patients with untreated oral cancer and 25 healthy subjects. Serum sialic acid (total and lipid bound) levels were measured spectrophotometrically. Tissue samples from all the patients were evaluated for dysplasia. Serum levels of total and lipid bound sialic acid were significantly elevated in patients with oral precancer and cancer when compared with healthy subjects. Analysis of variance test documented that there is progressive rise in serum levels of sialic acid with the degree of dysplastic changes in oral precancer patients. We observed positive correlation between serum levels of the markers and the extent of malignant disease (TNM Clinical staging) as well as histopathological grades. The results suggested that serum levels of TSA and LSA progressively increases with grades of dysplasia in precancerous groups and cancer group, when compared with healthy controls. These glycoconjugates, especially LSA has the clinical utility in indicating a premalignant change.

Development of capsular polysaccharide-based glycoconjugates for immunization against melioidosis and glanders.

PubMed

Burtnick, Mary N; Heiss, Christian; Roberts, Rosemary A; Schweizer, Herbert P; Azadi, Parastoo; Brett, Paul J

2012-01-01

Burkholderia pseudomallei and Burkholderia mallei, the etiologic agents of melioidosis and glanders, respectively, cause severe disease in humans and animals and are considered potential agents of biological warfare and terrorism. Diagnosis and treatment of infections caused by these pathogens can be challenging and, in the absence of chemotherapeutic intervention, acute disease is frequently fatal. At present, there are no human or veterinary vaccines available for immunization against these emerging/re-emerging infectious diseases. One of the long term objectives of our research, therefore, is to identify and characterize protective antigens expressed by B. pseudomallei and B. mallei and use them to develop efficacious vaccine candidates. Previous studies have demonstrated that the 6-deoxy-heptan capsular polysaccharide (CPS) expressed by these bacterial pathogens is both a virulence determinant and a protective antigen. Consequently, this carbohydrate moiety has become an important component of the various subunit vaccines that we are currently developing in our laboratory. In the present study, we describe a reliable method for isolating CPS antigens from O-polysaccharide (OPS) deficient strains of B. pseudomallei; including a derivative of the select agent excluded strain Bp82. Utilizing these purified CPS samples, we also describe a simple procedure for covalently linking these T-cell independent antigens to carrier proteins. In addition, we demonstrate that high titer IgG responses can be raised against the CPS component of such constructs. Collectively, these approaches provide a tangible starting point for the development of novel CPS-based glycoconjugates for immunization against melioidosis and glanders.

Co-administration of a meningococcal glycoconjugate ACWY vaccine with travel vaccines: a randomized, open-label, multi-center study.

PubMed

Alberer, Martin; Burchard, Gerd; Jelinek, Tomas; Reisinger, Emil; Beran, Jiri; Meyer, Seetha; Forleo-Neto, Eduardo; Gniel, Dieter; Dagnew, Alemnew F; Arora, Ashwani Kumar

2014-01-01

Potential interactions between vaccines may compromise the immunogenicity and/or safety of individual vaccines so must be assessed before concomitant administration is recommended. In this study, the immunogenicity and safety of travel vaccines against Japanese encephalitis (JEV) and rabies (PCECV) administered together with or without a quadrivalent meningococcal glycoconjugate ACWY-CRM vaccine were evaluated (NCT01466387). Healthy adults aged 18 to ≤60 years were randomized to one of four vaccine regimens: JEV + PCECV + MenACWY-CRM, JEV + PCECV, PCECV or MenACWY-CRM. Immunogenicity at baseline and 28 days post-complete vaccination was assessed by serum bactericidal assay using human complement or neutralization tests. Adverse events (AEs) were collected throughout the study period. JEV + PCECV + MenACWY-CRM was non-inferior to JEV + PCECV. Post-vaccination seroprotective neutralizing titers or concentrations were achieved in 98-99% (JE) and 100% (rabies) of subjects across the vaccine groups. Antibody responses to vaccine meningococcal serogroups were in the same range for MenACWY-CRM and JEV + PCECV + MenACWY-CRM. Rates of reporting of AEs were similar for JEV + PCECV and JEV + PCECV + MenACWY-CRM. MenACWY-CRM was administered with an inactivated adjuvanted JE and a purified chick embryo cell-culture rabies vaccine without compromising immunogenicity or safety of the individual vaccines. These data provide evidence that MenACWY-CRM could be effectively incorporated into travel vaccination programs. NCT01466387. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genetics of Capsular Polysaccharides and Cell Envelope (Glyco)lipids

PubMed Central

Daffé, Mamadou; Crick, Dean C.; Jackson, Mary

2014-01-01

This chapter summarizes what is currently known of the structures, physiological roles, involvement in pathogenicity and biogenesis of a variety of non-covalently bound cell envelope lipids and glycoconjugates of Mycobacterium tuberculosis and other Mycobacterium species. Topics addressed in this chapter include phospholipids; phosphatidylinositol mannosides; triglycerides; isoprenoids and related compounds (polyprenyl phosphate, menaquinones, carotenoids, non-carotenoid cyclic isoprenoids); acyltrehaloses (lipooligosaccharides, trehalose mono- and di-mycolates, sulfolipids, di- and poly-acyltrehaloses); mannosyl-beta-1-phosphomycoketides; glycopeptidolipids; phthiocerol dimycocerosates, para-hydroxybenzoic acids and phenolic glycolipids; mycobactins; mycolactones; and capsular polysaccharides. PMID:25485178

Hydroxyester disaccharides from fruits of cape gooseberry (Physalis peruviana).

PubMed

Mayorga, Humberto; Duque, Carmenza; Knapp, Holger; Winterhalter, Peter

2002-02-01

The 3-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside of ethyl 3-hydroxyoctanoate and the diastereomeric 3-O-alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranosides of (3R) and (3S)-butyl 3-hydroxybutanoate, respectively, were isolated by chromatographic methods from fruits of cape gooseberry (Physalis peruviana) harvested in Colombia. Their structures were identified by ESI-MS/MS and NMR spectroscopy. The three glycoconjugates can be considered as immediate precursors of ethyl 3-hydroxyoctanoate and butyl 3-hydroxybutanoate, which are important aroma volatiles found in the fruit.

PNA-encoded chemical libraries.

PubMed

Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

2015-06-01

Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70. Copyright © 2015 Elsevier Ltd. All rights reserved.

Disinfection of preexisting contamination of bacillus cereus on stainless steel when using glycoconjugate solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pavan, Casey; Tarasenko, Olga

Stainless steel is ubiquitous in our modern world, however it can become contaminated. This can endanger our health. The aim of our study is to disinfect stainless steel using Bacillus cereus as a model organism. Bacillus cereus is a microbe that is ubiquitous in nature, specifically soil. B. cereus is known to cause illness in humans. To prevent this, we propose to use a glycoconjugate solution (GS) for disinfection of stainless steel after it is contamination by B. cereus spores. In this study, two GS (9, 10) were tested for disinfection effectiveness on B. cereus spores on the surface ofmore » stainless steel foil (AISI-Series 200/300/400, THERMA-FOIL, Dayville, CT 0241). The disinfection rate of each GS was assessed by exposing the steel surface to B. cereus spores first and allowing them to settle for 24 hours. GS was used to treat the contaminated surface. The steel is washed and the resulting solution is plated on tryptic soy agar (TSA) plates. The GS with the fewest colony forming unit (CFU) formed on TSA is determined to be the most efficient during disinfection. Results show that both GS demonstrate a strong ability to disinfect B. cereus spores. Between the two, GS 9 shows the highest disinfection efficacy by killing approximately 99.5% of spores. This is a drastic improvement over the 0-20% disinfection of the control. Based on this we find that studied GS do have the capacity to act as a disinfectant on stainless steel.« less

Comparison of CRM197, diphtheria toxoid and tetanus toxoid as protein carriers for meningococcal glycoconjugate vaccines.

PubMed

Tontini, M; Berti, F; Romano, M R; Proietti, D; Zambonelli, C; Bottomley, M J; De Gregorio, E; Del Giudice, G; Rappuoli, R; Costantino, P; Brogioni, G; Balocchi, C; Biancucci, M; Malito, E

2013-10-01

Glycoconjugate vaccines are among the most effective and safest vaccines ever developed. Diphtheria toxoid (DT), tetanus toxoid (TT) and CRM197 have been mostly used as protein carriers in licensed vaccines. We evaluated the immunogenicity of serogroup A, C, W-135 and Y meningococcal oligosaccharides conjugated to CRM197, DT and TT in naïve mice. The three carriers were equally efficient in inducing an immune response against the carbohydrate moiety in immunologically naïve mice. The effect of previous exposure to different dosages of the carrier protein on the anti-carbohydrate response was studied using serogroup A meningococcal (MenA) saccharide conjugates as a model. CRM197 showed a strong propensity to positively prime the anti-carbohydrate response elicited by its conjugates or those with the antigenically related carrier DT. Conversely in any of the tested conditions TT priming did not result in enhancement of the anti-carbohydrate response elicited by the corresponding conjugates. Repeated exposure of mice to TT or to CRM197 before immunization with the respective MenA conjugates resulted in a drastic suppression of the anti-carbohydrate response in the case of TT conjugate and only in a slight reduction in the case of CRM197. The effect of carrier priming on the anti-MenA response of DT-based conjugates varied depending on their carbohydrate to protein ratio. These data may have implications for human vaccination since conjugate vaccines are widely used in individuals previously immunized with DT and TT carrier proteins. Copyright © 2013 Elsevier Ltd. All rights reserved.

Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Linxiao; Valentine, Jenny L.; Huang, Chung-Jr

The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. In this paper, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Usingmore » this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS–specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Finally, given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.« less

Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies

DOE PAGES

Chen, Linxiao; Valentine, Jenny L.; Huang, Chung-Jr; ...

2016-06-06

The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. In this paper, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Usingmore » this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS–specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Finally, given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.« less

6-O-Branched Oligo-β-glucan-Based Antifungal Glycoconjugate Vaccines.

PubMed

Liao, Guochao; Zhou, Zhifang; Liao, Jun; Zu, Luning; Wu, Qiuye; Guo, Zhongwu

2016-02-12

With the rapid growth in fungal infections and drug-resistant fungal strains, antifungal vaccines have become an especially attractive strategy to tackle this important health problem. β-Glucans, a class of extracellular carbohydrate antigens abundantly and consistently expressed on fungal cell surfaces, are intriguing epitopes for antifungal vaccine development. β-Glucans have a conserved β-1,3-glucan backbone with sporadic β-1,3- or β-1,6-linked short glucans as branches at the 6-O-positions, and the branches may play a critical role in their immunologic functions. To study the immunologic properties of branched β-glucans and develop β-glucan-based antifungal vaccines, three branched β-glucan oligosaccharides with 6-O-linked β-1,6-tetraglucose, β-1,3-diglucose, and β-1,3-tetraglucose branches on a β-1,3-nonaglucan backbone, which mimic the structural epitopes of natural β-glucans, were synthesized and coupled with keyhole limpet hemocyanin (KLH) to form novel synthetic conjugate vaccines. These glycoconjugates were proved to elicit strong IgG antibody responses in mice. It was also discovered that the number, size, and structure of branches linked to the β-glucan backbone had a significant impact on the immunologic property. Moreover, antibodies induced by the synthetic oligosaccharide-KLH conjugates were able to recognize and bind to natural β-glucans and fungal cells. Most importantly, these conjugates elicited effective protection against systemic Candida albicans infection in mice. Thus, branched oligo-β-glucans were identified as functional epitopes for antifungal vaccine design and the corresponding protein conjugates as promising antifungal vaccine candidates.

Morphological Changes in Skin Glands During Development in Rhinella Arenarum (Anura: Bufonidae).

PubMed

Regueira, Eleonora; Dávila, Camila; Hermida, Gladys N

2016-01-01

Avoiding predation is critical to survival of animals; chemical defenses represent a common strategy among amphibians. In this study, we examined histologically the morphology of skin glands and types of secretions related to chemical skin defense during ontogeny of Rhinella arenarum. Prior to metamorphic climax the epidermis contains typical bufonid giant cells producing a mucous substance supposedly involved in triggering a flight reaction of the tadpole school. An apical layer of alcianophilic mucus covers the epidermis, which could produce the unpleasant taste of bufonid tadpoles. Giant cells disappear by onset of metamorphic climax, when multicellular glands start developing, but the apical mucous layer remains. By the end of climax, neither the granular glands of the dorsum nor the parotoid regions are completely developed. Conversely, by the end of metamorphosis the mucous glands are partially developed and secrete mucus. Adults have at least three types of granular glands, which we designate type A (acidophilic), type B (basophilic) and ventral (mucous). Polymorphic granular glands distribute differently in the body: dorsal granular glands between warts and in the periphery of parotoids contain protein; granular glands of big warts and in the central region of parotoids contain catecholamines, lipids, and glycoconjugates, whereas ventral granular glands produce acidic glycoconjugates. Mucous glands produce both mucus and proteins. Results suggest that in early juveniles the chemical skin defense mechanisms are not functional. Topographical differences in adult skin secretions suggest that granular glands from the big warts in the skin produce similar toxins to the parotoid glands. © 2015 Wiley Periodicals, Inc.

Cell type-specific glycoconjugates of collecting duct cells during maturation of the rat kidney.

PubMed

Holthöfer, H

1988-08-01

The ontogeny of lectin-positive epithelial cell types and the maturation of polarized expression of the glycocalyx of the collecting ducts (CD) of the rat kidney were studied from samples of 18th-day fetal and neonatal kidneys of various ages. Lectins from Dolichos biflorus (DBA) and Vicia villosa (VVA), with preferential affinity to principal cells, stained virtually all CD cells of the fetal kidneys. However, within two days postnatally, the number of cells positive for DBA and VVA decreased to amounts found in the adult kidneys. Moreover, a characteristic change occurred rapidly after birth in the intracellular polarization of the reactive glycoconjugates, from a uniform plasmalemmal to a preferentially apical staining. In contrast, lectins from Arachis hypogaea (PNA), Maclura pomifera (MPA) and Lotus tetragonolobus (LTA), reacting indiscriminatively with principal and intercalated cells of adult kidneys, stained most CD cells in the fetal kidneys, and failed to show any postnatal change in the amount of positive cells or in the intracellular polarization. The immunocytochemical tests for (Na + K)-ATPase and carbonic anhydrase (CA II) revealed the characteristic postnatal decrease in the amount of principal cells and simultaneous increase in the amount of CA II rich intercalated cells. DBA and VVA reactive cells also decreased postnatally, paralleling the changes observed in the (Na + K)-ATPase positive principal cells. The present results suggest that the expression of the cell type-specific glycocalyx of principal and intercalated cells is developmentally regulated, undergoes profound changes during maturation, and is most likely associated with electrolyte transport phenomena.

Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

USGS Publications Warehouse

Gauthier, J.D.; Jenkins, J.A.; La Peyre, Jerome F.

2004-01-01

Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

NEU3 inhibitory effect of naringin suppresses cancer cell growth by attenuation of EGFR signaling through GM3 ganglioside accumulation.

PubMed

Yoshinaga, Ayana; Kajiya, Natsuki; Oishi, Kazuki; Kamada, Yuko; Ikeda, Asami; Chigwechokha, Petros Kingstone; Kibe, Toshiro; Kishida, Michiko; Kishida, Shosei; Komatsu, Masaharu; Shiozaki, Kazuhiro

2016-07-05

Naringin, which is one of the flavonoids contained in citrus fruits, is well known to possess various healthy functions to humans. It has been reported that naringin suppresses cancer cell growth in vitro and in vivo, although the underlying mechanisms are not fully understood. Recently, the roles of glycoconjugates, such as gangliosides, in cancer cells have been focused because of their regulatory effects of malignant phenotypes. Here, to clarify the roles of naringin in the negative-regulation of cancer cell growth, the alteration of glycoconjugates induced by naringin exposure and its significance on cell signaling were investigated. Human cancer cells, HeLa and A549, were exposed to various concentrations of naringin. Naringin treatment induced the suppression of cell growth toward HeLa and A549 cells accompanied with an increase of apoptotic cells. In naringin-exposed cells, GM3 ganglioside was drastically increased compared to the GM3 content prior to the treatment. Furthermore, naringin inhibited NEU3 sialidase, a GM3 degrading glycosidase. Similarly, NEU3 inhibition activities were also detected by other flavanone, such as hesperidin and neohesperidin dihydrocalcone, but their aglycones showed less inhibitions. Naringin-treated cancer cells showed suppressed EGFR and ERK phosphorylation levels. These results suggest a novel mechanism of naringin in the suppression of cancer cell growth through the alteration of glycolipids. NEU3 inhibitory effect of naringin induced GM3 accumulation in HeLa and A549 cells, leading the attenuation of EGFR/ERK signaling accompanied with a decrease in cell growth. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of novel O-polysaccharide based glycoconjugates for immunization against glanders.

PubMed

Burtnick, Mary N; Heiss, Christian; Schuler, A Michele; Azadi, Parastoo; Brett, Paul J

2012-01-01

Burkholderia mallei the etiologic agent of glanders, causes severe disease in humans and animals and is a potential agent of biological warfare and terrorism. Diagnosis and treatment of glanders can be challenging, and in the absence of chemotherapeutic intervention, acute human disease is invariably fatal. At present, there are no human or veterinary vaccines available for immunization against disease. One of the goals of our research, therefore, is to identify and characterize protective antigens expressed by B. mallei and use them to develop efficacious glanders vaccine candidates. Previous studies have demonstrated that the O-polysaccharide (OPS) expressed by B. mallei is both a virulence factor and a protective antigen. Recently, we demonstrated that Burkholderia thailandensis, a closely related but non-pathogenic species, can be genetically manipulated to express OPS antigens that are recognized by B. mallei OPS-specific monoclonal antibodies (mAbs). As a result, these antigens have become important components of the various OPS-based subunit vaccines that we are currently developing in our laboratory. In this study, we describe a method for isolating B. mallei-like OPS antigens from B. thailandensis oacA mutants. Utilizing these purified OPS antigens, we also describe a simple procedure for coupling the polysaccharides to protein carriers such as cationized bovine serum albumin, diphtheria toxin mutant CRM197 and cholera toxin B subunit. Additionally, we demonstrate that high titer IgG responses against purified B. mallei LPS can be generated by immunizing mice with the resulting constructs. Collectively, these approaches provide a rational starting point for the development of novel OPS-based glycoconjugates for immunization against glanders.

Development of novel O-polysaccharide based glycoconjugates for immunization against glanders

PubMed Central

Burtnick, Mary N.; Heiss, Christian; Schuler, A. Michele; Azadi, Parastoo; Brett, Paul J.

2012-01-01

Burkholderia mallei the etiologic agent of glanders, causes severe disease in humans and animals and is a potential agent of biological warfare and terrorism. Diagnosis and treatment of glanders can be challenging, and in the absence of chemotherapeutic intervention, acute human disease is invariably fatal. At present, there are no human or veterinary vaccines available for immunization against disease. One of the goals of our research, therefore, is to identify and characterize protective antigens expressed by B. mallei and use them to develop efficacious glanders vaccine candidates. Previous studies have demonstrated that the O-polysaccharide (OPS) expressed by B. mallei is both a virulence factor and a protective antigen. Recently, we demonstrated that Burkholderia thailandensis, a closely related but non-pathogenic species, can be genetically manipulated to express OPS antigens that are recognized by B. mallei OPS-specific monoclonal antibodies (mAbs). As a result, these antigens have become important components of the various OPS-based subunit vaccines that we are currently developing in our laboratory. In this study, we describe a method for isolating B. mallei-like OPS antigens from B. thailandensis oacA mutants. Utilizing these purified OPS antigens, we also describe a simple procedure for coupling the polysaccharides to protein carriers such as cationized bovine serum albumin, diphtheria toxin mutant CRM197 and cholera toxin B subunit. Additionally, we demonstrate that high titer IgG responses against purified B. mallei LPS can be generated by immunizing mice with the resulting constructs. Collectively, these approaches provide a rational starting point for the development of novel OPS-based glycoconjugates for immunization against glanders. PMID:23205347

Synthesis and Photochromic Properties of Configurationally Varied Azobenzene Glycosides

PubMed Central

Chandrasekaran, Vijayanand; Johannes, Eugen; Kobarg, Hauke; Sönnichsen, Frank D; Lindhorst, Thisbe K

2014-01-01

Spatial orientation of carbohydrates is a meaningful parameter in carbohydrate recognition processes. To vary orientation of sugars with temporal and spatial resolution, photosensitive glycoconjugates with favorable photochromic properties appear to be opportune. Here, a series of azobenzene glycosides were synthesized, employing glycoside synthesis and Mills reaction, to allow “switching” of carbohydrate orientation by reversible E/Z isomerization of the azobenzene N=N double bond. Their photochromic properties were tested and effects of azobenzene substitution as well as the effect of anomeric configuration and the orientation of the sugars 2-hydroxy group were evaluated. PMID:25050228

Immunologic mapping of glycomes: implications for cancer diagnosis and therapy.

PubMed

Zhou, Dapeng; Levery, Steven B; Hsu, Fong-Fu; Wang, Peng G; Teneberg, Susann; Almeida, Igor C; Li, Yunsen; Xu, Huaxi; Wang, Lai-Xi; Xia, Chengfeng; Ibrahim, Nuhad K; Michael, Katja

2011-06-01

Cancer associated glycoconjugates are important biomarkers, as exemplified by globo-H, CA125, CA15.3 and CA27.29. However, the exact chemical structures of many such biomarkers remain unknown because of technological limitations. In this article, we propose the "immunologic mapping" of cancer glycomes based on specific immune recognition of glycan structures, which can be hypothesized theoretically, produced chemically, and examined biologically by immuno-assays. Immunologic mapping of glycans not only provides a unique perspective on cancer glycomes, but also may lead to the invention of powerful reagents for diagnosis and therapy.

Immunologic mapping of glycomes: implications for cancer diagnosis and therapy

PubMed Central

Zhou, Dapeng; Levery, Steven B.; Hsu, Fong-Fu; Wang, Peng G.; Teneberg, Susann; Almeida, Igor C.; Li, Yunsen; Xu, Huaxi; Wang, Lai-Xi; Xia, Chengfeng; Ibrahim, Nuhad K; Michael, Katja

2013-01-01

Cancer associated glycoconjugates are important biomarkers, as exemplified by globo-H, CA125, CA15.3 and CA27.29. However, the exact chemical structures of many such biomarkers remain unknown because of technological limitations. In this article, we propose the “immunologic mapping” of cancer glycomes based on specific immune recognition of glycan structures, which can be hypothesized theoretically, produced chemically, and examined biologically by immuno-assays. Immunologic mapping of glycans not only provides a unique perspective on cancer glycomes, but also may lead to the invention of powerful reagents for diagnosis and therapy. PMID:21622287

Carbohydrate conjugation through microwave-assisted functionalization of single-walled carbon nanotubes using perfluorophenyl azides.

PubMed

Kong, Na; Shimpi, Manishkumar R; Ramström, Olof; Yan, Mingdi

2015-03-20

Carbohydrate-functionalized single-walled carbon nanotubes (SWNTs) were synthesized using microwave-assisted reaction of perfluorophenyl azide with the nanotubes. The results showed that microwave radiation provides a rapid and effective means to covalently attach carbohydrates to SWNTs, producing carbohydrate-SWNT conjugates for biorecognition. The carbohydrate-functionalized SWNTs were furthermore shown to interact specifically with cognate carbohydrate-specific proteins (lectins), resulting in predicted recognition patterns. The carbohydrate-presenting SWNTs constitute a new platform for sensitive protein- or cell recognition, which pave the way for glycoconjugated carbon nanomaterials in biorecognition applications. Copyright © 2014 Elsevier Ltd. All rights reserved.

Detection Method of TOXOPLASMA GONDII Tachyzoites

NASA Astrophysics Data System (ADS)

Eassa, Souzan; Bose, Chhanda; Alusta, Pierre; Tarasenko, Olga

2011-06-01

Tachyzoites are considered to be the most important stage of Toxoplasma gondii which causes toxoplasmosis. T. gondii is, an obligate intracellular parasite which infects a wide range of cells. The present study was designed to develop a method for an early detection of T. gondii tachyzoites. The method comprised of a binding assay which was analyzed using principal component and cluster analysis. Our data showed that glycoconjugates GC1, GC2, GC3 and GC10 exhibit a significantly higher binding affinity for T. gondii tachyzoites as compared to controls (T. gondii only, PAA only, GC 1, 2, 3, and 10 only).

Arabinogalactan-proteins and the research challenges for these enigmatic plant cell surface proteoglycans

PubMed Central

Tan, Li; Showalter, Allan M.; Egelund, Jack; Hernandez-Sanchez, Arianna; Doblin, Monika S.; Bacic, Antony

2012-01-01

Arabinogalactan-proteins (AGPs) are complex glycoconjugates that are commonly found at the cell surface and in secretions of plants. Their location and diversity of structures have made them attractive targets as modulators of plant development but definitive proof of their direct role(s) in biological processes remains elusive. Here we overview the current state of knowledge on AGPs, identify key challenges impeding progress in the field and propose approaches using modern bioinformatic, (bio)chemical, cell biological, molecular and genetic techniques that could be applied to redress these gaps in our knowledge. PMID:22754559

Distributional map of the terminal and sub-terminal sugar residues of the glycoconjugates in the prepubertal and postpubertal testis of a subject affected by complete androgen insensitivity syndrome (Morris's syndrome): lectin histochemical study.

PubMed

Gheri, G; Vannelli, G B; Marini, M; Zappoli Thyrion, G D; Gheri, R G; Sgambati, E

2004-01-01

In the present research we have investigated the distribution of the sugar residues of the glycoconjugates in the prepubertal and postpubertal testes of a subject with Morris's syndrome (CAIS, Complete Androgen Insensitivity Syndrome). For this purpose a battery of six horseradish peroxidase-conjugated lectins was used (SBA, PNA, WGA, ConA, LTA and UEAI). We have obtained a complete distributional map of the terminal and sub-terminal oligosaccharides in the tunica albuginea, interstitial tissue, lamina propria of the seminiferous tubules, Leydig cells, Sertoli cells, spermatogonia, mastocytes and endothelial cells. Furthermore the present study has shown that a large amount of sugar residues were detectable in the prepubertal and postpubertal testes but that some differences exist with particular regard to the Sertoli cells. The Sertoli cells and the Leydig cells of the retained prepubertal testis of the patient affected by Morris's syndrome were characterized by the presence of alpha-L-fucose, which was absent in the retained prepubertal testis of the normal subjects. Comparing the results on the postpubertal testis with those obtained on the same aged testis of healthy subjects we have demonstrated that alpha-L-fucose in the Sertoli and Leydig cells and D-galactose-N-acetyl-D-galactosamine in the Leydig cells are a unique feature of the subject affected by Morris's syndrome. D-galactose (ss1,3)-N-acetyl-D-galactosamine and sialic acid, which are present in the Leydig cells of the normal testis were never observed in the same cells of the postpubertal testis of the CAIS patient.

Immunogenicity and efficacy following sequential parenterally-administered doses of Salmonella Enteritidis COPS:FliC glycoconjugates in infant and adult mice.

PubMed

Baliban, Scott M; Curtis, Brittany; Toema, Deanna; Tennant, Sharon M; Levine, Myron M; Pasetti, Marcela F; Simon, Raphael

2018-05-23

In sub-Saharan Africa, invasive nontyphoidal Salmonella (iNTS) infections with serovars S. Enteritidis, S. Typhimurium and I 4,[5],12:i:- are widespread in children < 5 years old. Development of an efficacious vaccine would provide an important public health tool to prevent iNTS disease in this population. Glycoconjugates of S. Enteritidis core and O-polysaccharide (COPS) coupled to the homologous serovar phase 1 flagellin protein (FliC) were previously shown to be immunogenic and protected adult mice against death following challenge with a virulent Malian S. Enteritidis blood isolate. This study extends these observations to immunization of mice in early life and also assesses protection with partial and full regimens. Anti-COPS and anti-FliC serum IgG titers were assessed in infant and adult mice after immunization with 1, 2 or 3 doses of S. Enteritidis COPS:FliC alone or co-formulated with aluminum hydroxide or monophosphoryl lipid A (MPL) adjuvants. S. Enteritidis COPS:FliC was immunogenic in both age groups, although the immune responses were quantitatively lower in infants. Kinetics of antibody production were similar for the native and adjuvanted formulations after three doses; conjugates formulated with MPL elicited significantly increased anti-COPS IgG titers in adult but not infant mice. Nevertheless, robust protection against S. Enteritidis challenge was seen for all three formulations when three doses were given either during infancy or as adults. We further found that significant protection could be achieved with two COPS:FliC doses, despite elicitation of modest serum anti-COPS IgG antibody titers. These findings guide potential immunization strategies that may be translated to develop a human pediatric iNTS vaccine for sub-Saharan Africa.

Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM(197) conjugate vaccine co-localizes with MHC II on the antigen processing cell surface.

PubMed

Lai, Zengzu; Schreiber, John R

2009-05-21

Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM(197) induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHC II and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM(197) in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM(197) and 19F-CRM(197) was specifically labeled by Alexa Fluor 594 hydrazide (red). The CRM(197) was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHC II antibody labeled green by Alexa Fluor 488, followed by confocal microscopy. Labeled CRM(197) was presented on APC surface and co-localized with MHC II (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM(197) and 19F-CRM(197) was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM(197). Brefeldin A and chloroquine blocked both CRM(197) and PS labeled 14-CRM(197) and 19F-CRM(197) from co-localizing with MHC II. These data suggest that the PS component of the CRM(197) glycoconjugate enters the endosome, travels with CRM(197) peptides to the APC surface and co-localizes with MHC II.

Hypothalamic digoxin, hemispheric chemical dominance, and peptic ulcer disease.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2003-10-01

The isoprenoid pathway produces three key metabolites--endogenous digoxin-like factor (EDLF) (membrane sodium-potassium ATPase inhibitor and regulator of neurotransmitter transport), ubiquinone (free radical scavenger), and dolichol (regulator of glycoconjugate metabolism). The pathway was assessed in peptic ulcer and acid peptic disease and its relation to hemispheric dominance studied. The activity of HMG CoA reductase, serum levels of EDLF, magnesium, tryptophan catabolites, and tyrosine catabolites were measured in acid peptic disease, right hemispheric dominant, left hemispheric dominant, and bihemispheric dominant individuals. All the patients with peptic ulcer disease were right-handed/left hemispheric dominant by the dichotic listening test. The pathway was upregulated with increased EDLF synthesis in peptic ulcer disease (PUD). There was increase in tryptophan catabolites and reduction in tyrosine catabolites in these patients. The ubiquinone levels were low and free radical production increased. Dolichol and glycoconjugate levels were increased and lysosomal stability reduced in patients with acid peptic disease (APD). There was increase in cholesterol:phospholipid ratio with decreased glyco conjugate levels in membranes of patients with PUD. Acid peptic disease represents an elevated EDLF state which can modulate gastric acid secretion and the structure of the gastric mucous barrier. It can also lead to persistence of Helicobacter pylori infection. The biochemical pattern obtained in peptic ulcer disease is similar to those obtained in left-handed/right hemispheric chemically dominant individuals. But all the patients with peptic ulcer disease were right-handed/left hemispheric dominant by the dichotic listen ing test. Hemispheric chemical dominance has no correlation with handedness or the dichotic listening test. Peptic ulcer disease occurs in right hemispheric chemically dominant individuals and is a reflection of altered brain function.

Hypothalamic digoxin and hemispheric chemical dominance: relation to alcoholic addiction, alcoholic cirrhosis, and acquired hepatocerebral degeneration.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2003-08-01

The isoprenoid pathway produces three key metabolites--endogenous digoxin (modulate tryptophan/tyrosine transport), dolichol (important in N -glycosylation of proteins), and ubiquinone (free radical scavenger). It was considered pertinent to assess the pathway in alcoholic addiction, alcoholic cirrhosis, and acquired hepatocerebral degeneration. Since endogenous digoxin can regulate neurotransmitter transport, the pathway was also assessed in individuals with differing hemispheric dominance to find out the role of hemispheric dominance in its pathogenesis. In the patient group there was elevated digoxin synthesis, increased dolichol and glycoconjugate levels, and low ubiquinone and elevated free radical levels. There was also an increase in tryptophan catabolites and a reduction in tyrosine catabolites as reduced endogenous morphine synthesis from tyrosine. There was an increase in cholesterol:phospholipid ratio and a reduction in glycoconjugate level of RBC membrane in these groups of patients. The same patterns were obtained in individuals with right hemispheric chemical dominance. Alcoholic cirrhosis, alcoholic addiction, and acquired hepatocerebral degeneration are associated with an upregulated isoprenoid pathway and elevated digoxin secretion from the hypothalamus. This can contribute to NMDA excitotoxicity and altered connective tissue/lipid metabolism important in its pathogenesis. Endogenous morphine deficiency plays a role in alcoholic addiction. Alcoholic cirrhosis, addiction, and acquired hepato -cerebral degeneration occur in right hemispheric chemically dominant individuals. Ninety percent of the patients with alcoholic addiction, alcoholic cirrhosis, and acquired hepatocerebral degeneration were right-handed and left hemispheric dominant by the dichotic listening test. However, their biochemical patterns were similar to those obtained in right hemispheric chemical dominance. Hemispheric chemical dominance is a different entity and has no correlation with handedness or the dichotic listening test.

Glycoconjugates reveal diversity of human neural stem cells (hNSCs) derived from human induced pluripotent stem cells (hiPSCs).

PubMed

Kandasamy, Majury; Roll, Lars; Langenstroth, Daniel; Brüstle, Oliver; Faissner, Andreas

2017-06-01

Neural stem cells (NSCs) have the ability to self-renew and to differentiate into various cell types of the central nervous system. This potential can be recapitulated by human induced pluripotent stem cells (hiPSCs) in vitro. The differentiation capacity of hiPSCs is characterized by several stages with distinct morphologies and the expression of various marker molecules. We used the monoclonal antibodies (mAbs) 487 LeX , 5750 LeX and 473HD to analyze the expression pattern of particular carbohydrate motifs as potential markers at six differentiation stages of hiPSCs. Mouse ESCs were used as a comparison. At the pluripotent stage, 487 LeX -, 5750 LeX - and 473HD-related glycans were differently expressed. Later, cells of the three germ layers in embryoid bodies (hEBs) and, even after neuralization of hEBs, subpopulations of cells were labeled with these surface antibodies. At the human rosette-stage of NSCs (hR-NSC), LeX- and 473HD-related epitopes showed antibody-specific expression patterns. We also found evidence that these surface antibodies could be used to distinguish the hR-NSCs from the hSR-NSCs stages. Characterization of hNSCs FGF-2/EGF derived from hSR-NSCs revealed that both LeX antibodies and the 473HD antibody labeled subpopulations of hNSCs FGF-2/EGF . Finally, we identified potential LeX carrier molecules that were spatiotemporally regulated in early and late stages of differentiation. Our study provides new insights into the regulation of glycoconjugates during early human stem cell development. The mAbs 487 LeX , 5750 LeX and 473HD are promising tools for identifying distinct stages during neural differentiation.

Fulfilling Koch's postulates in glycoscience: HCELL, GPS and translational glycobiology.

PubMed

Sackstein, Robert

2016-06-01

Glycoscience-based research that is performed expressly to address medical necessity and improve patient outcomes is called "translational glycobiology". In the 19th century, Robert Koch proposed a set of postulates to rigorously establish causality in microbial pathogenesis, and these postulates can be reshaped to guide knowledge into how naturally-expressed glycoconjugates direct molecular processes critical to human well-being. Studies in the 1990s indicated that E-selectin, an endothelial lectin that binds sialofucosylated carbohydrate determinants, is constitutively expressed on marrow microvessels, and investigations in my laboratory indicated that human hematopoietic stem cells (HSCs) uniquely express high levels of a specialized glycoform of CD44 called "hematopoietic cell E-/L-selectin ligand" (HCELL) that functions as a highly potent E-selectin ligand. To assess the role of HCELL in directing HSC migration to marrow, a method called "glycosyltransferase-programmed stereosubstitution" (GPS) was developed to custom-modify CD44 glycans to enforce HCELL expression on viable cell surfaces. Human mesenchymal stem cells (MSCs) are devoid of E-selectin ligands, but GPS-based glycoengineering of CD44 on MSCs licenses homing of these cells to marrow in vivo, providing direct evidence that HCELL serves as a "bone marrow homing receptor". This review will discuss the molecular basis of cell migration in historical context, will describe the discovery of HCELL and its function as the bone marrow homing receptor, and will inform on how glycoengineering of CD44 serves as a model for adapting Koch's postulates to elucidate the key roles that glycoconjugates play in human biology and for realizing the immense impact of translational glycobiology in clinical medicine. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Tissue- and cell-specific localization of galectins, β-galactose-binding animal lectins, and their potential functions in health and disease.

PubMed

Nio-Kobayashi, Junko

2017-01-01

Fifteen galectins, β-galactose-binding animal lectins, are known to be distributed throughout the body. We herein summarize current knowledge on the tissue- and cell-specific localization of galectins and their potential functions in health and disease. Galectin-3 is widely distributed in epithelia, including the simple columnar epithelium in the gut, stratified squamous epithelium in the gut and skin, and transitional epithelium and several regions in nephrons in the urinary tract. Galectin-2 and galectin-4/6 are gut-specific, while galectin-7 is found in the stratified squamous epithelium in the gut and skin. The reproductive tract mainly contains galectin-1 and galectin-3, and their expression markedly changes during the estrous/menstrual cycle. The galectin subtype expressed in the corpus luteum (CL) changes in association with luteal function. The CL of women and cows displays a "galectin switch" with coordinated changes in the major galectin subtype and its ligand glycoconjugate structure. Macrophages express galectin-3, which may be involved in phagocytotic activity. Lymphoid tissues contain galectin-3-positive macrophages, which are not always stained with the macrophage marker, F4/80. Subsets of neurons in the brain and dorsal root ganglion express galectin-1 and galectin-3, which may contribute to the regeneration of damaged axons, stem cell differentiation, and pain control. The subtype-specific contribution of galectins to implantation, fibrosis, and diabetes are also discussed. The function of galectins may differ depending on the tissues or cells in which they act. The ligand glycoconjugate structures mediated by glycosyltransferases including MGAT5, ST6GAL1, and C2GnT are important for revealing the functions of galectins in healthy and disease states.

Immunization with recombinantly expressed glycan antigens from Schistosoma mansoni induces glycan-specific antibodies against the parasite

PubMed Central

Prasanphanich, Nina Salinger; Luyai, Anthony E; Song, Xuezheng; Heimburg-Molinaro, Jamie; Mandalasi, Msano; Mickum, Megan; Smith, David F; Nyame, A Kwame; Cummings, Richard D

2014-01-01

Schistosomiasis caused by infection with parasitic helminths of Schistosoma spp. is a major global health problem due to inadequate treatment and lack of a vaccine. The immune response to schistosomes includes glycan antigens, which could be valuable diagnostic markers and vaccine targets. However, no precedent exists for how to design vaccines targeting eukaryotic glycoconjugates. The di- and tri-saccharide motifs LacdiNAc (GalNAcβ1,4GlcNAc; LDN) and fucosylated LacdiNAc (GalNAcβ1,4(Fucα1-3)GlcNAc; LDNF) are the basis for several important schistosome glycan antigens. They occur in monomeric form or as repeating units (poly-LDNF) and as part of a variety of different glycoconjugates. Because chemical synthesis and conjugation of such antigens is exceedingly difficult, we sought to develop a recombinant expression system for parasite glycans. We hypothesized that presentation of parasite glycans on the cell surface would induce glycan-specific antibodies. We generated Chinese hamster ovary (CHO) Lec8 cell lines expressing poly-LDN (L8-GT) and poly-LDNF (L8-GTFT) abundantly on their membrane glycoproteins. Sera from Schistosoma mansoni-infected mice were highly cross-reactive with the cells and with cell-surface N-glycans. Immunizing mice with L8-GT and L8-GTFT cells induced glycan-specific antibodies. The L8-GTFT cells induced a sustained booster response, with antibodies that bound to S. mansoni lysates and recapitulated the exquisite specificity of the anti-parasite response for particular presentations of LDNF antigen. In summary, this recombinant expression system promotes successful generation of antibodies to the glycans of S. mansoni, and it can be adapted to study the role of glycan antigens and anti-glycan immune responses in many other infections and pathologies. PMID:24727440

Identification and characterization of a bile salt hydrolase from Lactobacillus salivarius for development of novel alternatives to antibiotic growth promoters.

PubMed

Wang, Zhong; Zeng, Ximin; Mo, Yiming; Smith, Katie; Guo, Yuming; Lin, Jun

2012-12-01

Antibiotic growth promoters (AGPs) have been used as feed additives to improve average body weight gain and feed efficiency in food animals for more than 5 decades. However, there is a worldwide trend to limit AGP use to protect food safety and public health, which raises an urgent need to discover effective alternatives to AGPs. The growth-promoting effect of AGPs has been shown to be highly correlated with the decreased activity of intestinal bile salt hydrolase (BSH), an enzyme that is produced by various gut microflora and involved in host lipid metabolism. Thus, BSH inhibitors are likely promising feed additives to AGPs to improve animal growth performance. In this study, the genome of Lactobacillus salivarius NRRL B-30514, a BSH-producing strain isolated from chicken, was sequenced by a 454 GS FLX sequencer. A BSH gene identified by genome analysis was cloned and expressed in an Escherichia coli expression system for enzymatic analyses. The BSH displayed efficient hydrolysis activity for both glycoconjugated and tauroconjugated bile salts, with slightly higher catalytic efficiencies (k(cat)/K(m)) on glycoconjugated bile salts. The optimal pH and temperature for the BSH activity were 5.5 and 41°C, respectively. Examination of a panel of dietary compounds using the purified BSH identified some potent BSH inhibitors, in which copper and zinc have been recently demonstrated to promote feed digestion and body weight gain in different food animals. In sum, this study identified and characterized a BSH with broad substrate specificity from a chicken L. salivarius strain and established a solid platform for us to discover novel BSH inhibitors, the promising feed additives to replace AGPs for enhancing the productivity and sustainability of food animals.

Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis.

PubMed

Moriuchi, Hiromi; Unno, Hideaki; Goda, Shuichiro; Tateno, Hiroaki; Hirabayashi, Jun; Hatakeyama, Tomomitsu

2015-07-01

CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin. Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis. Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17×10(3) M(-1) at 25°C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose. Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs. Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

History of meningococcal vaccines and their serological correlates of protection.

PubMed

Vipond, Caroline; Care, Rory; Feavers, Ian M

2012-05-30

For over a hundred years Neisseria meningitidis has been known to be one of the major causes of bacterial meningitis. However, effective vaccines were not developed until the latter part of the 20th century. The first of these were based on purified high molecular weight capsular polysaccharides and more recently the development of glycoconjugate vaccines has made paediatric immunisation programmes possible. The prevention of group B meningococcal disease has remained a challenge throughout this period. This review charts the history of the development of meningococcal vaccines and the importance of serological correlates of protection in their evaluation. Copyright © 2012 Elsevier Ltd. All rights reserved.

From chitin to bioactive chitooligosaccharides and conjugates: access to lipochitooligosaccharides and the TMG-chitotriomycin.

PubMed

Despras, Guillaume; Alix, Aurélien; Urban, Dominique; Vauzeilles, Boris; Beau, Jean-Marie

2014-10-27

The direct and chemoselective N-transacylation of peracetylated chitooligosaccharides (COSs), readily obtained from chitin, to give per-N-trifluoroacetyl derivatives offers an attractive route to size-defined COSs and derived glycoconjugates. It involves the use of various acceptor building blocks and trifluoromethyl oxazoline dimer donors prepared with efficiency and highly reactive in 1,2-trans glycosylation reactions. This method was applied to the preparation of the important symbiotic glycolipids which are highly active on plants and to the TMG-chitotriomycin, a potent and specific inhibitor of insect, fungal, and bacterial N-acetylglucosaminidases. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Selective binding and transcytosis of Ulex europaeus 1 lectin by mouse Peyer's patch M-cells in vivo.

PubMed

Clark, M A; Jepson, M A; Simmons, N L; Hirst, B H

1995-12-01

The in vivo interaction of the lectin Ulex europaeus agglutinin 1 with mouse Peyer's patch follicle-associated epithelial cells was studied in the mouse Peyer's patch gut loop model by immunofluorescence and electron microscopy. The lectin targets to mouse Peyer's patch M-cells and is rapidly endocytosed and transcytosed. These processes are accompanied by morphological changes in the M-cell microvilli and by redistribution of polymerised actin. The demonstration of selective binding and uptake of a lectin by intestinal M-cells in vivo suggests that M-cell-specific surface glycoconjugates might act as receptors for the selective adhesion/uptake of microorganisms.

Recent advances in synthesis of bacterial rare sugar building blocks and their applications.

PubMed

Emmadi, Madhu; Kulkarni, Suvarn S

2014-07-01

Covering: 1964 to 2013. Bacteria have unusual glycans on their surfaces which distinguish them from the host cells. These unique structures offer avenues for targeting bacteria with specific therapeutics and vaccine. However, these rare sugars are not accessible in acceptable purity and amounts by isolation from natural sources. Thus, procurement of orthogonally protected rare sugar building blocks through efficient chemical synthesis is regarded as a crucial step towards the development of glycoconjugate vaccines. This Highlight focuses on recent advances in the synthesis of the bacterial deoxy amino hexopyranoside building blocks and their application in constructing various biologically important bacterial O-glycans.

The use of the 2-aminobenzoic acid tag for oligosaccharide gel electrophoresis.

PubMed

Huang, Z; Prickett, T; Potts, M; Helm, R F

2000-08-18

Gel electrophoresis of fluorophore labeled saccharides provides a rapid and reliable method to screen enzymatic and/or chemical treatments of polysaccharides and glycoconjugates, as well as a sensitive and efficient microscale method to separate and purify oligosaccharides for further analysis. A simple and inexpensive method of derivatization and analysis using 2-aminobenzoic acid (anthranilic acid, AA) is described and applied to the extracellular polysaccharide released by the desiccation tolerant cyanobacterium Nostoc commune DRH-1. The results of these analyses suggest a possible protective functionality of two pendent groups, as well as a potential relationship between these groups and the desiccation tolerance of the organism.

An overview on Leishmania (Mundinia) enriettii: biology, immunopathology, LRV and extracellular vesicles during the host-parasite interaction.

PubMed

Paranaiba, Larissa F; Pinheiro, Lucélia J; Macedo, Diego H; Menezes-Neto, Armando; Torrecilhas, Ana C; Tafuri, Wagner L; Soares, Rodrigo P

2017-12-10

One of the Leishmania species known to be non-infective to humans is Leishmania (Mundinia) enriettii whose vertebrate host is the guinea pig Cavia porcellus. It is a good model for cutaneous leishmaniasis, chemotherapeutic and molecular studies. In the last years, an increased interest has emerged concerning the L. (Mundinia) subgenus after the finding of Leishmania (M.) macropodum in Australia and with the description of other new/putative species such as L. (M.) martiniquensis and 'L. (M.) siamensis'. This review focused on histopathology, glycoconjugates and innate immunity. The presence of Leishmania RNA virus and shedding of extracellular vesicles by the parasite were also evaluated.

Interaction of a lectin from Psathyrella velutina mushroom with N-acetylneuraminic acid.

PubMed

Ueda, H; Kojima, K; Saitoh, T; Ogawa, H

1999-04-01

A lectin from the fruiting body of Psathyrella velutina has been used as a specific probe for non-reducing terminal N-acetylglucosamine residues. We reveal in this report that P. velutina lectin recognizes a non-reducing terminal N-acetylneuraminic acid residue in glycoproteins and oligosaccharides. Binding of biotinyl P. velutina lectin to N-acetylneuraminic acid residues was prevented by desialylation of glycoconjugates and was distinguished from the binding to N-acetylglucosamine. Sialooligosaccharides were retarded or bound and eluted with N-acetylglucosamine on a P. velutina lectin column, being differentiated from each other and also from the oligosaccharides with non-reducing terminal N-acetylglucosamine which bound more strongly to the column.

Switching of bacterial adhesion to a glycosylated surface by reversible reorientation of the carbohydrate ligand.

PubMed

Weber, Theresa; Chandrasekaran, Vijayanand; Stamer, Insa; Thygesen, Mikkel B; Terfort, Andreas; Lindhorst, Thisbe K

2014-12-22

The surface recognition in many biological systems is guided by the interaction of carbohydrate-specific proteins (lectins) with carbohydrate epitopes (ligands) located within the unordered glycoconjugate layer (glycocalyx) of cells. Thus, for recognition, the respective ligand has to reorient for a successful matching event. Herein, we present for the first time a model system, in which only the orientation of the ligand is altered in a controlled manner without changing the recognition quality of the ligand itself. The key for this orientational control is the embedding into an interfacial system and the use of a photoswitchable mechanical joint, such as azobenzene. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improved conjugation and purification strategies for the preparation of protein-polysaccharide conjugates.

PubMed

Suárez, N; Massaldi, H; Franco Fraguas, L; Ferreira, F

2008-12-12

A glycoconjugate constituted by the Streptococcus pneumoniae serotype 14 capsular polysaccharide (CPS14) and bovine serum albumin (BSA) was prepared, and the unique properties of Sephadex LH-20 were used to separate the conjugate from the unconjugated material. The strength of this approach consists in its capacity to produce pure polysaccharide-protein conjugate in good yield and free from unconjugated material, a common residual contaminant of this type of immunobiologicals. The CPS14-BSA conjugate prepared via an improved 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP)-activation technique was characterized chemically and its immunogenicity was evaluated in mice. The purified conjugate, unlike the corresponding polysaccharide, produced a T-cell-dependent response in this species.

Chiral reagents in glycosylation and modification of carbohydrates.

PubMed

Wang, Hao-Yuan; Blaszczyk, Stephanie A; Xiao, Guozhi; Tang, Weiping

2018-02-05

Carbohydrates play a significant role in numerous biological events, and the chemical synthesis of carbohydrates is vital for further studies to understand their various biological functions. Due to the structural complexity of carbohydrates, the stereoselective formation of glycosidic linkages and the site-selective modification of hydroxyl groups are very challenging and at the same time extremely important. In recent years, the rapid development of chiral reagents including both chiral auxiliaries and chiral catalysts has significantly improved the stereoselectivity for glycosylation reactions and the site-selectivity for the modification of carbohydrates. These new tools will greatly facilitate the efficient synthesis of oligosaccharides, polysaccharides, and glycoconjugates. In this tutorial review, we will summarize these advances and highlight the most recent examples.

Structure of mycoside F, a family of trehalose-containing glycolipids of Mycobacterium fortuitum.

PubMed

Gautier, N; López Marín, L M; Lanéelle, M A; Daffé, M

1992-11-01

Nuclear magnetic resonance spectroscopy, fast-atom bombardment mass spectrometry, gas chromatography-mass spectrometry, as well as chemical degradations were used to elucidate the structure of the major glycolipids of Mycobacterium fortuitum. Three main glycoconjugates were detected and their structures established as 2,3-diacyl, 2,3,4- and 2,3,6-triacyl trehalose. The characteristic infrared spectrum which led to their original designation as mycoside F, a family of glycolipids limited in distribution to M. fortuitum, was due to the nature of the fatty acyl substiuents identified primarily as 2-methyl-octadecen-2-oyl. The antigenic glycolipids typified the biovar. fortuitum, thus allowing its easy recognition from the C-mycoside glycopeptidolipid-containing biovar. peregrinum.

Legume Lectins: Proteins with Diverse Applications

PubMed Central

Lagarda-Diaz, Irlanda; Guzman-Partida, Ana Maria; Vazquez-Moreno, Luz

2017-01-01

Lectins are a diverse class of proteins distributed extensively in nature. Among these proteins; legume lectins display a variety of interesting features including antimicrobial; insecticidal and antitumor activities. Because lectins recognize and bind to specific glycoconjugates present on the surface of cells and intracellular structures; they can serve as potential target molecules for developing practical applications in the fields of food; agriculture; health and pharmaceutical research. This review presents the current knowledge of the main structural characteristics of legume lectins and the relationship of structure to the exhibited specificities; provides an overview of their particular antimicrobial; insecticidal and antitumor biological activities and describes possible applications based on the pattern of recognized glyco-targets. PMID:28604616

Human Infant Memory B Cell and CD4+ T Cell Responses to HibMenCY-TT Glyco-Conjugate Vaccine

PubMed Central

Fuery, Angela; Richmond, Peter C.; Currie, Andrew J.

2015-01-01

Carrier-specific T cell and polysaccharide-specific B cell memory responses are not well characterised in infants following glyco-conjugate vaccination. We aimed to determine if the number of Meningococcal (Men) C- and Y- specific memory B cells and; number and quality of Tetanus Toxoid (TT) carrier-specific memory CD4+ T cells are associated with polysaccharide-specific IgG post HibMenCY-TT vaccination. Healthy infants received HibMenCY-TT vaccine at 2, 4 and 6 months with a booster at 12 months. Peripheral blood mononuclear cells were isolated and polysaccharide-specific memory B cells enumerated using ELISpot. TT-specific memory CD4+ T cells were detected and phenotyped based on CD154 expression and intracellular TNF-α, IL-2 and IFN-γ expression following stimulation. Functional polysaccharide-specific IgG titres were measured using the serum bactericidal activity (SBA) assay. Polysaccharide-specific Men C- but not Men Y- specific memory B cell frequencies pre-boost (12 months) were significantly associated with post-boost (13 months) SBA titres. Regression analysis showed no association between memory B cell frequencies post-priming (at 6 or 7 months) and SBA at 12 months or 13 months. TT-specific CD4+ T cells were detected at frequencies between 0.001 and 0.112 as a percentage of CD3+ T cells, but their numbers were not associated with SBA titres. There were significant negative associations between SBA titres at M13 and cytokine expression at M7 and M12. Conclusion: Induction of persistent polysaccharide-specific memory B cells prior to boosting is an important determinant of secondary IgG responses in infants. However, polysaccharide-specific functional IgG responses appear to be independent of the number and quality of circulating carrier-specific CD4+ T cells after priming. PMID:26191794

Hypothalamic digoxin, hemispheric chemical dominance, and oncogenesis: evidence from multiple myeloma.

PubMed

Kurup, Ravi Kumar; Kurup, Paramesware Achutha

2003-12-01

This study assessed the changes in the isoprenoid pathway and its metabolites digoxin, dolichol, and ubiquinone in multiple myeloma. The isoprenoid pathway and digoxin status were also studied for comparison in individuals of differing hemispheric dominance to find out the rote of cerebral dominance in the genesis of multiple myeloma and neoplasms. The following parameters were assessed: isoprenoid pathway metabolites, tyrosine and tryptophan catabolites, glycoconjugate metabolism, RBC membrane composition, and free radical metabolism--in multiple myeloma, as well as in individuals of differing hemispheric dominance. There was elevation in plasma HMG CoA reductase activity, serum digoxin, and dolichol, and a reduction in RBC membrane Na(+)-K+ ATPase activity, serum ubiquinone, and magnesium levels. Serum tryptophan, serotonin, nicotine, strychnine, and quinolinic acid were elevated, while tyrosine, dopamine, noradrenaline, and morphine were decreased. The total serum glycosaminoglycans and glycosaminoglycan fractions, the activity of GAG degrading enzymes and glycohydrolases, carbohydrate residues of glycoproteins, and serum glycolipids were elevated. The RBC membrane glycosaminoglycans, hexose, and fucose residues of glycoproteins, cholesterol, and phospholipids were reduced. The activity of all free-radical scavenging enzymes, concentration of glutathione, iron binding capacity, and ceruloplasmin decreased significantly, while the concentration of lipid peroxidation products and nitric oxide increased. Hyperdigoxinemia-related altered intracellular Ca++/Mg++ ratios mediated oncogene activation, dolichol-induced altered glycoconjugate metabolism, and ubiquinone deficiency-related mitochondrial dysfunction can contribute to the pathogenesis of multiple myeloma. The biochemical patterns obtained in multiple myeloma are similar to those obtained in left-handed/right hemispheric chemically dominant individuals by the dichotic listening test. But all the patients with multiple myeloma were right-handed/left hemispheric dominant by the dichotic listening test. Hemispheric chemical dominance has no correlation with handedness or the dichotic listening test. Multiple myeloma occurs in right hemispheric chemically dominant individuals and is a reflection of altered brain function.

Profiling monoterpenol glycoconjugation in Vitis vinifera L. cv. Muscat of Alexandria using a novel putative compound database approach, high resolution mass spectrometry and collision induced dissociation fragmentation analysis.

PubMed

Hjelmeland, Anna K; Zweigenbaum, Jerry; Ebeler, Susan E

2015-08-05

In this work we present a novel approach for the identification of plant metabolites using ultrahigh performance liquid chromatography coupled to accurate mass time-of-flight mass spectrometry. The workflow involves developing an in-house compound database consisting of exact masses of previously identified as well as putative compounds. The database is used to screen accurate mass spectrometry (MS) data to identify possible compound matches. Subsequent tandem MS data is acquired for possible matches and used for structural elucidation. The methodology is applied to profile monoterpene glycosides in Vitis vinifera cv. Muscat of Alexandria grape berries over three developmental stages. Monoterpenes are a subclass of terpenes, the largest class of plant secondary metabolites, and are found in two major forms in the plant, "bound" to one or more sugar moieties or "free" of said sugar moieties. In the free form, monoterpenes are noted for their fragrance and play important roles in plant defense and as attractants for pollinators. However, glycoconjugation renders these compounds odorless, and it is this form that the plant uses for monoterpene storage. In order to gain insight into monoterpene biochemistry and their fate in the plant an analysis of intact glycosides is essential. Eighteen monoterpene glycosides were identified including a monoterpene trisaccharide glycoside, which is tentatively identified here for this first time in any plant. Additionally, while previous studies have identified monoterpene malonylated glucosides in other grapevine tissue, we tentatively identify them for the first time in grape berries. This analytical approach can be readily applied to other plants and the workflow approach can also be used for other classes of compounds. This approach, in general, provides researchers with data to support the identification of putative compounds, which is especially useful when no standard is available. Copyright © 2015 Elsevier B.V. All rights reserved.

Differential release and phagocytosis of tegument glycoconjugates in neurocysticercosis: implications for immune evasion strategies.

PubMed

Alvarez, Jorge I; Rivera, Jennifer; Teale, Judy M

2008-04-09

Neurocysticercosis (NCC) is an infection of the central nervous system (CNS) by the metacestode of the helminth Taenia solium. The severity of the symptoms is associated with the intensity of the immune response. First, there is a long asymptomatic period where host immunity seems incapable of resolving the infection, followed by a chronic hypersensitivity reaction. Since little is known about the initial response to this infection, a murine model using the cestode Mesocestoides corti (syn. Mesocestoides vogae) was employed to analyze morphological changes in the parasite early in the infection. It was found that M. corti material is released from the tegument making close contact with the nervous tissue. These results were confirmed by infecting murine CNS with ex vivo-labeled parasites. Because more than 95% of NCC patients exhibit humoral responses against carbohydrate-based antigens, and the tegument is known to be rich in glycoconjugates (GCs), the expression of these types of molecules was analyzed in human, porcine, and murine NCC specimens. To determine the GCs present in the tegument, fluorochrome-labeled hydrazides as well as fluorochrome-labeled lectins with specificity to different carbohydrates were used. All the lectins utilized labeled the tegument. GCs bound by isolectinB4 were shed in the first days of infection and not resynthesized by the parasite, whereas GCs bound by wheat germ agglutinin and concavalinA were continuously released throughout the infectious process. GCs bound by these three lectins were taken up by host cells. Peanut lectin-binding GCs, in contrast, remained on the parasite and were not detected in host cells. The parasitic origin of the lectin-binding GCs found in host cells was confirmed using antibodies against T. solium and M. corti. We propose that both the rapid and persistent release of tegumental GCs plays a key role in the well-known immunomodulatory effects of helminths, including immune evasion and life-long inflammatory sequelae seen in many NCC patients.

Characterisation of glycoconjugate sugar residues in the vomeronasal organ of the armadillo Chaetophractus villosus (Mammalia, xenarthra)

PubMed Central

CARMANCHAHI, P. D.; FERRARI, C. C.; ALDANA MARCOS, H. J.; AFFANNI, J. M.; SONEZ, C. A.; PAZ, D. A.

2000-01-01

Conventional carbohydrate histochemistry and the binding patterns of 21 lectins were analysed to characterise the glycoconjugate content in the components of the vomeronasal organ of the armadillo Chaetophractus villosus. The mucomicrovillous complex of the sensory epithelium bound most of the lectins studied. No reaction was observed with Con A, PSA, S-Con A and SBA, and the sustentacular cells were stained with UEA-I, DSL, LEL, STL and Con A. The vomeronasal receptor neurons were labelled with S-WGA, WGA, PNA, UEA-I, STL, Con A, S-Con A, ECL and RCA120. The basal cell layer reacted with S-WGA, WGA, LCA, UEA-I, DSL, LEL, STL, Con A, JAC and VVA. The nonsensory epithelium exhibited a differential staining in relation to the different components. The mucociliary complex stained with ECL, DBA, JAC, RCA120, STL, LCA, PHA-E, PHA-L, LEL, BSL-I and VVA. However, SJA and UEA-I stained the mucus complex lining a subpopulation of columnar cells. The cytoplasm and cell membranes of columnar cells was labelled with DBA, DSL and LCA. The apical region of these cells exhibited moderate reactivity with LEL and SJA. None of the lectins bound specifically to secretory granules of the nonsecretory cells. Basal cells of the nonsensory epithelium were labelled with DSL, LEL, LCA, BSL-I and STL. The vomeronasal glands showed a positive reaction with WGA, DSL, LEL, LCA, DBA, PNA, RCA120 and SBA. Subpopulations of acinar cells were observed with ECL, S-WGA, Con A, S-Con A and DBA. PNA and RCA120 stained the cells lining the glandular ducts. In comparison with previous results obtained in the olfactory mucosa of the same group of armadillos, the carbohydrate composition of the vomeronasal organ sensory epithelium differed from the olfactory sensory epithelium. This is probably related to the different nature of molecules involved in the perireceptor processes. PMID:10853958

Multicomponent Reactions in Ligation and Bioconjugation Chemistry.

PubMed

Reguera, Leslie; Méndez, Yanira; Humpierre, Ana R; Valdés, Oscar; Rivera, Daniel G

2018-05-25

Multicomponent reactions (MCRs) encompass an exciting class of chemical transformations that have proven success in almost all fields of synthetic organic chemistry. These convergent procedures incorporate three or more reactants into a final product in one pot, thus combining high levels of complexity and diversity generation with low synthetic cost. Striking applications of these processes are found in heterocycle, peptidomimetic, and natural product syntheses. However, their potential in the preparation of large macro- and biomolecular constructs has been realized just recently. This Account describes the most relevant results of our group in the utilization of MCRs for ligation/conjugation of biomolecules along with significant contributions from other laboratories that validate the utility of this special class of bioconjugation process. Thus, MCRs have proven to be efficient in the ligation of lipids to peptides and oligosaccharides as well as the ligation of steroids, carbohydrates, and fluorescent and affinity tags to peptides and proteins. In the field of glycolipids, we highlight the power of isocyanide-based MCRs with the one-pot double lipidation of glycan fragments functionalized as either the carboxylic acid or amine. In peptide chemistry, the versatility of the multicomponent ligation strategy is demonstrated in both solution-phase lipidation protocols and solid-phase procedures enabling the simultaneous lipidation and biotinylation of peptides. In addition, we show that MCRs are powerful methods for synchronized lipidation/labeling and macrocyclization of peptides, thus accomplishing in one step what usually requires long sequences. In the realm of protein bioconjugation, MCRs have also proven to be effective in labeling, site-selective modification, immobilization, and glycoconjugation processes. For example, we illustrate a successful application of multicomponent polysaccharide-protein conjugation with the preparation of multivalent glycoconjugate vaccine candidates by the ligation of two antigenic capsular polysaccharides of a pathogenic bacterium to carrier proteins. By highlighting the ability to join several biomolecules in only one synthetic operation, we hope to encourage the biomolecular chemistry community to apply this powerful chemistry to novel biomedicinal challenges.

Cell Wall Modifications during Conidial Maturation of the Human Pathogenic Fungus Pseudallescheria boydii

PubMed Central

Ghamrawi, Sarah; Rénier, Gilles; Saulnier, Patrick; Cuenot, Stéphane; Zykwinska, Agata; Dutilh, Bas E.; Thornton, Christopher; Faure, Sébastien; Bouchara, Jean-Philippe

2014-01-01

Progress in extending the life expectancy of cystic fibrosis (CF) patients remains jeopardized by the increasing incidence of fungal respiratory infections. Pseudallescheria boydii (P. boydii), an emerging pathogen of humans, is a filamentous fungus frequently isolated from the respiratory secretions of CF patients. It is commonly believed that infection by this fungus occurs through inhalation of airborne conidia, but the mechanisms allowing the adherence of Pseudallescheria to the host epithelial cells and its escape from the host immune defenses remain largely unknown. Given that the cell wall orchestrates all these processes, we were interested in studying its dynamic changes in conidia as function of the age of cultures. We found that the surface hydrophobicity and electronegative charge of conidia increased with the age of culture. Melanin that can influence the cell surface properties, was extracted from conidia and estimated using UV-visible spectrophotometry. Cells were also directly examined and compared using electron paramagnetic resonance (EPR) that determines the production of free radicals. Consistent with the increased amount of melanin, the EPR signal intensity decreased suggesting polymerization of melanin. These results were confirmed by flow cytometry after studying the effect of melanin polymerization on the surface accessibility of mannose-containing glycoconjugates to fluorescent concanavalin A. In the absence of melanin, conidia showed a marked increase in fluorescence intensity as the age of culture increased. Using atomic force microscopy, we were unable to find rodlet-forming hydrophobins, molecules that can also affect conidial surface properties. In conclusion, the changes in surface properties and biochemical composition of the conidial wall with the age of culture highlight the process of conidial maturation. Mannose-containing glycoconjugates that are involved in immune recognition, are progressively masked by polymerization of melanin, an antioxidant that is commonly thought to allow fungal escape from the host immune defenses. PMID:24950099

Effect of endodontic irrigants on biofilm matrix polysaccharides.

PubMed

Tawakoli, P N; Ragnarsson, K T; Rechenberg, D K; Mohn, D; Zehnder, M

2017-02-01

To specifically investigate the effect of endodontic irrigants at their clinical concentration on matrix polysaccharides of cultured biofilms. Saccharolytic effects of 3% H 2 O 2 , 2% chlorhexidine (CHX), 17% EDTA, 5% NaOCl and 0.9% saline (control) were tested using agarose (α 1-3 and β 1-4 glycosidic bonds) blocks (n = 3) in a weight assay. The irrigants were also applied to three-species biofilms (Streptococcus mutans UAB 159, Streptococcus oralis OMZ 607 and Actinomyces oris OMZ 745) grown anaerobically on hydroxyapatite discs (n = 6). Glycoconjugates in the matrix and total bacterial cell volumes were determined using combined Concanavalin A-/Syto 59-staining and confocal laser-scanning microscopy. Volumes of each scanned area (triplicates/sample) were calculated using Imaris software. Data were compared between groups using one-way anova/Tukey HSD, α = 0.05. The weight assay revealed that NaOCl was the only irrigant under investigation capable of dissolving the agarose blocks. NaOCl eradicated stainable matrix and bacteria in cultured biofilms after 1 min of exposure (P < 0.05 compared to all groups, volumes in means ± standard deviation, 10 -3  mm 3 per 0.6 mm 2 disc; NaOCl matrix: 0.10 ± 0.08, bacteria: 0.03 ± 0.06; saline control matrix: 4.01 ± 1.14, bacteria: 11.56 ± 3.02). EDTA also appeared to have some effect on the biofilm matrix (EDTA matrix: 1.90 ± 0.33, bacteria: 9.26 ± 2.21), whilst H 2 O 2 and CHX merely reduced bacterial cell volumes. Sodium hypochlorite can break glycosidic bonds. It dissolves glycoconjugates in the biofilm matrix. It also lyses bacterial cells. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates.

PubMed

Bavaro, Teodora; Tengattini, Sara; Piubelli, Luciano; Mangione, Francesca; Bernardini, Roberta; Monzillo, Vincenzina; Calarota, Sandra; Marone, Piero; Amicosante, Massimo; Pollegioni, Loredano; Temporini, Caterina; Terreni, Marco

2017-06-29

Tuberculosis is still one of the most deadly infectious diseases worldwide, and the use of conjugated antigens, obtained by combining antigenic oligosaccharides, such as the lipoarabinomannane (LAM), with antigenic proteins from Mycobacterium tuberculosis (MTB), has been proposed as a new strategy for developing efficient vaccines. In this work, we investigated the effect of the chemical glycosylation on two recombinant MTB proteins produced in E. coli with an additional seven-amino acid tag (recombinant Ag85B and TB10.4). Different semi-synthetic glycoconjugated derivatives were prepared, starting from mannose and two disaccharide analogs. The glycans were activated at the anomeric position with a thiocyanomethyl group, as required for protein glycosylation by selective reaction with lysines. The glycosylation sites and the ex vivo evaluation of the immunogenic activity of the different neo- glycoproteins were investigated. Glycosylation does not modify the immunological activity of the TB10.4 protein. Similarly, Ag85B maintains its B-cell activity after glycosylation while showing a significant reduction in the T-cell response. The results were correlated with the putative B- and T-cell epitopes, predicted using a combination of in silico systems. In the recombinant TB10.4, the unique lysine is not included in any T-cell epitope. Lys30 of Ag85B, identified as the main glycosylation site, proved to be the most important site involved in the formation of T-cell epitopes, reasonably explaining why its glycosylation strongly influenced the T-cell activity. Furthermore, additional lysines included in different epitopes (Lys103, -123 and -282) are also glycosylated. In contrast, B-cell epitopic lysines of Ag85B were found to be poorly glycosylated and, thus, the antibody interaction of Ag85B was only marginally affected after coupling with mono- or disaccharides.

The loss of luteal progesterone production in women is associated with a galectin switch via α2,6-sialylation of glycoconjugates.

PubMed

Nio-Kobayashi, Junko; Boswell, Lyndsey; Amano, Maho; Iwanaga, Toshihiko; Duncan, W Colin

2014-12-01

Luteal progesterone is fundamental for reproduction, but the molecular regulation of the corpus luteum (CL) in women remains unclear. Galectin-1 and galectin-3 bind to the sugar chains on cells to control key biological processes including cell function and fate. The expression and localization of LGALS1 and LGALS3 were analyzed by quantitative PCR and histochemical analysis, with special reference to α2,6-sialylation of glycoconjugates in carefully dated human CL collected across the menstrual cycle and after exposure to human chorionic gonadotrophin (hCG) in vivo. The effects of hCG and prostaglandin E2 on the expression of galectins and an α2,6-sialyltransferase 1 (ST6GAL1) in granulosa lutein cells were analyzed in vitro. Galectin-1 was predominantly localized to healthy granulosa lutein cells and galectin-3 was localized to macrophages and regressing granulosa lutein cells. Acute exposure to luteotrophic hormones (hCG and prostaglandin E2) up-regulated LGALS1 expression (P < .001). ST6GAL1, which catalyzes α2,6-sialylation to block galectin-1 binding, increased during luteolysis (P < .05) as did LGALS3 (P < .05). Luteotrophic hormones reduced ST6GAL1 and LGALS3 in vivo (P < .05) and in vitro (P < .001). There was an inverse correlation between the expression of ST6GAL1 and HSD3B1 (P < .01) and a distinct cellular relationship among α2,6-sialylation, 3β-hydroxysteroid dehydrogenase, and galectin expression. Galectin-1 is a luteotrophic factor whose binding is inhibited by α2,6-sialylation in the human CL during luteolysis. ST6GAL1 and galectin-3 expression is increased during luteolysis and associated with a loss of progesterone synthesis. Luteotrophic hormones differentially regulate galectin-1 and galectin-3/α2,6-sialylation in granulosa lutein cells, suggesting a novel galectin switch regulated by luteotrophic stimuli during luteolysis and luteal rescue.

Antibody Kinetics and Response to Routine Vaccinations in Infants Born to Women Who Received an Investigational Trivalent Group B Streptococcus Polysaccharide CRM197-Conjugate Vaccine During Pregnancy

PubMed Central

Madhi, Shabir A; Koen, Anthonet; Cutland, Clare L; Jose, Lisa; Govender, Niresha; Wittke, Frederick; Olugbosi, Morounfolu; Sobanjo-ter Meulen, Ajoke; Baker, Sherryl; Dull, Peter M; Narasimhan, Vas; Slobod, Karen

2017-01-01

Abstract Background Maternal vaccination against group B Streptococcus (GBS) might provide protection against invasive GBS disease in infants. We investigated the kinetics of transplacentally transferred GBS serotype-specific capsular antibodies in the infants and their immune response to diphtheria toxoid and pneumococcal vaccination. Methods This phase 1b/2, observer-blind, single-center study (NCT01193920) enrolled infants born to women previously randomized (1:1:1:1) to receive either GBS vaccine at dosages of 0.5, 2.5, or 5.0 μg of each of 3 CRM197-glycoconjugates (serotypes Ia, Ib, and III), or placebo. Infants received routine immunization: combination diphtheria vaccine (diphtheria-tetanus-acellular pertussis–inactivated poliovirus/Haemophilus influenzae type b vaccine; age 6/10/ 14 weeks) and 13-valent pneumococcal CRM197-conjugate vaccine (PCV13; age 6/14 weeks and 9 months). Antibody levels were assessed at birth, day (D) 43, and D91 for GBS serotypes; 1 month postdose 3 (D127) for diphtheria; and 1 month postprimary (D127) and postbooster (D301) doses for pneumococcal serotypes. Results Of 317 infants enrolled, 295 completed the study. In infants of GBS vaccine recipients, GBS serotype-specific antibody geometric mean concentrations were significantly higher than in the placebo group at all timepoints and predictably decreased to 41%–61% and 26%–76% of birth levels by D43 and D91, respectively. Across all groups, ≥95% of infants were seroprotected against diphtheria at D127 and ≥91% of infants had seroprotective antibody levels against each PCV13 pneumococcal serotype at D301. Conclusions Maternal vaccination with an investigational CRM197-glycoconjugate GBS vaccine elicited higher GBS serotype-specific antibody levels in infants until 90 days of age, compared with a placebo group, and did not affect infant immune responses to diphtheria toxoid and pneumococcal vaccination. Clinical Trials Registration NCT01193920. PMID:29029127

Taming the Reactivity of Glycosyl Iodides To Achieve Stereoselective Glycosidation.

PubMed

Gervay-Hague, Jacquelyn

2016-01-19

Although glycosyl iodides have been known for more than 100 years, it was not until the 21st century that their full potential began to be harnessed for complex glycoconjugate synthesis. Mechanistic studies in the late 1990s probed glycosyl iodide formation by NMR spectroscopy and revealed important reactivity features embedded in protecting-group stereoelectronics. Differentially protected sugars having an anomeric acetate were reacted with trimethylsilyl iodide (TMSI) to generate the glycosyl iodides. In the absence of C-2 participation, generation of the glycosyl iodide proceeded by inversion of the starting anomeric acetate stereochemistry. Once formed, the glycosyl iodide readily underwent in situ anomerization, and in the presence of excess iodide, equilibrium concentrations of α- and β-iodides were established. Reactivity profiles depended upon the identity of the sugar and the protecting groups adorning it. Consistent with the modern idea of disarmed versus armed sugars, ester protecting groups diminished the reactivity of glycosyl iodides and ether protecting groups enhanced the reactivity. Thus, acetylated sugars were slower to form the iodide and anomerize than their benzylated analogues, and these disarmed glycosyl iodides could be isolated and purified, whereas armed ether-protected iodides could only be generated and reacted in situ. All other things being equal, the β-iodide was orders of magnitude more reactive than the thermodynamically more stable α-iodide, consistent with the idea of in situ anomerization introduced by Lemieux in the mid-20th century. Glycosyl iodides are far more reactive than the corresponding bromides, and with the increased reactivity comes increased stereocontrol, particularly when forming α-linked linear and branched oligosaccharides. Reactions with per-O-silylated glycosyl iodides are especially useful for the synthesis of α-linked glycoconjugates. Silyl ether protecting groups make the glycosyl iodide so reactive that even highly functionalized aglycon acceptors add. Following the coupling event, the TMS ethers are readily removed by methanolysis, and since all of the byproducts are volatile, multiple reactions can be performed in a single reaction vessel without isolation of intermediates. In this fashion, per-O-TMS monosaccharides can be converted to biologically relevant α-linked glycolipids in one pot. The stereochemical outcome of these reactions can also be switched to β-glycoside formation by addition of silver to chelate the iodide, thus favoring SN2 displacement of the α-iodide. While iodides derived from benzyl and silyl ether-protected oligosaccharides are susceptible to interglycosidic bond cleavage when treated with TMSI, the introduction of a single acetate protecting group prevents this unwanted side reaction. Partial acetylation of armed glycosyl iodides also attenuates HI elimination side reactions. Conversely, fully acetylated glycosyl iodides are deactivated and require metal catalysis in order for glycosidation to occur. Recent findings indicate that I2 activation of per-O-acetylated mono-, di-, and trisaccharides promotes glycosidation of cyclic ethers to give β-linked iodoalkyl glycoconjugates in one step. Products of these reactions have been converted into multivalent carbohydrate displays. With these synthetic pathways elucidated, chemical reactivity can be exquisitely controlled by the judicious selection of protecting groups to achieve high stereocontrol in step-economical processes.

Transition Metal Free Multicomponent approach to Stereo-enriched Cyclopentyl-isoxazoles via C-C Bond Cleavage.

PubMed

Kaliappan, Krishna Pillai; Subramanian, Parthasarathi

2018-06-19

An efficient multicomponent reaction leading to the synthesis of stereo-enriched cyclopentyl-isoxazoles from camphor derived α-oxime, alkynes and MeOH is reported. Our method involves a series of cascade transformations such as in situ generation of catalyst I(III) which catalyzes the addition MeOH into a sterically hindered ketone, oxime oxidation and α-hydroxyiminium ion rearrangement to generate in situ nitrile oxide which upon [3+2]-cycloaddition reaction with alkynes delivers regioselective products. The reaction is very selective to syn-oxime. This multicomponent approach has also been extended for the synthesis of a novel glycoconjugate, camphoric ester-isoxazole C-galactoside. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional network in posttranslational modifications: Glyco-Net in Glycoconjugate Data Bank.

PubMed

Miura, Nobuaki; Okada, Takuya; Murayama, Daisuke; Hirose, Kazuko; Sato, Taku; Hashimoto, Ryo; Fukushima, Nobuhiro

2015-01-01

Elucidating pathways related to posttranslational modifications (PTMs) such as glycosylation is 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. Glyco-Net (http://bibi.sci.hokudai.ac.jp/functions/) can dynamically make network figures among various biological molecules and biological events. A certain molecule or event is expressed with a node, and the relationship between the molecule and the event is indicated by arrows in the network figures. In this chapter, we mention the features and current status of the Glyco-Net and a simple example of the search with the Glyco-Net.

Human Milk Glycoproteins Protect Infants Against Human Pathogens

PubMed Central

Liu, Bo

2013-01-01

Abstract Breastfeeding protects the neonate against pathogen infection. Major mechanisms of protection include human milk glycoconjugates functioning as soluble receptor mimetics that inhibit pathogen binding to the mucosal cell surface, prebiotic stimulation of gut colonization by favorable microbiota, immunomodulation, and as a substrate for bacterial fermentation products in the gut. Human milk proteins are predominantly glycosylated, and some biological functions of these human milk glycoproteins (HMGPs) have been reported. HMGPs range in size from 14 kDa to 2,000 kDa and include mucins, secretory immunoglobulin A, bile salt-stimulated lipase, lactoferrin, butyrophilin, lactadherin, leptin, and adiponectin. This review summarizes known biological roles of HMGPs that may contribute to the ability of human milk to protect neonates from disease. PMID:23697737

Galactofuranose antigens, a target for diagnosis of fungal infections in humans

PubMed Central

Marino, Carla; Rinflerch, Adriana; de Lederkremer, Rosa M

2017-01-01

The use of biomarkers for the detection of fungal infections is of interest to complement histopathological and culture methods. Since the production of antibodies in immunocompromised patients is scarce, detection of a specific antigen could be effective for early diagnosis. D-Galactofuranose (Galf) is the antigenic epitope in glycoconjugates of several pathogenic fungi. Since Galf is not biosynthesized by mammals, it is an attractive candidate for diagnosis of infection. A monoclonal antibody that recognizes Galf is commercialized for detection of aspergillosis. The linkage of Galf in the natural glycans and the chemical structures of the synthesized Galf-containing oligosaccharides are described in this paper. The oligosaccharides could be used for the synthesis of artificial carbohydrate-based antigens, not enough exploited for diagnosis. PMID:28883999

TMG-chitotriomycin as a probe for the prediction of substrate specificity of β-N-acetylhexosaminidases.

PubMed

Shiota, Hiroto; Kanzaki, Hiroshi; Hatanaka, Tadashi; Nitoda, Teruhiko

2013-06-28

TMG-chitotriomycin (1) produced by the actinomycete Streptomyces annulatus NBRC13369 was examined as a probe for the prediction of substrate specificity of β-N-acetylhexosaminidases (HexNAcases). According to the results of inhibition assays, 14 GH20 HexNAcases from various organisms were divided into 1-sensitive and 1-insensitive enzymes. Three representatives of each group were investigated for their substrate specificity. The 1-sensitive HexNAcases hydrolyzed N-acetylchitooligosaccharides but not N-glycan-type oligosaccharides, whereas the 1-insensitive enzymes hydrolyzed N-glycan-type oligosaccharides but not N-acetylchitooligosaccharides, indicating that TMG-chitotriomycin can be used as a molecular probe to distinguish between chitin-degrading HexNAcases and glycoconjugate-processing HexNAcases. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Effect of thyroid hormones on the histotopography of lectin receptors in the rat salivary gland].

PubMed

Lutsik, A D; Iashchenko, A M; Detiuk, E S

1987-04-01

Using lectin-peroxidase technique, the influence of hypo- and hyperthyroidism on histotopography of glycoconjugates has been investigated in rat submandibular gland. The following lectins were used: peanut agglutinin (PNA), wheat germ agglutinin (WGA), Laburnum anagyroides lectin (LAL) and concanavalin A (con A). It has been demonstrated that hyperthyroidism is accompanied by the loss of con A, WGA and LAL receptor sites. Hypothyrodism enhanced con A binding to granular duct cells with a parallel reduction in WGA and LAL binding to these or other duct cells. Hypothyroidism as well as hyperthyroidism markedly enhanced PNA binding to duct epitheliocytes with redistribution of these lectin binding sites from the luminal surface of salivary ducts into the cytoplasm of duct cells. Possible interpretations of the observed phenomena are discussed.

Size determination of bacterial capsular oligosaccharides used to prepare conjugate vaccines.

PubMed

Ravenscroft, N; Averani, G; Bartoloni, A; Berti, S; Bigio, M; Carinci, V; Costantino, P; D'Ascenzi, S; Giannozzi, A; Norelli, F; Pennatini, C; Proietti, D; Ceccarini, C; Cescutti, P

1999-07-16

We recently described the use of ion exchange chromatography for analysis and the industrial scale preparation of pools of oligosaccharides of intermediate chain length from polysaccharides of Haemophilus influenzae type b (Hib) and Neisseria meningitidis groups A and C. These negatively charged "sized" oligosaccharides are activated and conjugated to the carrier protein (CRM197) to prepare the corresponding glycoconjugate vaccines. Characterization and accurate determination of the degree of polymerization (DP) of the pool of oligosaccharides is essential for the consistent production of these conjugate vaccines. This paper describes the colorimetric assays used for determination of the average DP of the Hib and meningococcal oligosaccharides, and the qualification of these assays achieved by size characterization of the respective oligosaccharides by use of physicochemical methods, including liquid chromatography, mass spectrometry (ionspray) and NMR spectroscopy.

A glycoporphyrin story: from chemistry to PDT treatment of cancer mouse models.

PubMed

Lupu, M; Maillard, Ph; Mispelter, J; Poyer, F; Thomas, C D

2018-06-01

Photodynamic therapy (PDT) represents a non-toxic and non-mutagenic antitumor therapy. The photosensitizer's (PS) chemo-physical properties are essential for the therapy, being responsible for the biological effects induced in the targeted tissues. In this study, we present the synthesis and development of some glycoconjugated porphyrins based on lectin-type receptor interaction. They were tested in vitro for finally choosing the most effective chemical structure for an optimum antitumor outcome. The most effective photosensitizer is substituted by three diethylene glycol α-d-mannosyl groups. In vivo studies allow firstly the determination of some characteristics of the biological processes triggered by the initial photochemical activation. Secondly, they make it possible to improve the therapeutic protocol in the function of the structural architecture of the targeted tumor tissue.

Cracking the Sugar Code by Mass Spectrometry - An Invited Perspective in Honor of Dr. Catherine E. Costello, Recipient of the 2017 ASMS Distinguished Contribution Award

NASA Astrophysics Data System (ADS)

Mirgorodskaya, Ekaterina; Karlsson, Niclas G.; Sihlbom, Carina; Larson, Göran; Nilsson, Carol L.

2018-04-01

The structural study of glycans and glycoconjugates is essential to assign their roles in homeostasis, health, and disease. Once dominated by nuclear magnetic resonance spectroscopy, mass spectrometric methods have become the preferred toolbox for the determination of glycan structures at high sensitivity. The patterns of such structures in different cellular states now allow us to interpret the sugar codes in health and disease, based on structure-function relationships. Dr. Catherine E. Costello was the 2017 recipient of the American Society for Mass Spectrometry's Distinguished Contribution Award. In this Perspective article, we describe her seminal work in a historical and geographical context and review the impact of her research accomplishments in the field. 8[Figure not available: see fulltext.

Galactofuranose antigens, a target for diagnosis of fungal infections in humans.

PubMed

Marino, Carla; Rinflerch, Adriana; de Lederkremer, Rosa M

2017-08-01

The use of biomarkers for the detection of fungal infections is of interest to complement histopathological and culture methods. Since the production of antibodies in immunocompromised patients is scarce, detection of a specific antigen could be effective for early diagnosis. D-Galactofuranose (Gal f ) is the antigenic epitope in glycoconjugates of several pathogenic fungi. Since Gal f is not biosynthesized by mammals, it is an attractive candidate for diagnosis of infection. A monoclonal antibody that recognizes Gal f is commercialized for detection of aspergillosis. The linkage of Gal f in the natural glycans and the chemical structures of the synthesized Gal f -containing oligosaccharides are described in this paper. The oligosaccharides could be used for the synthesis of artificial carbohydrate-based antigens, not enough exploited for diagnosis.

[Lectins, adhesins, and lectin-like substances of lactobacilli and bifidobacteria].

PubMed

Lakhtin, V M; Aleshkin, V A; Lakhtin, M V; Afanas'ev, S S; Pospelova, V V; Shenderov, B A

2006-01-01

Cell-surface adhesion factors of lactobacilli and bifidobacteria, such as lectin/adhesin proteins of S-layers, secreted lectin-like bacteriocins, and lectin-like complexes, are considered and classified in the article. Certain general and specific properties of these factors are noted, such as in vitro and in vivo adhesion, cell co(aggregation), participation in the forming of microbial biofilms and colonization of mammalian alimentary tract, as well as complexation with biopolymers and bioeffectors, specificity to glycanes and natural glycoconjugates, domain and spatial organization of adhesion factors, co-functioning with other cytokines (pro- and anti-inflammatory ones), regulation of target cell properties, and other biological and physiological activities. The authors also note possibilities of application of lectins and lectin-like proteins of probiotic strains of lactobacilli and bifidobacteria in medicine and biotechnology.

Effect of anticholinesterase agents on airway epithelial function. Annual report, 15 July 1888-14 August 1989

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marin, M.G.

1989-09-15

Irreversible anticholinesterase compounds have potential serious health effects when employed as chemical warfare agents. Intoxication with these agents will cause an accumulation of acetylcholine at nerve muscle and nerve-gland junctions. Because tracheal glands have rich cholinergic innervation, we hypothesized that exposure to anticholinesterase agents, such as soman, would stimulate glandular secretion. This would cause pathological changes in the important lung defense mechanism of mucociliary clearance. Initial work on this contract revealed a dose-related increase in mucociliary transport in the ferret in response to soman. This effect could be inhibited by atropine but not by pralidoxime. The investigation described in thismore » report relates to the effects of soman and its antidotes on glycoconjugate secretion of ferret trachea in vitro.« less

GLYDE-II: The GLYcan data exchange format

PubMed Central

Ranzinger, Rene; Kochut, Krys J.; Miller, John A.; Eavenson, Matthew; Lütteke, Thomas; York, William S.

2017-01-01

Summary The GLYcan Data Exchange (GLYDE) standard has been developed for the representation of the chemical structures of monosaccharides, glycans and glycoconjugates using a connection table formalism formatted in XML. This format allows structures, including those that do not exist in any database, to be unambiguously represented and shared by diverse computational tools. GLYDE implements a partonomy model based on human language along with rules that provide consistent structural representations, including a robust namespace for specifying monosaccharides. This approach facilitates the reuse of data processing software at the level of granularity that is most appropriate for extraction of the desired information. GLYDE-II has already been used as a key element of several glycoinformatics tools. The philosophical and technical underpinnings of GLYDE-II and recent implementation of its enhanced features are described. PMID:28955652

A minimalist approach to stereoselective glycosylation with unprotected donors.

PubMed

Le Mai Hoang, Kim; He, Jing-Xi; Báti, Gábor; Chan-Park, Mary B; Liu, Xue-Wei

2017-10-27

Mechanistic study of carbohydrate interactions in biological systems calls for the chemical synthesis of these complex structures. Owing to the specific stereo-configuration at each anomeric linkage and diversity in branching, significant breakthroughs in recent years have focused on either stereoselective glycosylation methods or facile assembly of glycan chains. Here, we introduce the unification approach that offers both stereoselective glycosidic bond formation and removal of protection/deprotection steps required for further elongation. Using dialkylboryl triflate as an in situ masking reagent, a wide array of glycosyl donors carrying one to three unprotected hydroxyl groups reacts with various glycosyl acceptors to furnish the desired products with good control over regioselectivity and stereoselectivity. This approach demonstrates the feasibility of straightforward access to important structural scaffolds for complex glycoconjugate synthesis.

Safety and immunogenicity of typhoid fever and yellow fever vaccines when administered concomitantly with quadrivalent meningococcal ACWY glycoconjugate vaccine in healthy adults.

PubMed

Alberer, Martin; Burchard, Gerd; Jelinek, Tomas; Reisinger, Emil; Beran, Jiri; Hlavata, Lucie Cerna; Forleo-Neto, Eduardo; Dagnew, Alemnew F; Arora, Ashwani K

2015-01-01

Compact and short pre-travel immunization schedules, which include several vaccinations in a single visit, are desirable for many travelers. However, concomitant vaccination could potentially compromise immunogenicity and/or safety of the individual vaccines and, therefore, possible vaccine interferences should be carefully assessed. This article discusses the immunogenicity and safety of travel vaccines for typhoid fever (TF) and yellow fever (YF), when administered with or without a quadrivalent meningococcal glycoconjugate ACWY-CRM vaccine (MenACWY-CRM). Healthy adults (18-≤60 years) were randomized to one of three vaccine regimens: TF + YF + MenACWY-CRM (group I; n = 100), TF + YF (group II; n = 101), or MenACWY-CRM (group III; n = 100). Immunogenicity at baseline and 4 weeks post-vaccination (day 29) was assessed by serum bactericidal assay using human complement (hSBA), enzyme-linked immunosorbent assay (ELISA), or a neutralization test. Adverse events (AEs) and serious adverse events (SAEs) were collected throughout the study period. Non-inferiority of post-vaccination geometric mean concentrations (GMCs) and geometric mean titers (GMTs) was established for TF and YF vaccines, respectively, when given concomitantly with MenACWY-CRM vaccine versus when given alone. The percentages of subjects with seroprotective neutralizing titers against YF on day 29 were similar in groups I and II. The antibody responses to meningococcal serogroups A, C, W-135, and Y were within the same range when MenACWY-CRM was given separately or together with TF and YF vaccines. The percentage of subjects reporting AEs was the same for TF and YF vaccines with or without MenACWY-CRM vaccine. There were no reports of SAEs or AEs leading to study withdrawals. These data provide evidence that MenACWY-CRM can be administered with typhoid Vi polysaccharide vaccine and live attenuated YF vaccine without compromising antibody responses stimulated by the individual vaccines. MenACWY-CRM can, therefore, be incorporated into travelers' vaccination programs without necessitating an additional clinic visit (NCT01466387). © 2014 International Society of Travel Medicine.

Characterizing the glycocalyx of poultry spermatozoa: III. Semen cryopreservation methods alter the carbohydrate component of rooster sperm membrane glycoconjugates.

PubMed

Peláez, J; Bongalhardo, D C; Long, J A

2011-02-01

The carbohydrate-rich zone on the sperm surface is essential for inmunoprotection in the female tract and early gamete interactions. We recently have shown the glycocalyx of chicken sperm to be extensively sialylated and to contain residues of mannose, glucose, galactose, fucose, N-acetyl-galactosamine, N-acetyl-glucosamine, and N-acetyl-lactosamine. Our objective here was to evaluate the effects of 3 different cryopreservation methods on the sperm glycocalyx. Semen from roosters was pooled, diluted, cooled to 5°C, and aliquoted for cryopreservation using 6% dimethylacetamide (DMA), 11% dimethylsulfoxide (DMSO), or 11% glycerol (GOH). For the DMA method, semen was equilibrated for 1 min with cryoprotectant and rapidly frozen by dropping 25-µL aliquots into liquid nitrogen. For the other methods, semen was equilibrated for either 1 min (DMSO) or 20 min (GOH), loaded into straws, and frozen with a programmable freezer. Thawing rates mimicked the freezing rates (e.g., rapid for DMA; moderate for DMSO and GOH). Aliquots of thawed and fresh, unfrozen semen were incubated with 1 of 12 fluorescein isothiocyanate-conjugated lectins and counterstained with propidium iodide, and mean fluorescence intensity (MFI) was assessed by flow cytometry. For each lectin, the MFI of propidium iodide-negative (viable sperm) was compared among the fresh and frozen-thawed treatments (n = 5). For sperm frozen with GOH and DMA, the MFI of most lectins was similar (P > 0.05) to that of fresh sperm, whereas only 5 of 12 lectins were similar between fresh and DMSO-frozen sperm. Sperm from all 3 methods had higher (P < 0.05) MFI for lectins specific for N-acetyl-glucosamine and β-galactose than did fresh sperm. Fewer sperm were damaged (P < 0.001) with GOH than with DMA or DMSO, and membrane integrity was correlated with MFI for 9 of 12 lectins (P < 0.05). These data indicate that surface carbohydrates are altered during cryopreservation, and that cryoprotectant type and freezing-thawing rates affect the degree of modification. Although the glycoconjugates have not yet been identified, it is likely that these cryopreservation-induced changes contribute to the reduced fertility of frozen-thawed chicken semen.

Hypothalamic digoxin, hemispheric chemical dominance, and the tridosha theory.

PubMed

Kurup, Ravi Kumar; Kurup, Parameswara Achutha

2003-05-01

Ayurveda, the traditional Indian System of Medicine, deals with the theory of the three tridosha states (both physical and psychological): Vata, Pitta, and Kapha. They are the three major human constitutional types that both depend on psychological and physical characteristics. The Pitta state is described as a critical, discriminative, and rational psychological state of mind, while the Kapha state is described as being dominant for emotional stimuli. The Vata state is an intermediate unstable shifting state. The Pitta types are of average height and built with well developed musculature. The Vata types are thin individuals with low body mass index. The Kapha types are short stocky individuals that tend toward obesity, and who are sedentary. The study assessed the biochemical differences between right hemispheric dominant, bihemispheric dominant, and left hemispheric dominant individuals, and then compared this with the patterns obtained in the Vata, Pitta, and Kapha states. The isoprenoid metabolites (digoxin, dolichol, and ubiquinone), glycoconjugate metabolism, free radical metabolism, and the RBC membrane composition were studied. The hemispheric chemical dominance in various systemic diseases and psychological states was also investigated. The results showed that right hemispheric chemically dominant/Kapha state had elevated digoxin levels, increased free radical production and reduced scavenging, increased tryptophan catabolites and reduced tyrosine catabolites, increased glycoconjugate levels and increased cholesterol: phospholipid ratio of RBC membranes. Left hemispheric chemically dominant/Pitta states had the opposite biochemical patterns. The patterns were normal or intermediate in the bihemispheric chemically dominant/Vata state. This pattern could be correlated with various systemic and neuropsychiatric diseases and personality traits. Right hemispheric chemical dominance/Kapha state represents a hyperdigoxinemic state with membrane sodium-potassium ATPase inhibition. Left hemispheric chemical dominance/Pitta state represents the reverse pattern with hypodigoxinemia and membrane sodium-potassium ATPase stimulation. The Vata state is the intermediate bihemispheric chemical dominant state. Ninety-five percent of the patients/individuals in the tridosha, pathological, and psychological groups were right-handed/left hemispheric dominant, however, their biochemical patterns were different--either left hemispheric chemical dominant or right hemispheric chemical dominant. Hemispheric chemical dominance/tridosha states had no correlation with cerebral dominance detected by handedness/dichotic listening test.

A New Family of Capsule Polymerases Generates Teichoic Acid-Like Capsule Polymers in Gram-Negative Pathogens.

PubMed

Litschko, Christa; Oldrini, Davide; Budde, Insa; Berger, Monika; Meens, Jochen; Gerardy-Schahn, Rita; Berti, Francesco; Schubert, Mario; Fiebig, Timm

2018-05-29

Group 2 capsule polymers represent crucial virulence factors of Gram-negative pathogenic bacteria. They are synthesized by enzymes called capsule polymerases. In this report, we describe a new family of polymerases that combine glycosyltransferase and hexose- and polyol-phosphate transferase activity to generate complex poly(oligosaccharide phosphate) and poly(glycosylpolyol phosphate) polymers, the latter of which display similarity to wall teichoic acid (WTA), a cell wall component of Gram-positive bacteria. Using modeling and multiple-sequence alignment, we showed homology between the predicted polymerase domains and WTA type I biosynthesis enzymes, creating a link between Gram-negative and Gram-positive cell wall biosynthesis processes. The polymerases of the new family are highly abundant and found in a variety of capsule-expressing pathogens such as Neisseria meningitidis , Actinobacillus pleuropneumoniae , Haemophilus influenzae , Bibersteinia trehalosi , and Escherichia coli with both human and animal hosts. Five representative candidates were purified, their activities were confirmed using nuclear magnetic resonance (NMR) spectroscopy, and their predicted folds were validated by site-directed mutagenesis. IMPORTANCE Bacterial capsules play an important role in the interaction between a pathogen and the immune system of its host. During the last decade, capsule polymerases have become attractive tools for the production of capsule polymers applied as antigens in glycoconjugate vaccine formulations. Conventional production of glycoconjugate vaccines requires the cultivation of the pathogen and thus the highest biosafety standards, leading to tremendous costs. With regard to animal husbandry, where vaccines could avoid the extensive use of antibiotics, conventional production is not sufficiently cost-effective. In contrast, enzymatic synthesis of capsule polymers is pathogen-free and fast, offers high stereo- and regioselectivity, and works with high efficacy. The new capsule polymerase family described here vastly increases the toolbox of enzymes available for biotechnology purposes. Representatives are abundantly found in human pathogens but also in animal pathogens, paving the way for the exploitation of polymerases for the development of a new generation of vaccines for animal husbandry. Copyright © 2018 Litschko et al.

Expression of prostate glycoconjugates in the stallion and castrated horse.

PubMed

Parillo, F; Mancuso, R; Vullo, C; Catone, G

2010-10-01

This work was undertaken to determine the glycoconjugates secreted by the epithelium of the prostate in the intact stallion and castrated horse using lectin histochemical procedures in conjunction with enzymatic digestion and deglycosylation treatments. Additionally, anti-5 and 13-16-cytokeratin antibodies were used to localize epithelial basal cells. In the stallion, lectin histochemistry showed the following sugar residues in the Golgi zone of the glandular cells: α-Glu/Man, α-Fuc and β-Gal included in both O- and N-linked oligosaccharides as well as β-GalNAc, GlcNAc and α-Gal, which belonged to O-glycoproteins. β-Gal and β-GalNAc moieties were also noted subterminal to sialyl residues. Sialic acid specific lectins identified Neu-5Ac(α2,3-6)-β-Gal or Neu5Ac(α2,6)-β-GalNAc sequences in both N- and O-bound glycoproteins. The prostatic glandular cells of the castrated horse expressed some of the same sugar moieties found in the stallions, such as α-Glu/Man, α-Gal and GlcNAc, but significant differences were also noted. In particular, β-D-GalNAc was only detected subterminal to sialic acid, β-D-Gal-(1-3)-D-GalNAc was found in N-linked glycans, whereas β-D-Gal-(1-4)-D-GlcNAc and Neu5Acα2,6Gal/GalNAc were noted only in O-glycoproteins. These results indicate that the lectin binding patterns in glandular cells may be modified by sex hormones. No specific lectin labelling of basal cells was found in either the stallion or the castrated horse even though they were immunostained with specific anti-cytokeratin antibodies. These cells stained more strongly in the castrated horse than in the intact stallion suggesting that they are androgen responsive. The glycomolecules detected in the equine prostate secretions may contribute to the remodelling of the sperm surface, which occurs during sperm transit through the male genital tract and also after ejaculation in the seminal plasma. These changes may be important in the understanding of the stallion fertility. © 2009 Blackwell Verlag GmbH.

Enteric neuromodulators and mucus discharge in a fish infected with the intestinal helminth Pomphorhynchus laevis.

PubMed

Bosi, Giampaolo; Shinn, Andrew Paul; Giari, Luisa; Sayyaf Dezfuli, Bahram

2015-07-08

In vertebrates, the presence of enteric worms can induce structural changes to the alimentary canal impacting on the neuroendocrine system, altering the proper functioning of the gastrointestinal tract and affecting the occurrence and relative density of endocrine cells (ECs). This account represents the first immunohistochemistry and ultrastructure-based study which documents the intimate relationship between the intestinal mucous cells and ECs in a fish-helminth system, investigating the potential effects of enteric neuromodulators on gut mucus secretion/discharge. A modified dual immunohisto- and histochemical staining technique was applied on intestinal sections from both infected and uninfected fish. Sections were incubated in antisera to a range of neuromodulators (i.e. leu-enkephalin, met-enkephalin, galanin and serotonin) and the glycoconjugate histochemistry of the mucous cells was determined using a subsequent alcian blue - periodic acid Schiff staining step. Dual fluorescent staining on sections prepared for confocal laser scanning microscopy and transmission electron microscopy were also used to document the relationship between ECs and mucous cells. From a total of 26 specimens of Squalius cephalus sampled from the River Paglia, 16 (i.e. 62 %) specimens were found to harbour an infection of the acanthocephalan Pomphorhynchus laevis (average intensity of infection 9.2 ± 0.8 parasites host(-1), mean ± standard error). When acanthocephalans were present, the numbers of mucous cells (most notably those containing acidic or mixed glycoconjugates) and ECs secreting leu-enkephalin, met-enkephalin, galanin, serotonin were significantly higher than those seen on sections from uninfected fish. The relationship between met-enkephalin-like or serotonin-like ECs and lectin DBA positive mucous cells was demonstrated through a dual fluorescent staining. The presence of tight connections and desmosomes between mucous and ECs in transmission electron micrographs provides further evidence of this intimate relationship. The presence of P. laevis induces an increase in the number of enteric ECs that are immunoreactive to leu- and met-enkephalin, galanin, and serotonin anti-sera. The mucous cells hyperplasia and enhanced mucus secretion in the helminth-infected intestines could be elicited by the increase in the number of ECs which release these regulatory substances.

Leishmania enriettii: biochemical characterisation of lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) and infectivity to Cavia porcellus.

PubMed

Paranaíba, Larissa Ferreira; de Assis, Rafael Ramiro; Nogueira, Paula Monalisa; Torrecilhas, Ana Claúdia; Campos, João Henrique; Silveira, Amanda Cardoso de Oliveira; Martins-Filho, Olindo Assis; Pessoa, Natalia Lima; Campos, Marco Antônio; Parreiras, Patrícia Martins; Melo, Maria Norma; Gontijo, Nelder de Figueiredo; Soares, Rodrigo Pedro Pinto

2015-01-17

Leishmania enriettii is a species non-infectious to man, whose reservoir is the guinea pig Cavia porcellus. Many aspects of the parasite-host interaction in this model are unknown, especially those involving parasite surface molecules. While lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) of Leishmania species from the Old and New World have already been described, glycoconjugates of L. enriettii and their importance are still unknown. Mice peritoneal macrophages from C57BL/6 and knock-out (TLR2 -/-, TLR4 -/-) were primed with IFN-γ and stimulated with purified LPG and GIPLs from both species. Nitric oxide and cytokine production were performed. MAPKs (p38 and JNK) and NF-kB activation were evaluated in J774.1 macrophages and CHO cells, respectively. LPGs were extracted, purified and analysed by western-blot, showing that LPG from L88 strain was longer than that of Cobaia strain. LPGs and GIPLs were depolymerised and their sugar content was determined. LPGs from both strains did not present side chains, having the common disaccharide Gal(β1,4)Man(α1)-PO4. The GIPL from L88 strain presented galactose in its structure, suggestive of type II GIPL. On the other hand, the GIPL of Cobaia strain presented an abundance of glucose, a characteristic not previously observed. Mice peritoneal macrophages from C57BL/6 and knock-outs (TLR2 -/- and TLR4 -/-) were primed with IFN-γ and stimulated with glycoconjugates and live parasites. No activation of NO or cytokines was observed with live parasites. On the other hand, LPGs and GIPLs were able to activate the production of NO, IL-6, IL-12 and TNF-α preferably via TRL2. However, in CHO cells, only GIPLs were able to activate TRL2 and TRL4. In vivo studies using male guinea pigs (Cavia porcellus) showed that only strain L88 was able to develop more severe ulcerated lesions especially in the presence of salivary gland extract (SGE). The two L. enriettii strains exhibited polymorphisms in their LPGs and GIPLs and those features may be related to a more pro-inflammatory profile in the L88 strain.

CAZyme discovery and design for sweet dreams.

PubMed

André, Isabelle; Potocki-Véronèse, Gabrielle; Barbe, Sophie; Moulis, Claire; Remaud-Siméon, Magali

2014-04-01

Development of synthetic routes to complex carbohydrates and glyco-conjugates is often hampered by the lack of enzymes with requisite properties or specificities. Indeed, assembly or degradation of carbohydrates requires carbohydrate-active enzymes (CAZymes) able to act on a vast range of glycosidic monomers, oligomers or polymers in a regio-specific or stereo-specific manner in order to produce the desired structure. Sequence-based analyses allow finding the most original enzymes. Novel screening methods have emerged that enable a more efficient exploitation of the CAZyme diversity found in the microbial world or generated by protein engineering. Computational biology methods also play a prominent role in the success of CAZyme design. Such progress allows circumventing current limitations of carbohydrate synthesis and opens new opportunities related to the synthetic biology field. Copyright © 2014 Elsevier Ltd. All rights reserved.

Fucose Migration in Intact Protonated Glycan Ions: A Universal Phenomenon in Mass Spectrometry.

PubMed

Mucha, Eike; Lettow, Maike; Marianski, Mateusz; Thomas, Daniel A; Struwe, Weston B; Harvey, David J; Meijer, Gerard; Seeberger, Peter H; von Helden, Gert; Pagel, Kevin

2018-06-18

Fucose is an essential deoxysugar that is found in a wide range of biologically relevant glycans and glycoconjugates. A recurring problem in mass spectrometric analyses of fucosylated glycans is the intramolecular migration of fucose units, which can lead to erroneous sequence assignments. This migration reaction is typically assigned to activation during collision-induced dissociation (CID) in tandem mass spectrometry (MS). In this work, we utilized cold-ion spectroscopy and show for the first time that fucose migration is not limited to fragments obtained in tandem MS and can also be observed in intact glycan ions. This observation suggests a possible low-energy barrier for this transfer reaction and generalizes fucose migration to an issue that may universally occur in any type of mass spectrometry experiment. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

How to Crack the Sugar Code.

PubMed

Gabius, H-J

2017-01-01

The known ubiquitous presence of glycans fulfils an essential prerequisite for fundamental roles in cell sociology. Since carbohydrates are chemically predestined to form biochemical messages of a maximum of structural diversity in a minimum of space, coding of biological information by sugars is the reason for the broad occurrence of cellular glycoconjugates. Their glycans originate from sophisticated enzymatic assembly and dynamically adaptable remodelling. These signals are read and translated into effects by receptors (lectins). The functional pairing between lectins and their counterreceptor(s) is highly specific, often orchestrated by intimate co-regulation of the receptor, the cognate glycan and the bioactive scaffold (e.g., an integrin). Bottom-up approaches, teaming up synthetic and supramolecular chemistry to prepare fully programmable nanoparticles as binding partners with systematic network analysis of lectins and rational design of variants, enable us to delineate the rules of the sugar code.

Visualization of predentine matrix components and endocytic structures in rat incisor odontoblasts with tannic acid.

PubMed

Goldberg, M; Septier, D

1989-12-01

Rat incisor odontoblasts and predentine fixed with tannic acid-glutaraldehyde-osmium tetroxide (Tago) were compared with those obtained by prior incubation in tannic acid-Ringer before conventional fixation with glutaraldehyde-osmium-tetroxide (Tari) The Tago method allowed visualization of complex glycoconjugates along the plasma membrane, in the pericellular spaces and in the intercellular predentine matrix. The non-collagenous proteins, proteoglycans and lipids were seen as granules and thin filaments located between the collagen fibers and at their surface. The collagen fibers themselves were also stained. The Tari method which was used to visualize exocytosis, mainly revealed endocytosis in the form of large intracellular vacuoles containing tannic acid and stained proteoglycans. It is suggested that tannic acid-Ringer incubation prior to fixation increases the endocytosis of the matrix components, which acculumates in these large vesicles.

Salivary exoglycosidases in gestational diabetes .

PubMed

Zalewska, Anna; Knaś, Małgorzata; Gumiężny, Grzegorz; Niczyporuk, Marek; Waszkiel, Danuta; Przystupa, Adrian Wojciech; Zarzycki, Wiesław

2013-04-19

As exoglycosidases have been described as potential markers of salivary gland pathology, we decided to check the possibility of the use of these enzymes in the detection of salivary gland involvement in gestational diabetes. For this purpose diabetic pregnant women were compared to pregnant and non-pregnant healthy women. The activities of total HEX as well as GLU in the saliva were determined in duplicate according to Marciniak et al. The activities of GAL, FUC, and MAN in the saliva were determined in duplicate according to Zwierz et al. It was found that the specific activities of exoglycosidases in the saliva of diabetic pregnant women significantly increased in comparison to healthy pregnant and non-pregnant women. Increased specific activity of exoglycosidases suggests that gestational diabetes provokes structural/functional alterations in salivary glands and changes in the salivary glycoconjugates metabolism.

Comparative study of structural models of Leishmania donovani and human GDP-mannose pyrophosphorylases.

PubMed

Daligaux, Pierre; Bernadat, Guillaume; Tran, Linh; Cavé, Christian; Loiseau, Philippe M; Pomel, Sébastien; Ha-Duong, Tâp

2016-01-01

Leishmania is the parasite responsible for the neglected disease leishmaniasis. Its virulence and survival require biosynthesis of glycoconjugates, whose guanosine diphospho-d-mannose pyrophosphorylase (GDP-MP) is a key player. However, experimentally resolved structures of this enzyme are still lacking. We herein propose structural models of the GDP-MP from human and Leishmania donovani. Based on a multiple sequences alignment, the models were built with MODELLER and then carefully refined with all atom molecular dynamics simulations in explicit solvent. Their quality was evaluated against several standard criteria, including their ability to bind GDP-mannose assessed by redocking calculations. Special attention was given in this study to interactions of the catalytic site residues with the enzyme substrate and competitive inhibitors, opening the perspective of medicinal chemistry developments. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Circular dichroism study of the carbohydrate-modified opioid peptides

NASA Astrophysics Data System (ADS)

Horvat, Štefica; Otvos, Laszlo; Urge, Laszlo; Horvat, Jaroslav; Čudić, Mare; Varga-Defterdarović, Lidija

1999-09-01

The conformational preferences of enkephalins and the related glycoconjugates in which free or protected carbohydrate moieties were linked to the opioid peptides through an ether, ester or amide bond were investigated by circular dichroism spectroscopy in water, trifluoroethanol and water-trifluoroethanol mixtures. The analysis of the spectra revealed that the conformation of the enkephalin molecule is very sensitive to slight changes in the peptide structure around the C-terminal region. It was found that the type II β-turn structures are populated in N-terminal tetrapeptide enkephalin fragment, while leucine-enkephalin amide feature a type I (III) β-turn structure in solution. Incorporation of the sugar moiety into opioid peptide compound did not significantly influence the overall conformation of the peptide backbone, although minor intensity changes may reflect shifts in the population of the different turn systems. These small structural alterations can be responsible for the receptor-subtype selectivity of the various carbohydrate-modified enkephalin analogs.

Tachykinin receptors mediating airway marcomolecular secretion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gentry, S.E.

1991-01-01

Three tachykinin receptor types, termed NK1, NK2, and NK3, can be distinguished by the relative potency of various peptides in eliciting tissue responses. Airway macromolecular secretion is stimulated by the tachykinin substance P (SP). The purposes of this study were to determine the tachykinin receptor subtype responsible for this stimulation, and to examine the possible involvement of other neurotransmitters in mediating this effect. Ferret tracheal explants maintained in organ culture were labeled with {sup 3}H-glucosamine, a precursor of high molecular weight glycoconjugates (HMWG) which are released by airway secretory cells. Secretion of labeled HMWG then was determined in the absencemore » and presence of the tachykinins SP, neurokinin A (NKA), neurokinin B (NKB), physalaemin (PHY), and eledoisin (ELE). To evaluate the possible contribution of other mediators, tachykinin stimulation was examined in the presence of several receptor blockers.« less

Anticancer substances of mushroom origin.

PubMed

Ivanova, T S; Krupodorova, T A; Barshteyn, V Y; Artamonova, A B; Shlyakhovenko, V A

2014-06-01

The present status of investigations about the anticancer activity which is inherent to medicinal mushrooms, as well as their biomedical potential and future prospects are discussed. Mushroom products and extracts possess promising immunomodulating and anticancer effects, so the main biologically active substances of mushrooms responsible for immunomodulation and direct cytoto-xicity toward cancer cell lines (including rarely mentioned groups of anticancer mushroom proteins), and the mechanisms of their antitumor action were analyzed. The existing to date clinical trials of mushroom substances are mentioned. Mushroom anticancer extracts, obtained by the different solvents, are outlined. Modern approaches of cancer treatment with implication of mushroom products, including DNA vaccinotherapy with mushroom immunomodulatory adjuvants, creation of prodrugs with mushroom lectins that can recognize glycoconjugates on the cancer cell surface, development of nanovectors etc. are discussed. The future prospects of mushroom anticancer substances application, including chemical modification of polysaccharides and terpenoids, gene engineering of proteins, and implementation of vaccines are reviewed.

Lectin histochemistry of goblet cell sugar residues in the gut of the chick embryo and of the newborn.

PubMed

Bryk, S G; Sgambati, E; Gheri Bryk, G

1999-04-01

The anlage of duodenum, ileum and colon were removed from chick embryos of day 8-21 of incubation and from 1-day-old chicks. A battery of seven different horseradish peroxidase-conjugated lectins (PNA, SBA, DBA, Con A, WGA, LTA and UEAI) was used to study the carbohydrate residues of the glycoconjugates in the goblet cells of the three parts of the intestine. The main results can be summarized as follows: differences in lectin binding were absent in the proximal and distal parts of the duodenum, ileum and colon. Lectin histochemistry showed differences among the three intestinal segments for the time of appearance of the oligosaccharides in the goblet mucus. In the colonic goblet cells of 1-day-old chicks, LTA and UEAI lectins showed two different types of linkage of alpha-L-fucose. This is the first demonstration of UEAI reactive sites in Gallus domesticus.

Structures of the glycopeptidolipid antigens of two animal pathogens: Mycobacterium senegalense and Mycobacterium porcinum.

PubMed

López Marín, L M; Lanéelle, M A; Promé, D; Daffé, M

1993-08-01

The structures of the major glycolipid antigens of two animal pathogens Mycobacterium senegalense and Mycobacterium porcinum were elucidated by a combination of fast-atom bombardment mass spectrometry, nuclear magnetic resonance spectroscopy, chemical analyses and radiolabeling experiments. Five glycoconjugates belonging to the class of C-mycoside glycopeptidolipids were characterized in each species. They shared with those recently described in M. peregrinum the same unusual distribution of the disaccharides on the alaninol end of the molecules. Both species showed the presence of the novel sulfated glycopeptidolipid. In addition, some acetylated forms of the glycolipids were also present in the species examined. Identical seroreactivities were observed between the glycolipid antigens extracted from M. senegalense, M. porcinum and M. peregrinum and an antiserum raised against the whole lipid antigens of M. peregrinum. These data reinforce the close taxonomic relationships between the three mycobacterial species and demonstrate the antigenicity of the new variants of mycobacterial glycopeptidolipids.

Podocalyxin as a major pluripotent marker and novel keratan sulfate proteoglycan in human embryonic and induced pluripotent stem cells.

PubMed

Toyoda, Hidenao; Nagai, Yuko; Kojima, Aya; Kinoshita-Toyoda, Akiko

2017-04-01

Podocalyxin (PC) was first identified as a heavily sialylated transmembrane protein of glomerular podocytes. Recent studies suggest that PC is a remarkable glycoconjugate that acts as a universal glyco-carrier. The glycoforms of PC are responsible for multiple functions in normal tissue, human cancer cells, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). PC is employed as a major pluripotent marker of hESCs and hiPSCs. Among the general antibodies for human PC, TRA-1-60 and TRA-1-81 recognize the keratan sulfate (KS)-related structures. Therefore, It is worthwhile to summarize the outstanding chemical characteristic of PC, including the KS-related structures. Here, we review the glycoforms of PC and discuss the potential of PC as a novel KS proteoglycan in undifferentiated hESCs and hiPSCs.

Reviews on Mechanisms of In Vitro Antioxidant Activity of Polysaccharides

PubMed Central

Wang, Junqiao; Hu, Shuzhen; Nie, Shaoping; Yu, Qiang; Xie, Mingyong

2016-01-01

It is widely acknowledged that the excessive reactive oxygen species (ROS) or reactive nitrogen species (RNS) induced oxidative stress will cause significant damage to cell structure and biomolecular function, directly or indirectly leading to a number of diseases. The overproduction of ROS/RNS will be balanced by nonenzymatic antioxidants and antioxidant enzymes. Polysaccharide or glycoconjugates derived from natural products are of considerable interest from the viewpoint of potent in vivo and in vitro antioxidant activities recently. Particularly, with regard to the in vitro antioxidant systems, polysaccharides are considered as effective free radical scavenger, reducing agent, and ferrous chelator in most of the reports. However, the underlying mechanisms of these antioxidant actions have not been illustrated systematically and sometimes controversial results appeared among various literatures. To address this issue, we summarized the latest discoveries and advancements in the study of antioxidative polysaccharides and gave a detailed description of the possible mechanisms. PMID:26682009

Regioselective Galactofuranosylation for the Synthesis of Disaccharide Patterns Found in Pathogenic Microorganisms.

PubMed

Legentil, Laurent; Cabezas, Yari; Tasseau, Olivier; Tellier, Charles; Daligault, Franck; Ferrières, Vincent

2017-07-21

Koenigs-Knorr glycosylation of acceptors with more than one free hydroxyl group by 2,3,5,6-tetrabenzoyl galactofuranosyl bromide was performed using diphenylborinic acid 2-aminoethyl ester (DPBA) as inducer of regioselectivity. High regioselectivity for the glycosylation on the equatorial hydroxyl group of the acceptor was obtained thanks to the transient formation of a borinate adduct of the corresponding 1,2-cis diol. Nevertheless formation of orthoester byproducts hampered the efficiency of the method. Interestingly electron-withdrawing groups on O-6 or on C-1 of the acceptor displaced the reaction in favor of the desired galactofuranosyl containing disaccharide. The best yield was obtained for the furanosylation of p-nitrophenyl 6-O-acetyl mannopyranoside. Precursors of other disaccharides, found in the glycocalix of some pathogens, were synthesized according to the same protocol with yields ranging from 45 to 86%. This is a good alternative for the synthesis of biologically relevant glycoconjugates.

Glycobiology of Reproductive Processes in Marine Animals: The State of the Art

PubMed Central

Gallo, Alessandra; Costantini, Maria

2012-01-01

Glycobiology is the study of complex carbohydrates in biological systems and represents a developing field of science that has made huge advances in the last half century. In fact, it combines all branches of biomedical research, revealing the vast and diverse forms of carbohydrate structures that exist in nature. Advances in structure determination have enabled scientists to study the function of complex carbohydrates in more depth and to determine the role that they play in a wide range of biological processes. Glycobiology research in marine systems has primarily focused on reproduction, in particular for what concern the chemical communication between the gametes. The current status of marine glycobiology is primarily descriptive, devoted to characterizing marine glycoconjugates with potential biomedical and biotechnological applications. In this review, we describe the current status of the glycobiology in the reproductive processes from gametogenesis to fertilization and embryo development of marine animals. PMID:23247316

Vaccines against invasive Salmonella disease

PubMed Central

MacLennan, Calman A; Martin, Laura B; Micoli, Francesca

2014-01-01

Though primarily enteric pathogens, Salmonellae are responsible for a considerable yet under-appreciated global burden of invasive disease. In South and South-East Asia, this manifests as enteric fever caused by serovars Typhi and Paratyphi A. In sub-Saharan Africa, a similar disease burden results from invasive nontyphoidal Salmonellae, principally serovars Typhimurium and Enteritidis. The existing Ty21a live-attenuated and Vi capsular polysaccharide vaccines target S. Typhi and are not effective in young children where the burden of invasive Salmonella disease is highest. After years of lack of investment in new Salmonella vaccines, recent times have seen increased interest in the area led by emerging-market manufacturers, global health vaccine institutes and academic partners. New glycoconjugate vaccines against S. Typhi are becoming available with similar vaccines against other invasive serovars in development. With other new vaccines under investigation, including live-attenuated, protein-based and GMMA vaccines, now is an exciting time for the Salmonella vaccine field. PMID:24804797

Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

2013-11-01

Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

Fluorescence polarization-based assay using N-glycan-conjugated quantum dots for screening in hemagglutinin blockers for influenza A viruses.

PubMed

Okamatsu, Masatoshi; Feng, Fei; Ohyanagi, Tatsuya; Nagahori, Noriko; Someya, Kazuhiko; Sakoda, Yoshihiro; Miura, Nobuaki; Nishimura, Shin-Ichiro; Kida, Hiroshi

2013-02-01

Attachment of influenza virus to susceptible cells is mediated by viral protein hemagglutinin (HA), which recognizes cell surface glycoconjugates that terminate in α-sialosides. To develop anti-influenza drugs based on inhibition of HA-mediated infection, novel fluorescent nanoparticles displaying multiple biantennary N-glycan chains with α-sialosides (A2-PC-QDs) that have high affinity for the HA were designed and constructed. The A2-PC-QDs enabled an easy and efficient fluorescence polarization (FP) assay for detection of interaction with the HA and competitive inhibition even by small molecule compounds against A2-PC-QDs-HA binding. The quantum dot (QD)-based FP assay established in the present study is a useful tool for high-throughput screening and to accelerate the development of novel and more effective blockers of the viral attachment of influenza virus. Copyright © 2012 Elsevier B.V. All rights reserved.

Quantification of residual EDU (N-ethyl-N'-(dimethylaminopropyl) carbodiimide (EDC) hydrolyzed urea derivative) and other residual by LC-MS/MS.

PubMed

Lei, Q Paula; Lamb, David H; Shannon, Anthony G; Cai, Xinxing; Heller, Ronald K; Huang, Michael; Zablackis, Earl; Ryall, Robert; Cash, Patricia

2004-12-25

An LC-MS/MS method for determination of the break down product of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) urea derivative, EDU, has been developed and validated for monitoring the residual coupling reagents. Results indicate that the method exhibits suitable specificity, sensitivity, precision, linearity and accuracy for quantification of residual EDU in the presence of meningococcal polysaccharide-diphtheria toxoid conjugate vaccine and other vaccine matrix compounds. The assay has been validated for a detection range of 10-100 ng/mL and then successfully transferred to quality control (QC) lab. This same method has also been applied to the determination of residual diaminohexane (DAH) in the presence of EDU. LC-MS/MS has proven to be useful as a quick and sensitive approach for simultaneous determination of multiple residual compounds in glycoconjugate vaccine samples.

Identification and functional analysis of two Golgi-localized UDP-galactofuranose transporters with overlapping functions in Aspergillus niger.

PubMed

Park, Joohae; Tefsen, Boris; Heemskerk, Marc J; Lagendijk, Ellen L; van den Hondel, Cees A M J J; van Die, Irma; Ram, Arthur F J

2015-11-02

Galactofuranose (Galf)-containing glycoconjugates are present in numerous microbes, including filamentous fungi where they are important for morphology, virulence and maintaining cell wall integrity. The incorporation of Galf-residues into galactomannan, galactomannoproteins and glycolipids is carried out by Golgi-localized Galf transferases. The nucleotide sugar donor used by these transferases (UDP-Galf) is produced in the cytoplasm and has to be transported to the lumen of the Golgi by a dedicated nucleotide sugar transporter. Based on homology with recently identified UDP-Galf-transporters in A. fumigatus and A. nidulans, two putative UDP-Galf-transporters in A. niger were found. Their function and localization was determined by gene deletions and GFP-tagging studies, respectively. The two putative UDP-Galf-transporters in A. niger are homologous to each other and are predicted to contain eleven transmembrane domains (UgtA) or ten transmembrane domains (UgtB) due to a reduced length of the C-terminal part of the UgtB protein. The presence of two putative UDP-Galf-transporters in the genome was not unique for A. niger. From the twenty Aspergillus species analysed, nine species contained two additional putative UDP-Galf-transporters. Three of the nine species were outside the Aspergillus section nigri, indication an early duplication of UDP-Galf-transporters and subsequent loss of the UgtB copy in several aspergilli. Deletion analysis of the single and double mutants in A. niger indicated that the two putative UDP-Galf-transporters (named UgtA and UgtB) have a redundant function in UDP-Galf-transport as only the double mutant displayed a Galf-negative phenotype. The Galf-negative phenotype of the double mutant could be complemented by expressing either CFP-UgtA or CFP-UgtB fusion proteins from their endogenous promoters, indicating that both CFP-tagged proteins are functional. Both Ugt proteins co-localize with each other as well as with the GDP-mannose nucleotide transporter, as was demonstrated by fluorescence microscopy, thereby confirming their predicted localization in the Golgi. A. niger contains two genes encoding UDP-Galf-transporters. Deletion and localization studies indicate that UgtA and UgtB have redundant functions in the biosynthesis of Galf-containing glycoconjugates.

The hatching gland cells of trout embryos: characterisation of N- and O-linked oligosaccharides

PubMed Central

DE GASPAR, IGNACIO; BLANQUEZ, MARIA JOSE; FRAILE, BENITO; PANIAGUA, RICARDO; ARENAS, MARIA ISABEL

1999-01-01

A histochemical, light and electron microscopy study of the hatching gland cells (HGCs) in incubated 50-d-old trout embryos is reported. The distribution of carbohydrate residues in the glycoconjugates of these cells was studied by means of a battery of 13 different lectins conjugated with horseradish peroxidase (PNA, ConA, LCA, WGA, SBA, UEA-I, HPA, DBA) or digoxigenin (DSA, MAA, AAA, SNA, GNA). Identification of N- and O-linked oligosaccharides in HGCs was performed by application of both chemical and enzymatic treatments. Present results suggest that HGCs are seromucous cells which store both high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE), and that their cytoplasmic granules, endoplasmic reticulum and Golgi complex contain additional sialic acid-rich glycoproteins. The negative charge of these glycoproteins might be responsible for the rapid expansion of mucin to form a highly hydrated gel, which would facilite the action of these enzymes in programmed cell death and might play a major role during the morphogenic events. PMID:10227672

Functional mechanics of the plant defensive Griffonia simplicifolia lectin II: resistance to proteolysis is independent of glycoconjugate binding in the insect gut.

PubMed

Zhu-Salzman, K; Salzman, R A

2001-10-01

Griffonia simplicifolia lectin II (GSII) is a plant defensive protein that significantly delays development of the cowpea bruchid Callosobruchus maculatus (F.). Previous structure/function analysis by site-directed mutagenesis indicated that carbohydrate binding and resistance to insect gut proteolysis are required for the anti-insect activity of this lectin. However, whether there is a causal link between carbohydrate binding and resistance to insect metabolism remains unknown. Two proteases principally responsible for digestive proteolysis in third and fourth instar larvae of C. maculatus were purified by activated thiol sepharose chromatography and resolved as cathepsin L-like proteases, based on N-terminal amino acid sequence analysis. Digestion of bacterially expressed recombinant GSII (rGSII) and its mutant protein variants with the purified gut proteases indicates that carbohydrate binding, presumably to a target ligand in insect gut, and proteolytic resistance are independent properties of rGSII, and that both facilitate its efficacy as a plant defensive molecule.

Golgi Glycosylation

PubMed Central

Stanley, Pamela

2011-01-01

Glycosylation is a very common modification of protein and lipid, and most glycosylation reactions occur in the Golgi. Although the transfer of initial sugar(s) to glycoproteins or glycolipids occurs in the ER or on the ER membrane, the subsequent addition of the many different sugars that make up a mature glycan is accomplished in the Golgi. Golgi membranes are studded with glycosyltransferases, glycosidases, and nucleotide sugar transporters arrayed in a generally ordered manner from the cis-Golgi to the trans-Golgi network (TGN), such that each activity is able to act on specific substrate(s) generated earlier in the pathway. The spectrum of glycosyltransferases and other activities that effect glycosylation may vary with cell type, and thus the final complement of glycans on glycoconjugates is variable. In addition, glycan synthesis is affected by Golgi pH, the integrity of Golgi peripheral membrane proteins, growth factor signaling, Golgi membrane dynamics, and cellular stress. Knowledge of Golgi glycosylation has fostered the development of assays to identify mechanisms of intracellular vesicular trafficking and facilitated glycosylation engineering of recombinant glycoproteins. PMID:21441588

Sugar residues content and distribution in atrophic and hyperplastic postmenopausal human endometrium: lectin histochemistry.

PubMed

Gheri, G; Bryk, S G; Taddei, G; Moncini, D; Noci, I

1996-10-01

A lectin histochemical study was performed to investigate the glycoconjugate saccharidic moieties of the human postmenopausal endometrium (14 atrophic and 15 hyperplastic). For this purpose a battery of seven horseradish peroxidase-conjugated lectins (PNA, SBA, DBA, WGA, ConA, LTA and UEA I) was used. No differences in lectin binding between atrophic and hyperplastic endometria were observed. This investigation allowed us to provide a basic picture of the oligosaccharidic distribution in postmenopausal endometria. The data on the saccharidic distribution at the postmenopausal endometria showed a large amount of sugar residues at all the investigated sites, i.e. the lining and glandular epithelium, the stroma and the vessels (capillary and large vessels). Furthermore, at the endometrial lining epithelium, at the glands and at the wall of the blood vessels of some postmenopausal women the presence of alpha-L-fucosyl residues which bind via alpha (1-6) linkage to penultimate glucosaminyl residues and/or difucosylated oligosaccharides was demonstrated for the first time.

Molecular interaction of Siglecs (sialic acid-binding Ig-like lectins) with sialylated ligands on Trypanosoma cruzi.

PubMed

Jacobs, Thomas; Erdmann, Hanna; Fleischer, Bernhard

2010-01-01

The protozoan parasite Trypanosoma cruzi (T. cruzi) is transmitted by blood-sucking insect vectors. After transmission, parasites circulate in the blood as trypomastigotes and invade a variety of cells to multiply intracellularly as amastigotes. The acute phase triggers an immune response that restricts the dissemination and proliferation of parasites. However, parasites are able to persist in different tissues for decades causing the pathology of Chagas' disease. T. cruzi expresses a trans-sialidase (TS). This unique enzyme transfers sialic acid from host glycoconjugates to mucin-like molecules on the parasite and is supposed to be a major virulence factor. TS and sialylated structures were implicated in the persistence of parasites. We discuss here the recent findings on the function of sialylated structures on the surface of T. cruzi with a special emphasis on their property to interact with sialic acid-binding Ig-like lectins, which may allow the parasite to modulate the immune system of the host. Copyright (c) 2009 Elsevier GmbH. All rights reserved.

High-Resolution Mass Spectrometers

NASA Astrophysics Data System (ADS)

Marshall, Alan G.; Hendrickson, Christopher L.

2008-07-01

Over the past decade, mass spectrometry has been revolutionized by access to instruments of increasingly high mass-resolving power. For small molecules up to ˜400 Da (e.g., drugs, metabolites, and various natural organic mixtures ranging from foods to petroleum), it is possible to determine elemental compositions (CcHhNnOoSsPp…) of thousands of chemical components simultaneously from accurate mass measurements (the same can be done up to 1000 Da if additional information is included). At higher mass, it becomes possible to identify proteins (including posttranslational modifications) from proteolytic peptides, as well as lipids, glycoconjugates, and other biological components. At even higher mass (˜100,000 Da or higher), it is possible to characterize posttranslational modifications of intact proteins and to map the binding surfaces of large biomolecule complexes. Here we review the principles and techniques of the highest-resolution analytical mass spectrometers (time-of-flight and Fourier transform ion cyclotron resonance and orbitrap mass analyzers) and describe some representative high-resolution applications.

Antigenic potential of a highly conserved Neisseria meningitidis lipopolysaccharide inner core structure defined by chemical synthesis.

PubMed

Reinhardt, Anika; Yang, You; Claus, Heike; Pereira, Claney L; Cox, Andrew D; Vogel, Ulrich; Anish, Chakkumkal; Seeberger, Peter H

2015-01-22

Neisseria meningitidis is a leading cause of bacterial meningitis worldwide. We studied the potential of synthetic lipopolysaccharide (LPS) inner core structures as broadly protective antigens against N. meningitidis. Based on the specific reactivity of human serum antibodies to synthetic LPS cores, we selected a highly conserved LPS core tetrasaccharide as a promising antigen. This LPS inner core tetrasaccharide induced a robust IgG response in mice when formulated as an immunogenic glycoconjugate. Binding of raised mouse serum to a broad collection of N. meningitidis strains demonstrated the accessibility of the LPS core on viable bacteria. The distal trisaccharide was identified as the crucial epitope, whereas the proximal Kdo moiety was immunodominant and induced mainly nonprotective antibodies that are responsible for lack of functional protection in polyclonal serum. Our results identified key antigenic determinants of LPS core glycan and, hence, may aid the design of a broadly protective immunization against N. meningitidis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Azaglycomimetics: Natural Occurrence, Biological Activity, and Application

NASA Astrophysics Data System (ADS)

Asano, Naoki

A large number of alkaloids mimicking the structures of monosaccharides or oligosaccharides have been isolated from plants and microorganisms. The sugar mimicking alkaloids with a nitrogen in the ring are called azasugars or iminosugars. Naturally occurring azasugars are classified into five structural classes: polyhydroxylated piperidines, pyrrolidines, indolizidines, pyrrolizidines, and nortropanes. They are easily soluble in water because of their polyhydroxylated structures and inhibit glycosidases because of a structural resemblance to the sugar moiety of the natural substrate. Glycosidases are involved in a wide range of anabolic and catabolic processes, such as digestion, lysosomal catabolism of glycoconjugates, biosynthesis of glycoproteins, and the endoplasmic reticulum (ER) quality control and ER-associated degradation of glycoproteins. Hence, modifying or blocking these processes in vivo by inhibitors is of great interest from a therapeutic point of view. Azasugars are an important class of glycosidase inhibitors and are arousing great interest for instance as antidiabetics, antiobesity drugs, antivirals, and therapeutic agents for some genetic disorders. This review describes the recent studies on isolation, characterization, glycosidase inhibitory activity, and therapeutic application of azaglycomimetics.

Regulation and function of endothelial glycocalyx layer in vascular diseases.

PubMed

Sieve, Irina; Münster-Kühnel, Anja K; Hilfiker-Kleiner, Denise

2018-01-01

In the vascular system, the endothelial surface layer (ESL) as the inner surface of blood vessels affects mechanotransduction, vascular permeability, rheology, thrombogenesis, and leukocyte adhesion. It creates barriers between endothelial cells and blood and neighbouring cells. The glycocalyx, composed of glycoconjugates and proteoglycans, is an integral component of the ESL and a key element in inter- and intracellular communication and tissue homeostasis. In pathophysiological conditions (atherosclerosis, infection, ischemia/reperfusion injury, diabetes, trauma and acute lung injury) glycocalyx-degrading factors, i.e. reactive oxygen and nitrogen species, matrix metalloproteinases, heparanase and sialidases, damage the ESL, thereby impairing endothelial functions. This leads to increased capillary permeability, leucocyte-endothelium interactions, thrombosis and vascular inflammation, the latter further driving glycocalyx destruction. The present review highlights current knowledge on the vasculoprotective role of the ESL, with specific emphasis on its remodelling in inflammatory vascular diseases and discusses its potential as a novel therapeutic target to treat vascular pathologies. Copyright © 2017 Elsevier Inc. All rights reserved.

Reflections on my career in analytical chemistry and biochemistry

PubMed Central

SWEELEY, Charles C.

2010-01-01

My career has been focused in two major areas, analytical chemistry and biochemistry of complex lipids and glycoconjugates. Included here are the pioneering work on the gas chromatography of long-chain sphingolipid bases, carbohydrates, steroids and urinary organic acids. Mass spectrometry was utilized extensively in structural studies of sphingolipids, fatty acids, carbohydrates, steroids, urinary organic acids, polyisoprenoid alcohols, and juvenile hormone. Computer systems were developed for the acquisition and analysis of mass spectra, and were used for development of automated metabolic profiling of complex mixtures of metabolites. Fabry’s disease was discovered to be a glycosphingolipidosis. Enzymes of lysosomal metabolism of glycosphingolipids were purified, characterized, and used in one of the first demonstrations of the feasibility of enzyme replacement therapy in a lysosomal storage disorder (Fabry’s disease). Extracellular sialidases were studied to evaluate the hypothesis that they might be involved in the regulation of membrane growth factor receptors. The enzyme for hematoside synthesis was purified and characterized. PMID:20948176

Collision Cross Sections and Ion Mobility Separation of Fragment Ions from Complex N-Glycans.

PubMed

Harvey, David J; Watanabe, Yasunori; Allen, Joel D; Rudd, Pauline; Pagel, Kevin; Crispin, Max; Struwe, Weston B

2018-06-01

Ion mobility mass spectrometry (IM-MS) holds great potential for structural glycobiology, in particular in its ability to resolve glycan isomers. Generally, IM-MS has largely been applied to intact glycoconjugate ions with reports focusing on the separation of different adduct types. Here, we explore IM separation and report the collision cross section (CCS) of complex type N-glycans and their fragments in negative ion mode following collision-induced dissociation (CID). CCSs of isomeric fragment ions were found, in some cases, to reveal structural details that were not present in CID spectra themselves. Many fragment ions were confirmed as possessing multiple structure, details of which could be obtained by comparing their drift time profiles to different glycans. By using fragmentation both before and after mobility separation, information was gathered on the fragmentation pathways producing some of the ions. These results help demonstrate the utility of IM and will contribute to the growing use of IM-MS for glycomics. Graphical Abstract ᅟ.

Sweet Polymers: Poly(2-ethyl-2-oxazoline) Glycopolymers by Reductive Amination.

PubMed

Mees, Maarten A; Effenberg, Christiane; Appelhans, Dietmar; Hoogenboom, Richard

2016-12-12

Carbohydrates are important in signaling, energy storage, and metabolism. Depending on their function, carbohydrates can be part of larger structures, such as glycoproteins, glycolipids, or other functionalities (glycoside). To this end, polymers can act as carriers of carbohydrates in so-called glycopolymers, which mimic the multivalent carbohydrate functionalities. We chose a biocompatible poly(2-ethyl-2-oxazoline) (PEtOx) as the basis for making glycopolymers. Via the partial hydrolysis of PEtOx, a copolymer of PEtOx and polyethylenimine (PEI) was obtained; the subsequent reductive amination with the linear forms of glucose and maltose yielded the glycopolymers. The ratios of PEtOx and carbohydrates were varied systematically, and the solution behaviors of the resulting glycoconjugates are discussed. Dynamic light scattering (DLS) revealed that, depending on the carbohydrate ratio, the glycopolymers were either fully water-soluble or formed agglomerates in a temperature-dependent manner. Finally, these polymers were tested for their biological availability by studying their lectin binding ability with Concanavalin A.

Collision Cross Sections and Ion Mobility Separation of Fragment Ions from Complex N-Glycans

NASA Astrophysics Data System (ADS)

Harvey, David J.; Watanabe, Yasunori; Allen, Joel D.; Rudd, Pauline; Pagel, Kevin; Crispin, Max; Struwe, Weston B.

2018-04-01

Ion mobility mass spectrometry (IM-MS) holds great potential for structural glycobiology, in particular in its ability to resolve glycan isomers. Generally, IM-MS has largely been applied to intact glycoconjugate ions with reports focusing on the separation of different adduct types. Here, we explore IM separation and report the collision cross section (CCS) of complex type N-glycans and their fragments in negative ion mode following collision-induced dissociation (CID). CCSs of isomeric fragment ions were found, in some cases, to reveal structural details that were not present in CID spectra themselves. Many fragment ions were confirmed as possessing multiple structure, details of which could be obtained by comparing their drift time profiles to different glycans. By using fragmentation both before and after mobility separation, information was gathered on the fragmentation pathways producing some of the ions. These results help demonstrate the utility of IM and will contribute to the growing use of IM-MS for glycomics. [Figure not available: see fulltext.

Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing

NASA Astrophysics Data System (ADS)

Both, P.; Green, A. P.; Gray, C. J.; Šardzík, R.; Voglmeir, J.; Fontana, C.; Austeri, M.; Rejzek, M.; Richardson, D.; Field, R. A.; Widmalm, G.; Flitsch, S. L.; Eyers, C. E.

2014-01-01

Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.

Phylogenetic-Derived Insights into the Evolution of Sialylation in Eukaryotes: Comprehensive Analysis of Vertebrate β-Galactoside α2,3/6-Sialyltransferases (ST3Gal and ST6Gal)

PubMed Central

Teppa, Roxana E.; Petit, Daniel; Plechakova, Olga; Cogez, Virginie; Harduin-Lepers, Anne

2016-01-01

Cell surface of eukaryotic cells is covered with a wide variety of sialylated molecules involved in diverse biological processes and taking part in cell–cell interactions. Although the physiological relevance of these sialylated glycoconjugates in vertebrates begins to be deciphered, the origin and evolution of the genetic machinery implicated in their biosynthetic pathway are poorly understood. Among the variety of actors involved in the sialylation machinery, sialyltransferases are key enzymes for the biosynthesis of sialylated molecules. This review focus on β-galactoside α2,3/6-sialyltransferases belonging to the ST3Gal and ST6Gal families. We propose here an outline of the evolutionary history of these two major ST families. Comparative genomics, molecular phylogeny and structural bioinformatics provided insights into the functional innovations in sialic acid metabolism and enabled to explore how ST-gene function evolved in vertebrates. PMID:27517905

MALDI TOF imaging mass spectrometry in clinical pathology: a valuable tool for cancer diagnostics (review).

PubMed

Kriegsmann, Jörg; Kriegsmann, Mark; Casadonte, Rita

2015-03-01

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) is an evolving technique in cancer diagnostics and combines the advantages of mass spectrometry (proteomics), detection of numerous molecules, and spatial resolution in histological tissue sections and cytological preparations. This method allows the detection of proteins, peptides, lipids, carbohydrates or glycoconjugates and small molecules.Formalin-fixed paraffin-embedded tissue can also be investigated by IMS, thus, this method seems to be an ideal tool for cancer diagnostics and biomarker discovery. It may add information to the identification of tumor margins and tumor heterogeneity. The technique allows tumor typing, especially identification of the tumor of origin in metastatic tissue, as well as grading and may provide prognostic information. IMS is a valuable method for the identification of biomarkers and can complement histology, immunohistology and molecular pathology in various fields of histopathological diagnostics, especially with regard to identification and grading of tumors.

Purification and characterization of Phaseolus vulgaris alpha-D-galactosidase isozymes.

PubMed

Dhar, M; Mitra, M; Hata, J; Butnariu, O; Smith, D

1994-11-01

A highly purified preparation of alpha-D-galactosidase [E.C. 3.2.1.22] isozymes was obtained from Phaseolus vulgaris (pinto bean) seeds by extraction, salt precipitation, ion exchange, and affinity chromatography. The final preparation was homogeneous by SDS-PAGE but revealed isozymes of relative mass of 38.3 and 39.6 kDa. The N-terminal sequence for both isozymes was identical, LANGLAKT (one letter code for amino acids). Relative native molecular mass was estimated at 149.3 kDa by Sephacryl S-200 chromatography. Activity was unaffected by ionic strength at high enzyme concentrations, and was specific for alpha-D-galactoside conjugates. No protease or hemagglutinin activity was detected, and activity was stable at 4 degrees C. Studies with soluble oligosaccharides demonstrated high activity against the selected straight and branched-chain substrates. The enzyme was active against terminal alpha 1-3 galactosyl residues on human and rabbit erythrocyte membranes. Because of its activity against membrane glycoconjugates, these isozymes may have potential utility for modifying membrane epitopes on native erythrocytes.

Expression, purification, crystallization and preliminary X-ray characterization of a putative glycosyltransferase of the GT-A fold found in mycobacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fulton, Zara; Crellin, Paul K.; Brammananth, Rajini

2008-05-28

Glycosidic bond formation is a ubiquitous enzyme-catalysed reaction. This glycosyltransferase-mediated process is responsible for the biosynthesis of innumerable oligosaccharides and glycoconjugates and is often organism- or cell-specific. However, despite the abundance of genomic information on glycosyltransferases (GTs), there is a lack of structural data for this versatile class of enzymes. Here, the cloning, expression, purification and crystallization of an essential 329-amino-acid (34.8 kDa) putative GT of the classic GT-A fold implicated in mycobacterial cell-wall biosynthesis are reported. Crystals of MAP2569c from Mycobacterium avium subsp. paratuberculosis were grown in 1.6 M monoammonium dihydrogen phosphate and 0.1 M sodium citrate pH 5.5.more » A complete data set was collected to 1.8 {angstrom} resolution using synchrotron radiation from a crystal belonging to space group P4{sub 1}2{sub 1}2.« less

Carbohydrates as T-cell antigens with implications in health and disease.

PubMed

Sun, Lina; Middleton, Dustin R; Wantuch, Paeton L; Ozdilek, Ahmet; Avci, Fikri Y

2016-10-01

Glycosylation is arguably the most ubiquitous post-translational modification on proteins in microbial and mammalian cells. During the past few years, there has been intensive research demonstrating that carbohydrates, either in pure forms or in conjunction with proteins or lipids, evoke and modulate adaptive immune responses. We now know that carbohydrates can be directly recognized by T cells or participate in T-cell stimulation as components of T-cell epitopes. T-cell recognition of carbohydrate antigens takes place via their presentation by major histocompatibility complex pathways on antigen-presenting cells. In this review, we summarize studies on carbohydrates as T-cell antigens modulating adaptive immune responses. Through discussion of glycan-containing antigens, such as glycoproteins, glycolipids, zwitterionic polysaccharides and carbohydrate-based glycoconjugate vaccines, we will illustrate the key molecular and cellular interactions between carbohydrate antigens and T cells and the implications of these interactions in health and disease. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Realizing the Promise of Chemical Glycobiology.

PubMed

Wang, Lai-Xi; Davis, Benjamin G

2013-09-01

Chemical glycobiology is emerging as one of the most uniquely powerful sub-disciplines of chemical biology. The previous scarcity of chemical strategies and the unparalleled structural diversity have created a uniquely fertile ground that is both rich in challenges and potentially very profound in implications. Glycans (oligosaccharides, polysaccharides, and glycoconjugates) are everywhere in biological systems and yet remain disproportionately neglected - reviews highlighting this 'Cinderella status' abound. Yet, the last two decades have witnessed tremendous progress, notably in chemical and chemoenzymatic synthesis, 'sequencing' and arraying, metabolic engineering and imaging. These vital steps serve to highlight not only the great potential but just how much more remains to be done. The vast chemical and functional space of glycans remains to be truly explored. Top-down full-scale glycomic and glycoproteomic studies coupled with hypothesis-driven, bottom-up innovative chemical strategies will be required to properly realize the potential impact of glycoscience on human health, energy, and economy. In this review, we cherry-pick far-sighted advances and use these to identify possible challenges, opportunities and avenues in chemical glycobiology.

Glyco-functionalized dinuclear rhenium(i) complexes for cell imaging.

PubMed

Palmioli, Alessandro; Aliprandi, Alessandro; Septiadi, Dedy; Mauro, Matteo; Bernardi, Anna; De Cola, Luisa; Panigati, Monica

2017-02-21

The design, synthesis and photophysical characterization of four new luminescent glycosylated luminophores based on dinuclear rhenium complexes, namely Glyco-Re, are described. The derivatives have the general formula [Re 2 (μ-Cl) 2 (CO) 6 (μ-pydz-R)] (R-pydz = functionalized 1,2-pyridazine), where a sugar residue (R) is covalently bound to the pyridazine ligand in the β position. Different synthetic pathways have been investigated including the so-called neo-glycorandomization procedure, affording stereoselectively glyco-conjugates containing glucose and maltose in a β anomeric configuration. A multivalent dinuclear rhenium glycodendron bearing three glucose units is also synthesized. All the Glyco-Re conjugates are comprehensively characterized and their photophysical properties and cellular internalization experiments on human cervical adenocarcinoma (HeLa) cells are reported. The results show that such Glyco-Re complexes display interesting bio-imaging properties, i.e. high cell permeability, organelle selectivity, low cytotoxicity and fast internalization. These findings make the presented Glyco-Re derivatives efficient phosphorescent probes suitable for cell imaging application.

Vaccine responsiveness in premature infants.

PubMed

Baxter, David

2010-06-01

The purpose of this review is to document adaptive immune responses in premature infants with a gestational age ≤32 weeks to the different vaccines used in the primary immunisation programme in the UK. Evidence suggests that these infants have impaired immune functioning that is consequent on maturational status and which resolve at variable time periods after birth - this impacts both on their risk of infection and response to vaccination. Assessing vaccine responsiveness can help establish whether the administration of additional vaccines is appropriate for a premature infant, and this may be determined either by vaccine immunogenicity or efficacy studies. The focus of the paper is immunogenicity studies for the following vaccines: tetanus, and diphtheria (toxoid vaccines), Haemophilus influenzae type b (Hib), meningococcal C (Men C) and pneumococcal (PnC) (subunit glycoconjugate vaccines), pertussis (subunit vaccine) and polio (inactivated vaccine). Data show that immunogenicity in premature infants is vaccine specific and whilst highly protective for the toxoid and inactivated preparations, responses to the subunit preparations are less optimal and consequently additional vaccinations or serology testing for ≤32 week gestation infants be considered.

Molecular cloning and functional expression of Lewis type α1,3/α1,4-fucosyltransferase cDNAs from Mangifera indica L.

PubMed

Okada, Takahiro; Ihara, Hideyuki; Ito, Ritsu; Ikeda, Yoshitaka

2017-12-01

In higher plants, complex type N-glycans contain characteristic carbohydrate moieties that are not found in mammals. In particular, the attachment of the Lewis a (Le a ) epitope is currently the only known outer chain elongation that is present in plant N-glycans. Such a modification is of great interest in terms of the biological function of complex type N-glycans in plant species. However, little is known regarding the exact molecular basis underlying their Le a expression. In the present study, we cloned two novel Lewis type fucosyltransferases (MiFUT13) from mango fruit, Mangifera indica L., heterologously expressed the proteins and structurally and functionally characterized them. Using an HPLC-based assay, we demonstrated that the recombinant MiFUT13 proteins mediate the α1,4-fucosylation of acceptor tetrasaccharides with a strict preference for type I-based structure to type II. The results and other findings suggest that MiFUT13s are involved in the biosynthesis of Le a containing glycoconjugates in mango fruits. Copyright © 2017 Elsevier Ltd. All rights reserved.

Randomized trial on the safety, tolerability, and immunogenicity of MenACWY-CRM, an investigational quadrivalent meningococcal glycoconjugate vaccine, administered concomitantly with a combined tetanus, reduced diphtheria, and acellular pertussis vaccine in adolescents and young adults.

PubMed

Gasparini, Roberto; Conversano, Michele; Bona, Gianni; Gabutti, Giovanni; Anemona, Alessandra; Dull, Peter M; Ceddia, Francesca

2010-04-01

This study evaluated the safety, tolerability, and immunogenicity of an investigational quadrivalent meningococcal conjugate vaccine, MenACWY-CRM, when administered concomitantly with a combined tetanus, reduced diphtheria, and acellular pertussis (Tdap) vaccine, in subjects aged 11 to 25 years. Subjects received either MenACWY-CRM and Tdap, MenACWY-CRM and saline placebo, or Tdap and saline placebo. No significant increase in reactogenicity and no clinically significant vaccine-related adverse events (AEs) occurred when MenACWY-CRM and Tdap were administered concomitantly. Similar immunogenic responses to diphtheria, tetanus, and meningococcal (serogroups A, C, W-135, and Y) antigens were observed, regardless of concomitant vaccine administration. Antipertussis antibody responses were comparable between vaccine groups for filamentous hemagglutinin and were slightly lower, although not clinically significantly, for pertussis toxoid and pertactin when the two vaccines were administered concomitantly. These results indicate that the investigational MenACWY-CRM vaccine is well tolerated and immunogenic and that it can be coadministered with Tdap to adolescents and young adults.

A histochemical study of the distribution of lectin binding sites in the developing branchial area of the trout Salmo trutta.

PubMed Central

Rojo, M C; Blánquez, M J; González, M E

1996-01-01

A histochemical study of the branchial area of brown trout embryos from 35 to 71 d of incubation is reported. A battery of 6 different horseradish peroxidase-labelled lectins, the PAS reaction and Alcian blue staining were used to study the distribution of carbohydrate residues in glycoconjugates along the pharyngeal and branchial epithelia. Con A and WGA reacted at every site of the branchial region thus showing the ubiquitous presence of alpha-D-mannose and N-acetyl-D-glucosamine. WGA, DBA and SBA were good markers for the hatching gland cells (HGCs) and mucous cells. Other lectins, such as PNA and UEA I, reacted only for a short time at some sites during the considered period of incubation. From 35 d until posthatching stages, a manifest strong reaction was noted both in the dorsal epithelium of branchial arches and the HGCs as shown by SBA reactivity. This may be significant with regard to the controversial origin of HGCs, which is thought to be endodermal. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8982837

Enhanced Imaging of Specific Cell-Surface Glycosylation Based on Multi-FRET.

PubMed

Yuan, Baoyin; Chen, Yuanyuan; Sun, Yuqiong; Guo, Qiuping; Huang, Jin; Liu, Jianbo; Meng, Xiangxian; Yang, Xiaohai; Wen, Xiaohong; Li, Zenghui; Li, Lie; Wang, Kemin

2018-05-15

Cell-surface glycosylation contains abundant biological information that reflects cell physiological state, and it is of great value to image cell-surface glycosylation to elucidate its functions. Here we present a hybridization chain reaction (HCR)-based multifluorescence resonance energy transfer (multi-FRET) method for specific imaging of cell-surface glycosylation. By installing donors through metabolic glycan labeling and acceptors through aptamer-tethered nanoassemblies on the same glycoconjugate, intramolecular multi-FRET occurs due to near donor-acceptor distance. Benefiting from amplified effect and spatial flexibility of the HCR nanoassemblies, enhanced multi-FRET imaging of specific cell-surface glycosylation can be obtained. With this HCR-based multi-FRET method, we achieved obvious contrast in imaging of protein-specific GalNAcylation on 7211 cell surfaces. In addition, we demonstrated the general applicability of this method by visualizing the protein-specific sialylation on CEM cell surfaces. Furthermore, the expression changes of CEM cell-surface protein-specific sialylation under drug treatment was accurately monitored. This developed imaging method may provide a powerful tool in researching glycosylation functions, discovering biomarkers, and screening drugs.

Genistein isoflavone glycoconjugates in sour cherry (Prunus cerasus L.) cultivars.

PubMed

Abrankó, László; Nagy, Ádám; Szilvássy, Blanka; Stefanovits-Bányai, Éva; Hegedűs, Attila

2015-01-01

The isoflavone genistein on the contrary to its well-established health-beneficial effects is not a major component of the Western diet, since soy consumption, considered as the main dietary source of genistein, in these populations is low. Genistein compounds in twelve commercial sour cherry (Prunus cerasus L.) cultivars grown in Hungary were studied. High performance liquid chromatography coupled to electrospray ionisation quadrupole/time-of-flight mass spectrometry (HPLC-ESI-qToF-MS) was used for screening and confirmatory analyses. Genistin and genistein were found in 'Pipacs1', 'Kántorjánosi', 'Debreceni bőtermő' and 'Éva', which are native cultivars to Hungary. Genistein content of the latter three were in the range of 0.4-0.6, while in 'Pipacs1' in total 4.4 mg genistein compounds were measured expressed as aglycone equivalents per 100g of fresh fruit flesh. These cultivars may play important role as complementary genistein sources in the Western diet. Especially 'Pipacs 1', may be best utilised in functional food products. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lipase-catalyzed acylation of microbial mannosylerythritol lipids (biosurfactants) and their characterization.

PubMed

Recke, Verena K; Beyrle, Catharina; Gerlitzki, Melanie; Hausmann, Rudolf; Syldatk, Christoph; Wray, Victor; Tokuda, Harukuni; Suzuki, Nobutaka; Lang, Siegmund

2013-05-24

Culturing Pseudozyma aphidis on glucose as main carbon source and soybean oil as co-substrate the mannosylerythritol lipids MEL-A and MEL-B were produced. Based on their excellent surface/interfacial active behavior they possess a high potential among all known biosurfactants. The components of a microbial MEL mixture were purified by medium pressure liquid chromatography (MPLC) and were used as substrates for in vitro enzymatic modifications. Lipase-catalyzed acylations of MEL-A and MEL-B with uncommon fatty acids from other microbial glycolipids-3-hydroxydecanoic acid from rhamnolipids and 17-hydroxyoctadecanoic acid from classical sophorolipids-yielded functionalized products at the C-1 position of the erythritol. The novel products were purified by MPLC and their structures elucidated by (1)H and (13)C nuclear magnetic resonance spectroscopy and mass spectrometry. In physicochemical characterization experiments two of the three new glycoconjugates lowered the surface tension of water from 72 mN m(-1) to 27-38 mN m(-1). Moreover the novel compounds inhibited the growth of gram-positive bacteria and showed a potential for anti-tumor-promoting activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cell-free biosynthesis of lipophosphoglycan from Leishmania donovani. Characterization of microsomal galactosyltransferase and mannosyltransferase activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carver, M.A.; Turco, S.J.

1991-06-15

Incubation of microsomal preparations from Leishmania donovani parasites with UDP-({sup 3}H)galactose or GDP-({sup 14}C)mannose resulted in incorporation of radiolabel into an endogenous product that exhibited the chemical and chromatographic characteristics of the parasite's major surface glycoconjugate, lipophosphoglycan. The ({sup 3}H)galactose- or ({sup 14}C)mannose-labeled product was (1) cleaved by phosphatidylinositol-specific phospholipase C; (2) deaminated by nitrous acid; and (3) degraded into radioactive, low molecular weight fragments upon hydrolysis with mild acid. Analysis of the products of mild acid hydrolysis revealed the presence of phosphorylated Gal-beta-Man as the major fragment with lesser amounts of mono-, tri-, and tetrasaccharides. The incorporation of themore » two isotopic precursors was neither stimulated by the addition of dolichylphosphate nor inhibited by amphomycin, indicating that dolichol-saccharide intermediates are not involved in assembly of the repeating units of lipophosphoglycan. Development of this cell-free glycosylating system will facilitate further studies on the pathway and enzymes involved in lipophosphoglycan biosynthesis.« less

Trichomonas vaginalis clinical isolates: cytoadherence and adherence to polystyrene, intrauterine device, and vaginal ring.

PubMed

Dos Santos, Odelta; Rigo, Graziela Vargas; Macedo, Alexandre José; Tasca, Tiana

2017-12-01

The parasitism by Trichomonas vaginalis is complex and in part is mediated by cytoadherence accomplished via five surface proteins named adhesins and a glycoconjugate called lipophosphoglycan (TvLPG). In this study, we evaluated the ability of T. vaginalis isolates to adhere to cells, plastic (polystyrene microplates), intrauterine device (IUD), and vaginal ring. Of 32 T. vaginalis isolates, 4 (12.5%) were strong adherent. The T. vaginalis isolates TV-LACM6 and TV-LACM14 (strong polystyrene-adherent) were also able to adhere to IUD and vaginal ring. Following chemical treatments, results demonstrated that the T. vaginalis components, lipophosphoglycan, cytoskeletal proteins, and surface molecules, were involved in both adherence to polystyrene and cytoadherence. The gene expression level from four adhesion proteins was highest in trophozoites adhered to cells than trophozoites adhered to the abiotic surface (polystyrene microplate). Our data indicate the major involvement of TvLPG in adherence to polystyrene, and that adhesins are important for cytoadherence. Furthermore, to our knowledge, this is the first report showing the T. vaginalis adherence to contraceptive devices, reaffirming its importance as pathogen among women in reproductive age.

Over forty years of bladder cancer glycobiology: Where do glycans stand facing precision oncology?

PubMed Central

Azevedo, Rita; Peixoto, Andreia; Gaiteiro, Cristiana; Fernandes, Elisabete; Neves, Manuel; Lima, Luís; Santos, Lúcio Lara; Ferreira, José Alexandre

2017-01-01

The high molecular heterogeneity of bladder tumours is responsible for significant variations in disease course, as well as elevated recurrence and progression rates, thereby hampering the introduction of more effective targeted therapeutics. The implementation of precision oncology settings supported by robust molecular models for individualization of patient management is warranted. This effort requires a comprehensive integration of large sets of panomics data that is yet to be fully achieved. Contributing to this goal, over 40 years of bladder cancer glycobiology have disclosed a plethora of cancer-specific glycans and glycoconjugates (glycoproteins, glycolipids, proteoglycans) accompanying disease progressions and dissemination. This review comprehensively addresses the main structural findings in the field and consequent biological and clinical implications. Given the cell surface and secreted nature of these molecules, we further discuss their potential for non-invasive detection and therapeutic development. Moreover, we highlight novel mass-spectrometry-based high-throughput analytical and bioinformatics tools to interrogate the glycome in the postgenomic era. Ultimately, we outline a roadmap to guide future developments in glycomics envisaging clinical implementation. PMID:29207682

Copper-free click chemistry in living animals

PubMed Central

Chang, Pamela V.; Prescher, Jennifer A.; Sletten, Ellen M.; Baskin, Jeremy M.; Miller, Isaac A.; Agard, Nicholas J.; Lo, Anderson; Bertozzi, Carolyn R.

2010-01-01

Chemical reactions that enable selective biomolecule labeling in living organisms offer a means to probe biological processes in vivo. Very few reactions possess the requisite bioorthogonality, and, among these, only the Staudinger ligation between azides and triarylphosphines has been employed for direct covalent modification of biomolecules with probes in the mouse, an important model organism for studies of human disease. Here we explore an alternative bioorthogonal reaction, the 1,3-dipolar cycloaddition of azides and cyclooctynes, also known as “Cu-free click chemistry,” for labeling biomolecules in live mice. Mice were administered peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to metabolically label cell-surface sialic acids with azides. After subsequent injection with cyclooctyne reagents, glycoconjugate labeling was observed on isolated splenocytes and in a variety of tissues including the intestines, heart, and liver, with no apparent toxicity. The cyclooctynes tested displayed various labeling efficiencies that likely reflect the combined influence of intrinsic reactivity and bioavailability. These studies establish Cu-free click chemistry as a bioorthogonal reaction that can be executed in the physiologically relevant context of a mouse. PMID:20080615

Burkholderia pseudomallei and Burkholderia mallei vaccines: Are we close to clinical trials?

PubMed

Titball, Richard W; Burtnick, Mary N; Bancroft, Gregory J; Brett, Paul

2017-10-20

B. pseudomallei is the cause of melioidosis, a serious an often fatal disease of humans and animals. The closely related bacterium B. mallei, which cases glanders, is considered to be a clonal derivative of B. pseudomallei. Both B. pseudomallei and B. mallei were evaluated by the United States and the former USSR as potential bioweapons. Much of the effort to devise biodefence vaccines in the past decade has been directed towards the identification and formulation of sub-unit vaccines which could protect against both melioidosis and glanders. A wide range of proteins and polysaccharides have been identified which protective immunity in mice. In this review we highlight the significant progress that has been made in developing glycoconjugates as sub-unit vaccines. We also consider some of the important the criteria for licensing, including the suitability of the "animal rule" for assessing vaccine efficacy, the protection required from a vaccine and the how correlates of protection will be identified. Vaccines developed for biodefence purposes could also be used in regions of the world where naturally occurring disease is endemic. Copyright © 2017 Elsevier Ltd. All rights reserved.

Investigations of Scope and Mechanism of Nickel-Catalyzed Transformations of Glycosyl Trichloroacetimidates to Glycosyl Trichloroacetamides and Subsequent, Atom-Economical, One-Step Conversion to α-Urea-Glycosides

PubMed Central

McKay, Matthew J.; Park, Nathaniel H.; Nguyen, Hien M.

2014-01-01

The development and mechanistic investigation of a highly stereoselective methodology for preparing α-linked-urea neo-glycoconjugates and pseudo-oligosaccharides is described. This two-step procedure begins with the selective nickel-catalyzed conversion of glycosyl trichloroacetimidates to the corresponding α-trichloroacetamides. The α-selective nature of the conversion is controlled with a cationic nickel(II) catalyst, Ni(dppe)(OTf)2. Mechanistic studies have identified the coordination of the nickel catalyst with the equatorial C2-ether functionality of the α-glycosyl trichloroacetimidate to be paramount for achieving an α-stereoselective transformation. A cross-over experiment has indicated that the reaction does not proceed in an exclusively-intramolecular fashion. The second step in this sequence is the direct conversion of α-glycosyl trichloroacetamide products into the corresponding α-urea glycosides by reacting them with a wide variety of amine nucleophiles in presence of cesium carbonate. Only α-urea-product formation is observed, as the reaction proceeds with complete retention of stereochemical integrity at the anomeric C-N bond. PMID:24905328

Real-time analysis of the carbohydrates on cell surfaces using a QCM biosensor: a lectin-based approach.

PubMed

Pei, Zhichao; Saint-Guirons, Julien; Käck, Camilla; Ingemarsson, Björn; Aastrup, Teodor

2012-05-15

A novel approach to the study of molecular interactions on the surface of mammalian cells using a QCM biosensor was developed. For this study, an epidermoid carcinoma cell line (A-431) and a breast adenocarcinoma cell line (MDA-MB-468) were immobilized onto polystyrene-coated quartz crystals. The binding and dissociation between the lectin Con A and the cells as well as the inhibition of the binding by monosaccharides were monitored in real time and provided an insight into the complex avidic recognition of cell glycoconjugates. The real-time lectin screening of a range of lectins, including Con A, DBA, PNA and UEA-I, enabled the accurate study of the glycosylation changes between cells, such as changes associated with cancer progression and development. Furthermore, the kinetic parameters of the interaction of Con A with MDA-MB-468 cells were studied. This application provides investigators in the field of glycobiology with a novel tool to study cell surface glycosylation and may also have impacts on drug discovery. Copyright © 2012 Elsevier B.V. All rights reserved.

Carbohydrates and T cells: A sweet twosome

PubMed Central

Avci, Fikri Y.; Li, Xiangming; Tsuji, Moriya; Kasper, Dennis L.

2013-01-01

Carbohydrates as T cell-activating antigens have been generating significant interest. For many years, carbohydrates were thought of as T-independent antigens, however, more recent research had demonstrated that mono- or oligosaccharides glycosidically-linked to peptides can be recognized by T cells. T cell recognition of these glycopeptides depends on the structure of both peptide and glycan portions of the antigen. Subsequently, it was discovered that natural killer T cells recognized glycolipids when presented by the antigen presenting molecule CD1d. A transformative insight into glycan-recognition by T cells occurred when zwitterionic polysaccharides were discovered to bind to and be presented by MHCII to CD4+ T cells. Based on this latter observation, the role that carbohydrate epitopes generated from glycoconjugate vaccines had in activating helper T cells was explored and it was found that these epitopes are presented to specific carbohydrate recognizing T cells through a unique mechanism. Here we review the key interactions between carbohydrate antigens and the adaptive immune system at the molecular, cellular and systems levels exploring the significant biological implications in health and disease. PMID:23757291

Lipopolysaccharides in diazotrophic bacteria.

PubMed

Serrato, Rodrigo V

2014-01-01

Biological nitrogen fixation (BNF) is a process in which the atmospheric nitrogen (N2) is transformed into ammonia (NH3) by a select group of nitrogen-fixing organisms, or diazotrophic bacteria. In order to furnish the biologically useful nitrogen to plants, these bacteria must be in constant molecular communication with their host plants. Some of these molecular plant-microbe interactions are very specific, resulting in a symbiotic relationship between the diazotroph and the host. Others are found between associative diazotrophs and plants, resulting in plant infection and colonization of internal tissues. Independent of the type of ecological interaction, glycans, and glycoconjugates produced by these bacteria play an important role in the molecular communication prior and during colonization. Even though exopolysaccharides (EPS) and lipochitooligosaccharides (LCO) produced by diazotrophic bacteria and released onto the environment have their importance in the microbe-plant interaction, it is the lipopolysaccharides (LPS), anchored on the external membrane of these bacteria, that mediates the direct contact of the diazotroph with the host cells. These molecules are extremely variable among the several species of nitrogen fixing-bacteria, and there are evidences of the mechanisms of infection being closely related to their structure.

Hexose rearrangements upon fragmentation of N-glycopeptides and reductively aminated N-glycans.

PubMed

Wuhrer, Manfred; Koeleman, Carolien A M; Deelder, André M

2009-06-01

Tandem mass spectrometry of glycans and glycoconjugates in protonated form is known to result in rearrangement reactions leading to internal residue loss. Here we studied the occurrence of hexose rearrangements in tandem mass spectrometry of N-glycopeptides and reductively aminated N-glycans by MALDI-TOF/TOF-MS/MS and ESI-ion trap-MS/MS. Fragmentation of proton adducts of oligomannosidic N-glycans of ribonuclease B that were labeled with 2-aminobenzamide and 2-aminobenzoic acid resulted in transfer of one to five hexose residues to the fluorescently tagged innermost N-acetylglucosamine. Glycopeptides from various biological sources with oligomannosidic glycans were likewise shown to undergo hexose rearrangement reactions, resulting in chitobiose cleavage products that have acquired one or two hexose moieties. Tryptic immunoglobulin G Fc-glycopeptides with biantennary N-glycans likewise showed hexose rearrangements resulting in hexose transfer to the peptide moiety retaining the innermost N-acetylglucosamine. Thus, as a general phenomenon, tandem mass spectrometry of reductively aminated glycans as well as glycopeptides may result in hexose rearrangements. This characteristic of glycopeptide MS/MS has to be considered when developing tools for de novo glycopeptide structural analysis.

Lectin histochemistry and alkaline phosphatase activity in the pia mater vessels of spontaneously hypertensive rats (SHR).

PubMed

Szumańska, G; Gadamski, R

1992-01-01

Some lectins were used to study the localization of sugar residues on the endothelial cell surface in the pia mater blood vessels of control (WKY) and hypertensive rats (SHR). The lectins tested recognized the following residues: beta-D-galactosyl (Ricinus communis agglutinin 120, RCA-1), alpha-L-fucosyl (Ulex europaeus agglutinin, UEA-1), N-acetylglucosaminyl and sialyl (Wheat germ agglutinin, WGA), N-glycolyl-neuraminic acid (Limax flavus agglutinin, LFA), and N-acetyl-D-galactosaminyl (Helix pomatia agglutinin, HPA). Several differences were revealed in the presence of sugar receptors on the surface of endothelial cells between the control and the hypertensive rats. Our studies showed also differences in the localization of the tested glycoconjugates between pial capillaries, small, medium-size and large pial arteries. The histochemical evaluation of alkaline phosphatase revealed an increased activity of the enzyme in the pial vessels of SHRs as compared with control rats with a similar localization of the enzyme activity. Some differences in the distribution of lectin binding sites and alkaline phosphatase activity could be associated with the different functions of particular segments of the pial vascular network.

Novel interactions of complex carbohydrates with peanut (PNA), Ricinus communis (RCA-I), Sambucus nigra (SNA-I) and wheat germ (WGA) agglutinins as revealed by the binding specificities of these lectins towards mucin core-2 O-linked and N-linked glycans and related structures.

PubMed

Chandrasekaran, E V; Xue, Jun; Xia, Jie; Khaja, Siraj D; Piskorz, Conrad F; Locke, Robert D; Neelamegham, Sriram; Matta, Khushi L

2016-10-01

Plant lectins through their multivalent quaternary structures bind intrinsically flexible oligosaccharides. They recognize fine structural differences in carbohydrates and interact with different sequences in mucin core 2 or complex-type N-glycan chain and also in healthy and malignant tissues. They are used in characterizing cellular and extracellular glycoconjugates modified in pathological processes. We study here, the complex carbohydrate-lectin interactions by determining the effects of substituents in mucin core 2 tetrasaccharide Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα-O-R and fetuin glycopeptides on their binding to agarose-immobilized lectins PNA, RCA-I, SNA-I and WGA. Briefly, in mucin core 2 tetrasaccharide (i) structures modified by α2-3/6-Sialyl LacNAc, LewisX and α1-3-Galactosyl LacNAc resulted in regular binding to PNA whereas compounds with 6-sulfo LacNAc displayed no-binding; (ii) strucures bearing α2-6-sialyl 6-sulfo LacNAc, or 6-sialyl LacdiNAc carbohydrates displayed strong binding to SNA-I; (iii) structures with α2-3/6-sialyl, α1-3Gal LacNAc or LewisX were non-binder to RCA-I and compounds with 6-sulfo LacNAc only displayed weak binding; (iv) structures containing LewisX, 6-Sulfo LewisX, α2-3/6-sialyl LacNAc, α2-3/6-sialyl 6-sulfo LacNAc and GalNAc Lewis-a were non-binding to WGA, those with α1-2Fucosyl, α1-3-Galactosyl LacNAc, α2-3-sialyl T-hapten plus 3'/6'sulfo LacNAc displayed weak binding, and compounds with α2-3-sialyl T-hapten, α2.6-Sialyl LacdiNAc, α2-3-sialyl D-Fucβ1-3 GalNAc and Fucα-1-2 D-Fucβ-1-3GalNAc displaying regular binding and GalNAc LewisX and LacdiNAc plus D-Fuc β-1-3 GalNAcα resulting in tight binding. RCA-I binds Fetuin triantennary asialoglycopeptide 100 % after α-2-3 and 25 % after α-2-6 sialylation, 30 % after α-1-2 and 100 % after α-1-3 fucosylation, and 50 % after α-1-3 galactosylation. WGA binds 3-but not 6-Fucosyl chitobiose core. Thus, information on the influence of complex carbohydrate chain constituents on lectin binding is apparently essential for the potential application of lectins in glycoconjugate research.

Synthesis of antifungal vaccines by conjugation of β-1,2 trimannosides with T-cell peptides and covalent anchoring of neoglycopeptide to tetanus toxoid.

PubMed

Cartmell, Jonathan; Paszkiewicz, Eugenia; Dziadek, Sebastian; Tam, Pui-Hang; Luu, Thanh; Sarkar, Susmita; Lipinski, Tomasz; Bundle, David R

2015-02-11

Selective strategies for the construction of novel three component glycoconjugate vaccines presenting Candida albicans cell wall glycan (β-1,2 mannoside) and polypeptide fragments on a tetanus toxoid carrier are described. The first of two conjugation strategies employed peptides bearing an N-terminal thiopropionyl residue for conjugation to a trisaccharide equipped with an acrylate linker and a C-terminal S-acetyl thioglycolyl moiety for subsequent linking of neoglycopeptide to bromoacetylated tetanus toxoid. Michael addition of acrylate trisaccharides to peptide thiol under mildly basic conditions gave a mixture of N- and C- terminal glyco-peptide thioethers. An adaptation of this strategy coordinated S-acyl protection with anticipated thioester exchange equilibria. This furnished a single chemically defined fully synthetic neoglycopeptide conjugate that could be anchored to a tetanus toxoid carrier and avoids the introduction of exogenous antigenic groups. The second strategy retained the N-terminal thiopropionyl residue but replaced the C-terminal S-acetate functionality with an azido group that allowed efficient, selective formation of neoglycopeptide thioethers and subsequent conjugation of these with propargylated tetanus toxoid, but introduced potentially antigenic triazole linkages. Copyright © 2014 Elsevier Ltd. All rights reserved.

Contribution of galectin-1, a glycan-binding protein, to gastrointestinal tumor progression.

PubMed

Bacigalupo, María L; Carabias, Pablo; Troncoso, María F

2017-08-07

Gastrointestinal cancer is a group of tumors that affect multiple sites of the digestive system, including the stomach, liver, colon and pancreas. These cancers are very aggressive and rapidly metastasize, thus identifying effective targets is crucial for treatment. Galectin-1 (Gal-1) belongs to a family of glycan-binding proteins, or lectins, with the ability to cross-link specific glycoconjugates. A variety of biological activities have been attributed to Gal-1 at different steps of tumor progression. Herein, we summarize the current literature regarding the roles of Gal-1 in gastrointestinal malignancies. Accumulating evidence shows that Gal-1 is drastically up-regulated in human gastric cancer, hepatocellular carcinoma, colorectal cancer and pancreatic ductal adenocarcinoma tissues, both in tumor epithelial and tumor-associated stromal cells. Moreover, Gal-1 makes a crucial contribution to the pathogenesis of gastrointestinal malignancies, favoring tumor development, aggressiveness, metastasis, immunosuppression and angiogenesis. We also highlight that alterations in Gal-1-specific glycoepitopes may be relevant for gastrointestinal cancer progression. Despite the findings obtained so far, further functional studies are still required. Elucidating the precise molecular mechanisms modulated by Gal-1 underlying gastrointestinal tumor progression, might lead to the development of novel Gal-1-based diagnostic methods and/or therapies.

Transglycosidase-like activity of Mucor hiemalis endoglycosidase mutants enabling the synthesis of glycoconjugates using a natural glycan donor.

PubMed

Sakaguchi, Kouta; Katoh, Toshihiko; Yamamoto, Kenji

2016-11-01

Glycan conversion of glycoprotein via the transglycosylation activity of endo-β-N-acetylglucosaminidase is a promising chemoenzymatic technology for the production of glycoproteins including bio-medicines with a homogeneous glycoform. Although Endo-M is a key enzyme in this process, its product undergoes rehydrolysis, which leads to a lower yield, and limits the practical application of this enzyme. We developed several Endo-M mutant enzymes including N175Q with glycosynthase-like activity and/or transglycosidase-like activity. We found that the Endo-M N175H mutant showed glycosynthase-like activity comparable to N175Q as well as transglycosidase-like activity superior to N175Q. Using a natural sialylglycopeptide as a donor substrate, N175H readily transferred the sialo-glycan onto an N-acetylglucosamine residue attached to bovine ribonuclease B (RNase B), yielding a nonnative sialoglycosylated RNase B. These results demonstrate that use of Endo-M N175H is an alternative glycoengineering technique, which provides a relatively high yield of transglycosylation product and avoids the laborious synthesis of a sugar oxazoline as a donor substrate. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Sialyl-lactotetra, a novel cell surface marker of undifferentiated human pluripotent stem cells.

PubMed

Barone, Angela; Säljö, Karin; Benktander, John; Blomqvist, Maria; Månsson, Jan-Eric; Johansson, Bengt R; Mölne, Johan; Aspegren, Anders; Björquist, Petter; Breimer, Michael E; Teneberg, Susann

2014-07-04

Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10(9) cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

pKa cycling of the general acid/base in glycoside hydrolase families 33 and 34.

PubMed

Yu, Haibo; Griffiths, Thomas M

2014-03-28

Glycoside hydrolase families 33 and 34 catalyse the hydrolysis of terminal sialic acid residues from sialyl oligosaccharides and glycoconjugates with a net retention of the stereochemistry at the anomeric centre. It is generally believed that the conserved aspartic acid in the active site functions as a general acid to protonate the hydroxyl group of the departing aglycone during glycosylation, and then as a general base to facilitate the nucleophilic attack of the water molecule on the intermediate state during the deglycosylation reaction. The dual role of the general acid/base places specific demands upon its protonation state, and thus pKa values. However, it is not fully understood how this catalytic residue can achieve such pKa cycling during catalysis. We present both MM and combined QM/MM simulations to characterise the pKa values of the proposed catalytic general acid/base in the glycoside hydrolase families 33 and 34. Collectively, our study suggests that the binding of anionic substrates and the local solvation properties along with the neutralisation of the nearby glutamic acid upon glycosylation modulate the electrostatic environment around the general acid/base to achieve its proper protonation states.

Bacterial recognition of thermal glycation products derived from porcine serum albumin with lactose.

PubMed

Sarabia-Sainz, Andre-I; Ramos-Clamont, Gabriela; Winzerling, Joy; Vázquez-Moreno, Luz

2011-01-01

Recently, glyco-therapy is proposed to prevent the interaction of bacterial lectins with host ligands (glycoconjugates). This interaction represents the first step in infection. Neoglycans referred to as PSA-Lac (PSA-Glu (β1-4) Gal) were obtained by conjugation of porcine serum albumin (PSA) with lactose at 80 °C, 100 °C and 120 ºC. Characterization studies of the products showed that PSA could contain 1, 38 or 41 added lactoses, depending on the reaction temperature. These neoglycans were approximately 10 times more glycated than PSA-Lac obtained in previous work. Lactose conjugation occurred only at lysines and PSA-Lac contained terminal galactoses as confirmed by Ricinus communis lectin recognition. Furthermore, Escherichia coli K88+, K88ab, K88ac and K88ad adhesins showed affinity toward all PSA-Lac neoglycans, and the most effective was the PSA-Lac obtained after 100 ºC treatment. In vitro, this neoglycan partially inhibited the adhesion of E. coli K88+ to piglet mucin (its natural ligand). These results provide support for the hypothesis that glycated proteins can be used as an alternative for bioactive compounds for disease prevention.

Tyramine Pathways in Citrus Plant Defense: Glycoconjugates of Tyramine and Its N-Methylated Derivatives.

PubMed

Servillo, Luigi; Castaldo, Domenico; Giovane, Alfonso; Casale, Rosario; D'Onofrio, Nunzia; Cautela, Domenico; Balestrieri, Maria Luisa

2017-02-01

Glucosylated forms of tyramine and some of its N-methylated derivatives are here reported for the first time to occur in Citrus genus plants. The compounds tyramine-O-β-d-glucoside, N-methyltyramine-O-β-d-glucoside, and N,N-dimethyltyramine-O-β-d-glucoside were detected in juice and leaves of sweet orange, bitter orange, bergamot, citron, lemon, mandarin, and pomelo. The compounds were identified by mass spectrometric analysis, enzymatic synthesis, and comparison with extracts of Stapelia hirsuta L., a plant belonging to the Apocynaceae family in which N,N-dimethyltyramine-O-β-d-glucoside was identified by others. Interestingly, in Stapelia hirsuta we discovered also tyramine-O-β-d-glucoside, N-methyltyramine-O-β-d-glucoside, and the tyramine metabolite, N,N,N-trimethyltyramine-O-β-glucoside. However, the latter tyramine metabolite, never described before, was not detected in any of the Citrus plants included in this study. The presence of N-methylated tyramine derivatives and their glucosylated forms in Citrus plants, together with octopamine and synephrine, also deriving from tyramine, supports the hypothesis of specific biosynthetic pathways of adrenergic compounds aimed to defend against biotic stress.

Comparison of the antimicrobial adhesion potential of human body fluid glycoconjugates using fucose-binding lectin (PA-IIL) of Pseudomonas aeruginosa and Ulex europaeus lectin (UEA-I).

PubMed

Lerrer, Batia; Lesman-Movshovich, Efrat; Gilboa-Garber, Nechama

2005-09-01

Pseudomonas aeruginosa produces a fucose-binding lectin (PA-IIL) which strongly binds to human cells. This lectin was shown to be highly sensitive to inhibition by fucose-bearing human milk glycoproteins. Since the glycans of these glycoproteins mimic human cell receptors, they may function as decoys in blocking lectin-dependent pathogen adhesion to the host cells. Human saliva and seminal fluid also contain such compounds, and body fluids of individuals who are "secretors" express additional fucosylated (alpha 1,2) residues. The latter are selectively detected by Ulex europaeus lectin UEA-I. The aim of the present research was to compare the PA-IIL and UEA-I interactions with human salivas and seminal fluids of "secretors" and "nonsecretors" with those obtained with the respective milks. Using hemagglutination inhibition and Western blot analyses, we showed that PA-IIL interactions with the saliva and seminal fluid glycoproteins were somewhat weaker than those obtained with the milk and that "nonsecretor" body fluids were not less efficient than those of "secretors" in PA-IIL blocking. UEA-I, which interacted only with the "secretors" glycoproteins, was most sensitive to those of the seminal fluids.

The role of novel chitin-like polysaccharides in Alzheimer disease.

PubMed

Castellani, Rudy J; Perry, George; Smith, Mark A

2007-12-01

While controversy over the role of carbohydrates in amyloidosis has existed since the initial recognition of amyloid, current understanding of the role of polysaccharides in the pathogenesis of amyloid deposition of Alzheimer disease and other amyloidoses is limited to studies of glyco-conjugates such as heparin sulfate proteoglycan. We hypothesized that polysaccharides may play a broader role in light of 1) the impaired glucose utilization in Alzheimer disease; 2) the demonstration of amylose in the Alzheimer disease brain; 3) the role of amyloid in Alzheimer disease pathogenesis. Specifically, as with glucose polymers (amyloid), we wanted to explore whether glucosamine polymers such as chitin were being synthesized and deposited as a result of impaired glucose utilization and aberrant hexosamine pathway activation. To this end, using calcofluor histochemistry, we recently demonstrated that amyloid plaques and blood vessels affected by amyloid angiopathy in subjects with sporadic and familial Alzheimer disease elicit chitin-type characteristics. Since chitin is a highly insoluble molecule and a substrate for glycan-protein interactions, chitin-like polysaccharides within the Alzheimer disease brain could provide a scaffolding for amyloid-beta deposition. As such, glucosamine may facilitate the process of amyloidosis, and /or provide neuroprotection in the Alzheimer disease brain.

Galectin-1-asialofetuin interaction is inhibited by peptides containing the tyr-xxx-tyr motif acting on the glycoprotein.

PubMed

Wéber, Edit; Hetényi, Anasztázia; Váczi, Balázs; Szolnoki, Eva; Fajka-Boja, Roberta; Tubak, Vilmos; Monostori, Eva; Martinek, Tamás A

2010-01-25

Galectin-1 (Gal-1), a ubiquitous beta-galactoside-binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal-1 depend on its affinity for beta-galactoside-containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr-Xxx-Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal-1-asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr-Xxx-Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr-Xxx-Tyr peptides studied do not bind to Gal-1, whereas their binding to ASF is clearly detected. (15)N,(1)H HSQC titrations with (15)N-labeled Gal-1 confirm the absence of any peptide-Gal-1 interaction. These data indicate that the Tyr-Xxx-Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.

Expression of Lectins in Heterologous Systems

PubMed Central

Martínez-Alarcón, Dania; Blanco-Labra, Alejandro

2018-01-01

Lectins are proteins that have the ability to recognize and bind in a reversible and specific way to free carbohydrates or glycoconjugates of cell membranes. For these reasons, they have been extensively used in a wide range of industrial and pharmacological applications. Currently, there is great interest in their production on a large scale. Unfortunately, conventional techniques do not provide the appropriate platform for this purpose and therefore, the heterologous production of lectins in different organisms has become the preferred method in many cases. Such systems have the advantage of providing better yields as well as more homogeneous and better-defined properties for the resultant products. However, an inappropriate choice of the expression system can cause important structural alterations that have repercussions on their biological activity since the specificity may lay in their post-translational processing, which depends largely on the producing organism. The present review aims to examine the most representative studies in the area, exposing the four most frequently used systems (bacteria, yeasts, plants and animal cells), with the intention of providing the necessary information to determine the strategy to follow in each case as well as their respective advantages and disadvantages. PMID:29466298

Application of surface plasmon resonance for the detection of carbohydrates, glycoconjugates, and measurement of the carbohydrate-specific interactions: a comparison with conventional analytical techniques. A critical review.

PubMed

Safina, Gulnara

2012-01-27

Carbohydrates (glycans) and their conjugates with proteins and lipids contribute significantly to many biological processes. That makes these compounds important targets to be detected, monitored and identified. The identification of the carbohydrate content in their conjugates with proteins and lipids (glycoforms) is often a challenging task. Most of the conventional instrumental analytical techniques are time-consuming and require tedious sample pretreatment and utilising various labeling agents. Surface plasmon resonance (SPR) has been intensively developed during last two decades and has received the increasing attention for different applications, from the real-time monitoring of affinity bindings to biosensors. SPR does not require any labels and is capable of direct measurement of biospecific interaction occurring on the sensing surface. This review provides a critical comparison of modern analytical instrumental techniques with SPR in terms of their analytical capabilities to detect carbohydrates, their conjugates with proteins and lipids and to study the carbohydrate-specific bindings. A few selected examples of the SPR approaches developed during 2004-2011 for the biosensing of glycoforms and for glycan-protein affinity studies are comprehensively discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

Deciphering minimal antigenic epitopes associated with Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharide O-antigens.

PubMed

Tamigney Kenfack, Marielle; Mazur, Marcelina; Nualnoi, Teerapat; Shaffer, Teresa L; Ngassimou, Abba; Blériot, Yves; Marrot, Jérôme; Marchetti, Roberta; Sintiprungrat, Kitisak; Chantratita, Narisara; Silipo, Alba; Molinaro, Antonio; AuCoin, David P; Burtnick, Mary N; Brett, Paul J; Gauthier, Charles

2017-07-24

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), the etiologic agents of melioidosis and glanders, respectively, cause severe disease in both humans and animals. Studies have highlighted the importance of Bp and Bm lipopolysaccharides (LPS) as vaccine candidates. Here we describe the synthesis of seven oligosaccharides as the minimal structures featuring all of the reported acetylation/methylation patterns associated with Bp and Bm LPS O-antigens (OAgs). Our approach is based on the conversion of an L-rhamnose into a 6-deoxy-L-talose residue at a late stage of the synthetic sequence. Using biochemical and biophysical methods, we demonstrate the binding of several Bp and Bm LPS-specific monoclonal antibodies with terminal OAg residues. Mice immunized with terminal disaccharide-CRM197 constructs produced high-titer antibody responses that crossreacted with Bm-like OAgs. Collectively, these studies serve as foundation for the development of novel therapeutics, diagnostics, and vaccine candidates to combat diseases caused by Bp and Bm.Melioidosis and glanders are multifaceted infections caused by gram-negative bacteria. Here, the authors synthesize a series of oligosaccharides that mimic the lipopolysaccharides present on the pathogens' surface and use them to develop novel glycoconjugates for vaccine development.

Expression, purification, crystallization and preliminary X-ray characterization of a putative glycosyltransferase of the GT-A fold found in mycobacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fulton, Zara; Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800; Crellin, Paul K.

2008-05-01

MAP2569c from M. avium subsp. paratuberculosis, a putative glycosyltransferase implicated in mycobacterial cell-wall biosynthesis, was cloned, expressed, purified and crystallized. X-ray diffraction data were collected to 1.8 Å resolution. Glycosidic bond formation is a ubiquitous enzyme-catalysed reaction. This glycosyltransferase-mediated process is responsible for the biosynthesis of innumerable oligosaccharides and glycoconjugates and is often organism- or cell-specific. However, despite the abundance of genomic information on glycosyltransferases (GTs), there is a lack of structural data for this versatile class of enzymes. Here, the cloning, expression, purification and crystallization of an essential 329-amino-acid (34.8 kDa) putative GT of the classic GT-A fold implicatedmore » in mycobacterial cell-wall biosynthesis are reported. Crystals of MAP2569c from Mycobacterium avium subsp. paratuberculosis were grown in 1.6 M monoammonium dihydrogen phosphate and 0.1 M sodium citrate pH 5.5. A complete data set was collected to 1.8 Å resolution using synchrotron radiation from a crystal belonging to space group P4{sub 1}2{sub 1}2.« less

Lipoxin A4 Counter-regulates Histamine-stimulated Glycoconjugate Secretion in Conjunctival Goblet Cells.

PubMed

Hodges, Robin R; Li, Dayu; Shatos, Marie A; Serhan, Charles N; Dartt, Darlene A

2016-11-08

Conjunctival goblet cells synthesize and secrete mucins which play an important role in protecting the ocular surface. Pro-resolution mediators, such as lipoxin A 4 (LXA 4 ), are produced during inflammation returning the tissue to homeostasis and are also produced in non-inflamed tissues. The purpose of this study was to determine the actions of LXA 4 on cultured human conjunctival goblet cell mucin secretion and increase in intracellular [Ca 2+ ] ([Ca 2+ ] i ) and on histamine-stimulated responses. LXA 4 increased mucin secretion and [Ca 2+ ] i , and activated ERK1/2 in human goblet cells. Addition of LXA 4 before resolvin D1 (RvD1) decreased RvD1 responses though RvD1 did not block LXA 4 responses. LXA 4 inhibited histamine-stimulated increases in mucin secretion, [Ca 2+ ] i , and ERK1/2 activation through activation of β-adrenergic receptor kinase 1. We conclude that conjunctival goblet cells respond to LXA 4 through the ALX/FPR2 receptor to maintain homeostasis of the ocular surface and regulate histamine responses and could provide a new therapeutic approach for allergic conjunctivitis and dry eye diseases.

Lipoxin A4 Counter-regulates Histamine-stimulated Glycoconjugate Secretion in Conjunctival Goblet Cells

PubMed Central

Hodges, Robin R.; Li, Dayu; Shatos, Marie A.; Serhan, Charles N.; Dartt, Darlene A.

2016-01-01

Conjunctival goblet cells synthesize and secrete mucins which play an important role in protecting the ocular surface. Pro-resolution mediators, such as lipoxin A4 (LXA4), are produced during inflammation returning the tissue to homeostasis and are also produced in non-inflamed tissues. The purpose of this study was to determine the actions of LXA4 on cultured human conjunctival goblet cell mucin secretion and increase in intracellular [Ca2+] ([Ca2+]i) and on histamine-stimulated responses. LXA4 increased mucin secretion and [Ca2+]i, and activated ERK1/2 in human goblet cells. Addition of LXA4 before resolvin D1 (RvD1) decreased RvD1 responses though RvD1 did not block LXA4 responses. LXA4 inhibited histamine-stimulated increases in mucin secretion, [Ca2+]i, and ERK1/2 activation through activation of β-adrenergic receptor kinase 1. We conclude that conjunctival goblet cells respond to LXA4 through the ALX/FPR2 receptor to maintain homeostasis of the ocular surface and regulate histamine responses and could provide a new therapeutic approach for allergic conjunctivitis and dry eye diseases. PMID:27824117

De Novo Sequencing of a Sparassis latifolia Genome and Its Associated Comparative Analyses

PubMed Central

Ma, Lu; Yang, Chi; Ying, Zhenghe; Jiang, Xiaoling

2018-01-01

Known to be rich in β-glucan, Sparassis latifolia (S. latifolia) is a valuable edible fungus cultivated in East Asia. A few studies have suggested that S. latifolia is effective on antidiabetic, antihypertension, antitumor, and antiallergen medications. However, it is still unclear genetically why the fungus has these medical effects, which has become a key bottleneck for its further applications. To provide a better understanding of this fungus, we sequenced its whole genome, which has a total size of 48.13 megabases (Mb) and contains 12,471 predicted gene models. We then performed comparative and phylogenetic analyses, which indicate that S. latifolia is closely related to a few species in the antrodia clade including Fomitopsis pinicola, Wolfiporia cocos, Postia placenta, and Antrodia sinuosa. Finally, we annotated the predicted genes. Interestingly, the S. latifolia genome encodes most enzymes involved in carbohydrate and glycoconjugate metabolism and is also enriched in genes encoding enzymes critical to secondary metabolite biosynthesis and involved in indole, terpene, and type I polyketide pathways. As a conclusion, the genome content of S. latifolia sheds light on its genetic basis of the reported medicinal properties and could also be used as a reference genome for comparative studies on fungi. PMID:29682127

Supercharging Reagent for Enhanced Liquid Chromatographic Separation and Charging of Sialylated and High-Molecular-Weight Glycopeptides for NanoHPLC-ESI-MS/MS Analysis.

PubMed

Lin, Chia-Wei; Haeuptle, Micha A; Aebi, Markus

2016-09-06

Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)-based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides. Using commercial glycoproteins, we demonstrated that in the presence of m-NBA the charge state of sialylated glycopeptides increased and the chromatographic separation of neutral and acidic glycopeptides revealed a remarkable improvement. Next, we applied this system to the characterization of a glycoconjugate vaccine candidate consisting of a genetically detoxified exotoxin A of Pseudomonas aeruginosa covalently linked to Shigella flexneri type 2a O-antigen (Sf2E) produced by engineered Escherichia coli. The addition of m-NBA, allowed us to identify peptides with glycan chains of unprecedented size, up to 20 repeat units (98 monosaccharides). Our results indicated that incorporation of m-NBA into reversed-phase liquid chromatography (LC) solvents improves sensitivity, charging, and chromatographic resolution for acidic and hMW glycopeptides.

Lipopolysaccharides in diazotrophic bacteria

PubMed Central

Serrato, Rodrigo V.

2014-01-01

Biological nitrogen fixation (BNF) is a process in which the atmospheric nitrogen (N2) is transformed into ammonia (NH3) by a select group of nitrogen-fixing organisms, or diazotrophic bacteria. In order to furnish the biologically useful nitrogen to plants, these bacteria must be in constant molecular communication with their host plants. Some of these molecular plant-microbe interactions are very specific, resulting in a symbiotic relationship between the diazotroph and the host. Others are found between associative diazotrophs and plants, resulting in plant infection and colonization of internal tissues. Independent of the type of ecological interaction, glycans, and glycoconjugates produced by these bacteria play an important role in the molecular communication prior and during colonization. Even though exopolysaccharides (EPS) and lipochitooligosaccharides (LCO) produced by diazotrophic bacteria and released onto the environment have their importance in the microbe-plant interaction, it is the lipopolysaccharides (LPS), anchored on the external membrane of these bacteria, that mediates the direct contact of the diazotroph with the host cells. These molecules are extremely variable among the several species of nitrogen fixing-bacteria, and there are evidences of the mechanisms of infection being closely related to their structure. PMID:25232535

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

PubMed Central

Itakura, Yoko; Nakamura-Tsuruta, Sachiko; Kominami, Junko; Tateno, Hiroaki; Hirabayashi, Jun

2017-01-01

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations. PMID:28556796

Creating Knock-outs of Conserved Oligomeric Golgi complex subunits using CRISPR-mediated gene editing paired with a selection strategy based on glycosylation defects associated with impaired COG complex function

PubMed Central

Blackburn, Jessica Bailey; Lupashin, Vladimir V.

2017-01-01

Summary The Conserved Oligomeric Golgi (COG) complex is a key evolutionally conserved multisubunit protein machinery that regulates tethering and fusion of intra-Golgi transport vesicles. The Golgi apparatus specifically promotes sorting and complex glycosylation of glycoconjugates. Without proper glycosylation and processing, proteins and lipids will be mislocalized and/or have impaired function. The Golgi glycosylation machinery is kept in homeostasis by a careful balance of anterograde and retrograde trafficking to ensure proper localization of the glycosylation enzymes and their substrates. This balance, like other steps of membrane trafficking, is maintained by vesicle trafficking machinery that includes COPI vesicular coat proteins, SNAREs, Rabs, and both coiled-coil and multi-subunit vesicular tethers. COG complex interacts with other membrane trafficking components and is essential for proper localization of Golgi glycosylation machinery. Here we describe using CRISPR-mediated gene editing coupled with a phenotype-based selection strategy directly linked to the COG complex’s role in glycosylation homeostasis to obtain COG complex subunit knock-outs (KOs). This has resulted in clonal KOs for each COG subunit in HEK293T cells and gives the ability to further probe the role of the COG complex in Golgi homeostasis. PMID:27632008

Revealing the glycation sites in synthetic neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1, serotype Ogawa with the BSA protein carrier using LC-ESI-QqTOF-MS/MS

PubMed Central

Jahouh, Farid; Saksena, Rina; Kováč, Pavol; Banoub, Joseph

2012-01-01

In this manuscript, we present the determination of glycation sites in synthetic neoglycoconjugates formed by conjugation of the antigenic monosaccharide hapten of Vibrio cholerae O1 serotype Ogawa to BSA using nano- liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectroscopy (LC-ESI-QqTOF-MS/MS). The matrix-assisted laser desorption/ionization-TOF/TOF-MS/MS analyses of the tryptic digests of the glycoconjugates having a hapten:BSA ratio of 4.3:1, 6.6:1 and 13.2:1 revealed only three glycation sites, on the following lysine residues: Lys 235, Lys 437 and Lys 455. Digestion of the neoglycoconjugates with the proteases trypsin and GluC V8 gave complementary structural information and was shown to maximize the number of recognized glycation sites. Here, we report identification of 20, 27 and 33 glycation sites using LC-ESI-QqTOF-MS/MS analysis of a series of synthetic neoglycoconjugates with a hapten:BSA ratio of, respectively, 4.3:1, 6.6:1 and 13.2:1. We also tentatively propose that all the glycated lysine residues are located mainly near the outer surface of the protein. PMID:22791257

Determinants of ABH expression on human blood platelets.

PubMed

Cooling, Laura L W; Kelly, Kathleen; Barton, James; Hwang, Debbie; Koerner, Theodore A W; Olson, John D

2005-04-15

Platelets express ABH antigens, which can adversely effect platelet transfusion recovery and survival in ABH-incompatible recipients. To date, there has been no large, comprehensive study comparing specific donor factors with ABH expression on platelet membranes and glycoconjugates. We studied ABH expression in 166 group A apheresis platelet donors by flow cytometry, Western blotting, and thin layer chromatography relative to donor age, sex, A1/A2 subgroup, and Lewis phenotype. Overall, A antigen on platelet membranes, glycoproteins, and glycosphingolipids was linked to an A1 red blood cell (RBC) phenotype. Among A1 donors, platelet ABH varied significantly between donors (0%-87%). Intradonor variability, however, was minimal, suggesting that platelet ABH expression is a stable, donor-specific characteristic, with 5% of A1 donors typing as either ABH high- or low-expressers. Group A2 donors, in contrast, possessed a Bombay-like phenotype, lacking both A and H antigens. Unlike RBCs, ABH expression on platelets may be determined primarily by H-glycosyltransferase (FUT1) activity. Identification of A2 and A1 low expressers may increase the availability and selection of crossmatched and HLA-matched platelets. Platelets from group A2 may also be a superior product for patients undergoing A/O major mismatch allogeneic progenitor cell transplantation.

The distribution of lectin receptor sites in human breast lesions.

PubMed

Skutelsky, E; Hoenig, S; Griffel, B; Alroy, J

1988-08-01

Conflicting data regarding the status of A, B, H and T antigens in epithelium of normal, mastopathies, fibroadenomas and carcinomas of the breast stimulated us to re-examine the carbohydrate residues in these condition. Currently, we extended the number of carbohydrate residues studied by using ten different biotinylated lectins as probes and avidin-biotin-peroxidase complex (ABC) as a visualant. In addition, the pattern of lectin staining of cancerous cells in primary and metastatic sites was compared. In primary and metastatic breast carcinomas, lectin receptor sites were stained more intensely with Concanavalia ensiformi agglutinin (*Con A), Ricinus communis agglutinin-I (RCA-I) and wheat germ agglutinin (WGA), than in normal breast, in mastopathies or in fibroadenomas. Cryptic receptor sites for peanut agglutinin (PNA) were stained in all cases of breast carcinomas, while free PNA sites stained only in a few cases of well-differentiated carcinomas. Receptors sites for Ulex europaeus agglutinin-I (UEA-I) stained non-malignant epithelium of patients with blood group H but did not stain malignant cells. The results show significant differences in lectin-binding patterns and staining intensities between normal and non-malignant, and malignant epithelial breast cells. Furthermore, these results indicate that in malignant cells, there is an increased content of sialic acid-rich carbohydrates but not of asialylated glycoconjugates.

Screening glycosynthase libraries with a fluoride chemosensor assay independently of enzyme specificity: identification of a transitional hydrolase to synthase mutant.

PubMed

Andrés, Eduardo; Aragunde, Hugo; Planas, Antoni

2014-03-01

Glycosynthases have become efficient tools for the enzymatic synthesis of oligosaccharides, glycoconjugates and polysaccharides. Enzyme-directed evolution approaches are applied to improve the performance of current glycosynthases and engineer specificity for non-natural substrates. However, simple and general screening methods are required since most of the reported assays are specific for each particular enzyme. In the present paper, we report a general screening assay that is independent of enzyme specificity, and implemented in an HTS (high-throughput screening) format for the screening of cell extracts in directed evolution experiments. Fluoride ion is a general by-product released in all glycosynthase reactions with glycosyl fluoride donors. The new assay is based on the use of a specific chemical sensor (a silyl ether of a fluorogenic methylumbelliferone) to transduce fluoride concentration into a fluorescence signal. As a proof-of-concept, it has been applied to a nucleophile saturation mutant library of Bacillus licheniformis 1,3-1,4-β-glucanase. Beyond the expected mutations at the glutamic acid (catalytic) nucleophile, other variants have been shown to acquire glycosynthase activity. Surprisingly, an aspartic acid for glutamic acid replacement renders a highly active glycosynthase, but still retains low hydrolase activity. It appears as an intermediate state between glycosyl hydrolase and glycosynthase.

Selective and slow-binding inhibition of shikonin derivatives isolated from Lithospermum erythrorhizon on glycosyl hydrolase 33 and 34 sialidases.

PubMed

Kim, Ji Young; Jeong, Hyung Jae; Park, Ji-Young; Kim, Young Min; Park, Su-Jin; Cho, Jung Keun; Park, Ki Hun; Ryu, Young Bae; Lee, Woo Song

2012-03-01

Sialidases are enzymes that catalyze the hydrolysis of sialic acid residues from various glycoconjugates, which are widely found in a number of viral and microbial pathogens. In this study, we investigated the biological evaluation of isolated six shikonins (1-6) and three shikonofurans (7-9) from Lithospermum erythrorhizon. The nine isolated compounds 1-9 showed strong and selective inhibition of glycosyl hydrolase (GH) 33 and -34 sialidases activities. In GH33 bacterial-sialidase inhibition assay, the inhibitory activities against GH33 siadliase of all shikonofuran derivatives (7-9) were greater than shikonin derivatives (1-6). Shikonofuran E (8) exhibited the most potent inhibitory activity toward GH33 sialidases (IC(50)=0.24μM). Moreover, our detailed kinetic analysis of these species unveiled that they are all competitive and simple reversible slow-binding inhibitors. Otherwise, they showed different inhibitory capacities and kinetic modes to GH34 viral-sialidase activity. All the naphthoquinone derivatives (1-6) were of almost equal efficiency with IC(50) value of 40μM and shikonofurans (7-9) did not show the significant inhibitory effect to GH34 sialidase. Kinetic analyses indicated that naphthoquinones acted via a noncompetitive mechanism. Copyright © 2012 Elsevier Ltd. All rights reserved.

In vitro evidence for the participation of Drosophila melanogaster sperm β-N-acetylglucosaminidases in the interactions with glycans carrying terminal N-acetylglucosamine residues on the egg's envelopes.

PubMed

Intra, Jari; Veltri, Concetta; De Caro, Daniela; Perotti, Maria Elisa; Pasini, Maria Enrica

2017-09-01

Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm-associated β-N-acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid-phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well-defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β-N-acetylglucosamine, but not core β-N-acetylglucosamine residues. The capacity of sperm β-N-acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β-N-acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β-N-acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm-egg interactions in Drosophila. © 2017 Wiley Periodicals, Inc.

Sequence diversity of NanA manifests in distinct enzyme kinetics and inhibitor susceptibility

NASA Astrophysics Data System (ADS)

Xu, Zhongli; von Grafenstein, Susanne; Walther, Elisabeth; Fuchs, Julian E.; Liedl, Klaus R.; Sauerbrei, Andreas; Schmidtke, Michaela

2016-04-01

Streptococcus pneumoniae is the leading pathogen causing bacterial pneumonia and meningitis. Its surface-associated virulence factor neuraminidase A (NanA) promotes the bacterial colonization by removing the terminal sialyl residues from glycoconjugates on eukaryotic cell surface. The predominant role of NanA in the pathogenesis of pneumococci renders it an attractive target for therapeutic intervention. Despite the highly conserved activity of NanA, our alignment of the 11 NanAs revealed the evolutionary diversity of this enzyme. The amino acid substitutions we identified, particularly those in the lectin domain and in the insertion domain next to the catalytic centre triggered our special interest. We synthesised the representative NanAs and the mutagenized derivatives from E. coli for enzyme kinetics study and neuraminidase inhibitor susceptibility test. Via molecular docking we got a deeper insight into the differences between the two major variants of NanA and their influence on the ligand-target interactions. In addition, our molecular dynamics simulations revealed a prominent intrinsic flexibility of the linker between the active site and the insertion domain, which influences the inhibitor binding. Our findings for the first time associated the primary sequence diversity of NanA with the biochemical properties of the enzyme and with the inhibitory efficiency of neuraminidase inhibitors.

Morphological study on the olfactory systems of the snapping turtle, Chelydra serpentina.

PubMed

Nakamuta, Nobuaki; Nakamuta, Shoko; Kato, Hideaki; Yamamoto, Yoshio

2016-06-01

In this study, the olfactory system of a semi-aquatic turtle, the snapping turtle, has been morphologically investigated by electron microscopy, immunohistochemistry, and lectin histochemistry. The nasal cavity of snapping turtle was divided into the upper and lower chambers, lined by the sensory epithelium containing ciliated and non-ciliated olfactory receptor neurons, respectively. Each neuron expressed both Gαolf, the α-subunit of G-proteins coupling to the odorant receptors, and Gαo, the α-subunit of G-proteins coupling to the type 2 vomeronasal receptors. The axons originating from the upper chamber epithelium projected to the ventral part of the olfactory bulb, while those from the lower chamber epithelium to the dorsal part of the olfactory bulb. Despite the identical expression of G-protein α-subunits in the olfactory receptor neurons, these two projections were clearly distinguished from each other by the differential expression of glycoconjugates. In conclusion, these data indicate the presence of two types of olfactory systems in the snapping turtle. Topographic arrangement of the upper and lower chambers and lack of the associated glands in the lower chamber epithelium suggest their possible involvement in the detection of odorants: upper chamber epithelium in the air and the lower chamber epithelium in the water. Copyright © 2016 Elsevier Ltd. All rights reserved.

Polyphenolic-polysaccharide conjugates from plants of Rosaceae/Asteraceae family as potential radioprotectors.

PubMed

Zbikowska, Halina Malgorzata; Szejk, Magdalena; Saluk, Joanna; Pawlaczyk-Graja, Izabela; Gancarz, Roman; Olejnik, Alicja Klaudia

2016-05-01

Polyphenolic-polysaccharide macromolecular, water-soluble glycoconjugates, isolated from the selected medicinal plants of Rosaceae/Asteraceae family: from leaves of Fragaria vesca L., Rubus plicatus Whe. et N. E., and from flowering parts of Sanguisorba officinalis L., and Erigeron canadensis L., were investigated for their ability to protect proteins and lipids of human plasma against γ-radiation-induced oxidative damage. Treatment of plasma with plant conjugates (6, 30, 150 μg/ml) prior exposure to 100 Gy radiation resulted in a significant inhibition of lipid peroxidation, evaluated by TBARS levels; conjugates isolated from E. canadensis and R. plicatus and a reference flavonoid quercetin showed similar high potential (approx. 70% inhibition, at 6 μg/ml). The conjugates prevented radiation-induced oxidation of protein thiols and significantly improved plasma total antioxidant capacity, estimated with Ellman's reagent and ABTS(.+) assay, respectively. The results demonstrate by the first time a significant radioprotective capability of the polyphenolic-polysaccharide conjugates isolated from E. canadensis, R. plicatus, S. officinalis and to the less extent from F. vesca. The abilities of these substances to inhibit radiation-induced lipid peroxidation and thiol oxidation in plasma seems to be mediated, but not limited to ROS scavenging activity. Copyright © 2016 Elsevier B.V. All rights reserved.

Immobilization of sugars in supermacroporous cryogels for the purification of lectins by affinity chromatography.

PubMed

Gonçalves, Gabriel Ramos Ferreira; Gandolfi, Olga Reinert Ramos; Santos, Leandro Soares; Bonomo, Renata Cristina Ferreira; Veloso, Cristiane Martins; Veríssimo, Lizzy Ayra Alcântara; Fontan, Rafael da Costa Ilhéu

2017-11-15

Lectins are glycoproteins that bind to carbohydrates or glycoconjugates by specific interactions. The specificity of lectins to various carbohydrates is a determinant factor in the choice of ligand for the chromatographic matrix when using chromatography as a lectin purification technique. In this work, the immobilization of three different aminated carbohydrates on the surface of macroporous polymeric cryogels was evaluated. Carbohydrates were immobilized on cryogel surfaces via the glutaraldehyde method to create spacer arms, reducing steric hindrance. The immobilized N-acetyl-d-glucosamine and N-acetyl-d-mannosamine concentrations contained approximately 130mg of carbohydrate/g dehydrated cryogel, while the N-acetyl-d-galactosamine contained 105mg of carbohydrate/g dehydrated cryogel. Scanning electron microscopy showed that the physical structure and porosity of the chromatographic columns were not affected by the immobilization process, maintaining an elevated hydration capacity and the macroporous structure of the cryogels. Adsorption of concanavalin A on cryogels functionalized with N-acetyl-d-glucosamine (cryo-d-GlcNAc) was tested, as well as its reuse capability. After 5 cycles of use, cryo-d-GlcNAc was shown to be stable, with an adsorptive capacity of around 50mg/g. Carbohydrate immobilization in polyacrylamide cryogels was satisfactory, with promise for applications in lectin purification processes. Copyright © 2017 Elsevier B.V. All rights reserved.

Patterns of binding of aluminum-containing adjuvants to Haemophilus influenzae type b and meningococcal group C conjugate vaccines and components

PubMed Central

Otto, Robert B.D.; Burkin, Karena; Amir, Saba Erum; Crane, Dennis T.; Bolgiano, Barbara

2015-01-01

The basis of Haemophilus influenzae type b (Hib) and Neisseria meningitidis serogroup C (MenC) glycoconjugates binding to aluminum-containing adjuvants was studied. By measuring the amount of polysaccharide and protein in the non-adsorbed supernatant, the adjuvant, aluminum phosphate, AlPO4, was found to be less efficient than aluminum hydroxide, Al(OH)3 at binding to the conjugates, at concentrations relevant to licensed vaccine formulations and when equimolar. At neutral pH, binding of TT conjugates to AlPO4 was facilitated through the carrier protein, with only weak binding of AlPO4 to CRM197 being observed. There was slightly higher binding of either adjuvant to tetanus toxoid conjugates, than to CRM197 conjugates. This was verified in AlPO4 formulations containing DTwP–Hib, where the adsorption of TT-conjugated Hib was higher than CRM197-conjugated Hib. At neutral pH, the anionic Hib and MenC polysaccharides did not appreciably bind to AlPO4, but did bind to Al(OH)3, due to electrostatic interactions. Phosphate ions reduced the binding of the conjugates to the adjuvants. These patterns of adjuvant adsorption can form the basis for future formulation studies with individual and combination vaccines containing saccharide-protein conjugates. PMID:26194164

Rice Os9BGlu31 Is a Transglucosidase with the Capacity to Equilibrate Phenylpropanoid, Flavonoid, and Phytohormone Glycoconjugates*

PubMed Central

Luang, Sukanya; Cho, Jung-Il; Mahong, Bancha; Opassiri, Rodjana; Akiyama, Takashi; Phasai, Kannika; Komvongsa, Juthamath; Sasaki, Nobuhiro; Hua, Yan-ling; Matsuba, Yuki; Ozeki, Yoshihiro; Jeon, Jong-Seong; Cairns, James R. Ketudat

2013-01-01

Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu169), catalytic nucleophile residues (Glu387), and His386, the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism. PMID:23430256

Immunologic Memory Induced by a Glycoconjugate Vaccine in a Murine Adoptive Lymphocyte Transfer Model

PubMed Central

Guttormsen, Hilde-Kari; Wetzler, Lee M.; Finberg, Robert W.; Kasper, Dennis L.

1998-01-01

We have developed an adoptive cell transfer model in mice to study the ability of a glycoprotein conjugate vaccine to induce immunologic memory for the polysaccharide moiety. We used type III capsular polysaccharide from the clinically relevant pathogen group B streptococci conjugated to tetanus toxoid (GBSIII-TT) as our model vaccine. GBS are a major cause of neonatal infections in humans, and type-specific antibodies to the capsular polysaccharide protect against invasive disease. Adoptive transfer of splenocytes from mice immunized with the GBSIII-TT conjugate vaccine conferred anti-polysaccharide immunologic memory to naive recipient mice. The transfer of memory occurred in a dose-dependent manner. The observed anamnestic immune response was characterized by (i) more rapid kinetics, (ii) isotype switching from immunoglobulin M (IgM) to IgG, and (iii) 10-fold-higher levels of type III-specific IgG antibody than for the primary response in animals with cells transferred from placebo-immunized mice. The adoptive cell transfer model described in this paper can be used for at least two purposes: (i) to evaluate conjugate vaccines with different physicochemical properties for their ability to induce immunologic memory and (ii) to study the cellular interactions required for an immune response to these molecules. PMID:9573085

Lectins for gastrointestinal targeting--15 years on.

PubMed

Woodley, J F

2000-01-01

In the mid-1980s, the concept of bioadhesion using synthetic polymers emerged, and brought with it the promise of improved efficiency for the delivery of drugs via mucosal surfaces. Studies in the author's laboratory concentrated on 'biological' bioadhesion using the naturally-occurring proteins, lectins, which recognise and bind sugars in glycoconjugates, such as those found on the surfaces of cells. Tomato Lectin (TL) was extensively studied as a putative non-toxic lectin with potential for drug targeting/delivery to the gastrointestinal (GI) tract. In vitro, the TL displayed impressive binding to the intestinal mucosa, but in vivo failed to significantly modify intestinal transit. A number of research groups have coupled the TL to microparticles, and significant systemic uptake of these has been observed in animal studies. Polymers with pendant sugars have also been shown to be bioadhesive, by interacting with endogenous lectins present on the cells of the GI tract. The use of lectins to target to Peyer's patches and diseased tissues in the colon is an interesting development, but much work remains to be done. Lectins also have potential in mucosal vaccines. Before advanced drug delivery systems using lectins can be realised, rigorous evaluation of their toxicity and immunogenicity will be required, but they clearly offer a number of possibilities for GI drug targeting systems in the future.

Chondroitin sulphate-mediated fusion of brain neural folds in rat embryos.

PubMed

Alonso, M I; Moro, J A; Martín, C; de la Mano, A; Carnicero, E; Martínez-Alvarez, C; Navarro, N; Cordero, J; Gato, A

2009-01-01

Previous studies have demonstrated that during neural fold fusion in different species, an apical extracellular material rich in glycoconjugates is involved. However, the composition and the biological role of this material remain undetermined. In this paper, we show that this extracellular matrix in rat increases notably prior to contact between the neural folds, suggesting the dynamic behaviour of the secretory process. Immunostaining has allowed us to demonstrate that this extracellular matrix contains chondroitin sulphate proteoglycan (CSPG), with a spatio-temporal distribution pattern, suggesting a direct relationship with the process of adhesion. The degree of CSPG involvement in cephalic neural fold fusion in rat embryos was determined by treatment with specific glycosidases.In vitro rat embryo culture and microinjection techniques were employed to carry out selective digestion, with chondroitinase AC, of the CSPG on the apical surface of the neural folds; this was done immediately prior to the bonding of the cephalic neural folds. In all the treated embryos, cephalic defects of neural fold fusion could be detected. These results show that CSPG plays an important role in the fusion of the cephalic neural folds in rat embryos, which implies that this proteoglycan could be involved in cellular recognition and adhesion. (c) 2008 S. Karger AG, Basel.

Acylation-dependent protein export in Leishmania.

PubMed

Denny, P W; Gokool, S; Russell, D G; Field, M C; Smith, D F

2000-04-14

The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "non-classical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism.

The growing galectin network in colon cancer and clinical relevance of cytoplasmic galectin-3 reactivity.

PubMed

Dawson, Heather; André, Sabine; Karamitopoulou, Eva; Zlobec, Inti; Gabius, Hans-Joachim

2013-08-01

Human lectins translate sugar-encoded signals of cell surface glycoconjugates into biological effects, and this is what is known for the adhesion/growth-regulatory galectins. In addition, the multifunctional members of this group can be intracellular, binding to distinct proteins. The presence of galectins and galectin reactivity were exemplarily studied in the present article. We combined immuno- and lectin histochemical monitoring in colon cancer on tissue arrays. Intracellular presence of galectins-7 and -9 in colon cancer is detected, extending the previously known set of five expressed lectins this tumor type. The assumed significance of intracellular galectin presence, e.g. for an interplay with BCL2, β-catenin, oncogenic KRAS or synexin, is underscored by respective staining with labeled galectin-3. Statistical significance was obtained for galectin-3 staining with respect to tumor differentiation (p=0.0376), lymph node metastasis (p=0.0069) and lymphatic invasion (p=0.0156). Survival was correlated to staining, galectin-3 reactivity indicating a favorable prognosis (p=0.0183), albeit not as an independent marker. No correlation to KRAS/BRAF status was detected. These results encourage further testing of labeled human galectins as probes and immunohistochemical fingerprinting instead of measuring single or few activities, in colon cancer and other tumor types.

Biosynthesis and Degradation of Mono-, Oligo-, and Polysaccharides: Introduction

NASA Astrophysics Data System (ADS)

Wilson, Iain B. H.

Glycomolecules, whether they be mono-, oligo-, or polysaccharides or simple glycosides, are—as any biological molecules—the products of biosynthetic processes; on the other hand, at the end of their lifespan, they are also subject to degradation. The beginning point, biochemically, is the fixation of carbon by photosynthesis; subsequent metabolism in plants and other organisms results in the generation of the various monosaccharides. These must be activated—typically as nucleotide sugars or lipid-phosphosugars—before transfer by glycosyltransferases can take place in order to produce the wide variety of oligo- and polysaccharides seen in Nature; complicated remodelling processes may take place—depending on the pathway—which result in partial trimming of a precursor by glycosidases prior to the addition of further monosaccharide units. Upon completion of the 'life' of a glycoconjugate, glycosidases will degrade the macromolecule finally into monosaccharide units which can be metabolized or salvaged for incorporation into new glycan chains. In modern glycoscience, a wide variety of methods—genetic, biochemical, analytical—are being employed in order to understand these various pathways and to place them within their biological and medical context. In this chapter, these processes and relevant concepts and methods are introduced, prior to elaboration in the subsequent more specialized chapters on biosynthesis and degradation of mono-, oligo-, and polysaccharides.

Substance P and neurokinin A in human nasal mucosa.

PubMed

Baraniuk, J N; Lundgren, J D; Okayama, M; Goff, J; Mullol, J; Merida, M; Shelhamer, J H; Kaliner, M A

1991-03-01

The tachykinins substance P (SP) and neurokinin A (NKA) were studied in human inferior turbinate nasal mucosa by radioimmunoassay, immunohistochemistry, and autoradiography and for their effect upon mucus release in an in vitro culture system in order to infer their potential functions in the upper respiratory tract. Similar amounts of SP (1.03 +/- 0.12 pmol/g wet weight; mean +/- SEM; n = 26) and NKA (0.76 +/- 0.23; n = 7) were found. NKA and SP immunoreactive nerve fibers were found in the walls of arterioles, venules, and sinusoids and as individual fibers in gland acini, near the basement membrane, and in the epithelium. [125I]SP bound to arterioles, venules, and glands. [125I]NKA bound only to arterioles. In short-term explant culture of fragments of human nasal mucosa, both 1 microM SP and 1 microM NKA stimulated release of [3H]glucosamine-labeled respiratory glycoconjugates. These results indicate that SP and NKA have similar distributions in nociceptive sensory nerves in human nasal mucosa. The distribution of [125I]SP binding sites is consistent with a role for SP as a vasodilator and mucous secretagogue. The presence of [125I] NKA binding sites on vessels suggests a primary role for NKA in regulating vasomotor tone.

Structure of a protective epitope of group B Streptococcus type III capsular polysaccharide.

PubMed

Carboni, Filippo; Adamo, Roberto; Fabbrini, Monica; De Ricco, Riccardo; Cattaneo, Vittorio; Brogioni, Barbara; Veggi, Daniele; Pinto, Vittoria; Passalacqua, Irene; Oldrini, Davide; Rappuoli, Rino; Malito, Enrico; Margarit, Immaculada Y Ros; Berti, Francesco

2017-05-09

Despite substantial progress in the prevention of group B Streptococcus (GBS) disease with the introduction of intrapartum antibiotic prophylaxis, this pathogen remains a leading cause of neonatal infection. Capsular polysaccharide conjugate vaccines have been tested in phase I/II clinical studies, showing promise for further development. Mapping of epitopes recognized by protective antibodies is crucial for understanding the mechanism of action of vaccines and for enabling antigen design. In this study, we report the structure of the epitope recognized by a monoclonal antibody with opsonophagocytic activity and representative of the protective response against type III GBS polysaccharide. The structure and the atomic-level interactions were determined by saturation transfer difference (STD)-NMR and X-ray crystallography using oligosaccharides obtained by synthetic and depolymerization procedures. The GBS PSIII epitope is made by six sugars. Four of them derive from two adjacent repeating units of the PSIII backbone and two of them from the branched galactose-sialic acid disaccharide contained in this sequence. The sialic acid residue establishes direct binding interactions with the functional antibody. The crystal structure provides insight into the molecular basis of antibody-carbohydrate interactions and confirms that the conformational epitope is not required for antigen recognition. Understanding the structural basis of immune recognition of capsular polysaccharide epitopes can aid in the design of novel glycoconjugate vaccines.

Glycoconjugate distribution in early human notochord and axial mesenchyme.

PubMed

Götz, W; Quondamatteo, F

2001-02-01

Glycosylation patterns of cells and tissues give insights into spatially and temporally regulated developmental processes and can be detected histochemically using plant lectins with specific affinities for sugar moieties. The early development of the vertebral column in man is a process which has never been investigated by lectin histochemistry. Therefore, we studied binding of several lectins (AIA, Con A, GSA II, LFA, LTA, PNA, RCA I, SBA, SNA, WGA) in formaldehyde-fixed sections of the axial mesenchyme of 5 human embryos in Carnegie stages 12-15. During these developmental stages, an unsegmented mesenchyme covers the notochord. Staining patterns did not show striking temporal variations except for SBA which stained the cranial axial mesenchyme only in the early stage 12 embryo and for PNA, of which the staining intensity in the mesenchyme decreased with age. The notochord appeared as a highly glycosylated tissue. Carbohydrates detected may correspond to adhesion molecules or to secreted substances like proteoglycans or proteins which could play an inductive role, for example, for the neural tube. The axial perinotochordal unsegmented mesenchyme showed strong PNA binding. Therefore, its function as a PNA-positive "barrier" tissue is discussed. The endoderm of the primitive gut showed a lectin-binding pattern that was similar to that of the notochord, which may correlate with interactions between these tissues during earlier developmental stages.

Histochemistry of lectin-binding sites in Halicryptus spinulosus (Priapulida).

PubMed

Busch, A; Schumacher, U; Storch, V

2001-02-01

Priapulida represent one of the phylogenetically oldest multicellular animal groups. In multicellular animals (Metazoa) cell-to-cell and cell-to-matrix interactions are often mediated by carbohydrate residues of glycoconjugates. To analyze the carbohydrate composition of a phylogenetically old species, lectin histochemistry was employed on 5 specimens of the priapulid Halicryptus spinulosus. Many lectins bound to the chitin-containing cuticle, including those specific for carbohydrates other than N-acetylglucosamine, the principle building block of chitin. The connective tissue of the animals contained both N-acetylglucosamine and N-acetylgalactosamine. Mannose residues were widely distributed with the exception of the cuticle, but complex type carbohydrates were not present in the entire animal. Sialic acid residues were only detected in the cuticle and brush border of the intestinal epithelium, while fucose was limited to the cuticle. Thus, the lectin-binding pattern indicated that sugars typical for the linking region of both N- and O-glycoproteins in mammals are also present in H. spinulosus. Carbohydrate residues that are typical for the complex type of N-linked glycans in vertebrates are not present as are carbohydrate residues typical for the termination of O-linked carbohydrate chains. Hence, a truncated form of both N- and O-linked glycosylation is present in H. spinulosus indicating that more complex patterns of glycosylation developed later during evolution.

Metabolic Glyco-Engineering in Eukaryotic Cells and Selected Applications.

PubMed

Piller, Friedrich; Mongis, Aline; Piller, Véronique

2015-01-01

By metabolic glyco-engineering cellular glycoconjugates are modified through the incorporation of synthetic monosaccharides which are usually analogues of naturally present sugars. In order to get incorporated, the monosaccharides need to enter the cytoplasm and to be substrates for the enzymes necessary for their transformation into activated sugars, most often nucleotide sugars. These have to be substrates for glycosyltransferases which finally catalyze their incorporation into glycans. Such pathways are difficult to reconstitute in vitro and therefore new monosaccharide analogues have to be tested in tissue culture for their suitability in metabolic glyco-engineering. For this, glycosylation mutants are the most appropriate since they are unable to synthesize specific glycans but through the introduction of the monosaccharide analogues they may express some glycans at the cell surface with the unnatural sugar incorporated. The presence of those glycans can be easily and quantitatively detected by lectin binding or by chemical methods identifying specific sugars. Monosaccharide analogues can also block the pathways leading to sugar incorporation, thus inhibiting the synthesis of glycan structures which is also easily detectable at the cell surface by lectin labeling. The most useful and most frequently employed application of metabolic glyco-engineering is the introduction of reactive groups which can undergo bio-orthogonal click reactions for the efficient labeling of glycans at the surface of live cells.

An organophosphonate strategy for functionalizing silicon photonic biosensors

PubMed Central

Shang, Jing; Cheng, Fang; Dubey, Manish; Kaplan, Justin M.; Rawal, Meghana; Jiang, Xi; Newburg, David S.; Sullivan, Philip A.; Andrade, Rodrigo B.; Ratner, Daniel M.

2012-01-01

Silicon photonic microring resonators have established their potential for label-free and low-cost biosensing applications. However, the long-term performance of this optical sensing platform requires robust surface modification and biofunctionalization. Herein, we demonstrate a conjugation strategy based on an organophosphonate surface coating and vinyl sulfone linker to biofunctionalize silicon resonators for biomolecular sensing. To validate this method, a series of glycans, including carbohydrates and glycoconjugates, were immobilized on divinyl sulfone (DVS)/organophosphonate-modified microrings and used to characterize carbohydrate-protein and norovirus particle interactions. This biofunctional platform was able to orthogonally detect multiple specific carbohydrate-protein interactions simultaneously. Additionally, the platform was capable of reproducible binding after multiple regenerations by high-salt, high-pH or low-pH solutions and after 1-month storage in ambient conditions. This remarkable stability and durability of the organophosphonate immobilization strategy will facilitate the application of silicon microring resonators in various sensing conditions, prolong their lifetime, and minimize the cost for storage and delivery; these characteristics are requisite for developing biosensors for point-of-care and distributed diagnostics and other biomedical applications. In addition, the platform demonstrated its ability to characterize carbohydrate-mediated host-virus interactions, providing a facile method for discovering new anti-viral agents to prevent infectious disease. PMID:22220731

One-pot multienzyme (OPME) systems for chemoenzymatic synthesis of carbohydrates.

PubMed

Yu, Hai; Chen, Xi

2016-03-14

Glycosyltransferase-catalyzed enzymatic and chemoenzymatic syntheses are powerful approaches for the production of oligosaccharides, polysaccharides, glycoconjugates, and their derivatives. Enzymes involved in the biosynthesis of sugar nucleotide donors can be combined with glycosyltransferases in one pot for efficient production of the target glycans from simple monosaccharides and acceptors. The identification of enzymes involved in the salvage pathway of sugar nucleotide generation has greatly facilitated the development of simplified and efficient one-pot multienzyme (OPME) systems for synthesizing major glycan epitopes in mammalian glycomes. The applications of OPME methods are steadily gaining popularity mainly due to the increasing availability of wild-type and engineered enzymes. Substrate promiscuity of these enzymes and their mutants allows OPME synthesis of carbohydrates with naturally occurring post-glycosylational modifications (PGMs) and their non-natural derivatives using modified monosaccharides as precursors. The OPME systems can be applied in sequence for synthesizing complex carbohydrates. The sequence of the sequential OPME processes, the glycosyltransferase used, and the substrate specificities of the glycosyltransferases define the structures of the products. The OPME and sequential OPME strategies can be extended to diverse glycans in other glycomes when suitable enzymes with substrate promiscuity become available. This Perspective summarizes the work of the authors and collaborators on the development of glycosyltransferase-based OPME systems for carbohydrate synthesis. Future directions are also discussed.

One-pot multienzyme (OPME) systems for chemoenzymatic synthesis of carbohydrates

PubMed Central

Yu, Hai; Chen, Xi

2016-01-01

Glycosyltransferase-catalyzed enzymatic and chemoenzymatic syntheses are powerful approaches for the production of oligosaccharides, polysaccharides, glycoconjugates, and their derivatives. Enzymes involved in the biosynthesis of sugar nucleotide donors can be combined with the glycosyltransferases in one pot for efficient production of target glycans from simple monosaccharides and accpetors. The identification of enzymes involved in the salvage pathway of sugar nucleotide generation has greatly facilitate the development of simplified and efficient one-pot multienzyme (OPME) systems for synthesizing major glycan epitopes in mammalian glycomes. The applications of OPME methods are steadily gaining popularity mainly due to the increasing availability of wild-type and engineered enzymes. Substrate promiscuity of these enzymes and their mutants allows OPME synthesis of carbohydrates with naturally occurring post-glycosylational modificiation (PGMs) and their non-natural derivatives using modified monosaccharides as precursors. The OPME systems can be applied in sequential for synthesizing complex carbohydrates. The sequence of the sequential OPME processes, the glycosyltransferase used, and the substrate specificities of glycosyltransferasese define the structures of the products. The OPME and sequential OPME strategies can be extended to diverse glycans in other glycomes when suitable enzymes with substrate promiscuity become available. The Perspective summariezes the work of the authors and collaborators on the development of glycosyltransferase-based OPME systems for carbohydrate synthesis. Future directions are also discussed. PMID:26881499

Photoacoustic analysis of the solubilization kinetics of pulmonary secretions from cystic fibrosis patients - secretor and non-secretor phenotypes

NASA Astrophysics Data System (ADS)

Barja, P. R.; Coelho, C. C.; Paiva, R. F.; Barboza, M. A.; Matos, L. C.; Matos, C. C. B.; Oliveira, L. V. F.

2010-03-01

Cystic fibrosis (CF) is an autosomal recessive inherited disease that increases viscoelasticity of pulmonary secretions. Affected patients are required to use therapeutic aerosols continuously. The expression of ABH glycoconjugates in exocrine secretions determines the nature of part of the carbohydrates present in these secretions, allowing the classification of individuals into the so-called "secretor" and "non secretor" phenotypes. The aim of this work was to employ photoacoustic (PA) measurements to monitor the solubilization kinetics of pulmonary secretions from CF patients, analyzing the influence of the secretor status in the solubilization kinetics of samples nebulized with different therapeutic aerosols. Sputum samples were obtained by spontaneous expectoration from positive and negative secretor CF patients. Each sample was nebulized with i) tobramycin, ii) alpha dornase, and iii) N-acetylcysteine in a PA cell; fitting of the data with the Boltzmann equation led to the determination of t0 (typical interaction time) and Δt (solubilization interval) for each curve. Differences between the secretor and non-secretor phenotypes were statistically significant in the groups for tobramycin and alpha dornase, but not for N-acetylcysteine. Results show that the secretor status influences the solubilization of pulmonary mucus of CF patients nebulized with tobramycin and alpha dornase.

Host Glycan Sugar-Specific Pathways in Streptococcus pneumonia: Galactose as a Key Sugar in Colonisation and Infection

PubMed Central

Paixão, Laura; Oliveira, Joana; Veríssimo, André; Vinga, Susana; Lourenço, Eva C.; Ventura, M. Rita; Kjos, Morten; Veening, Jan-Willem; Fernandes, Vitor E.; Andrew, Peter W.; Yesilkaya, Hasan; Neves, Ana Rute

2015-01-01

The human pathogen Streptococcus pneumoniae is a strictly fermentative organism that relies on glycolytic metabolism to obtain energy. In the human nasopharynx S. pneumoniae encounters glycoconjugates composed of a variety of monosaccharides, which can potentially be used as nutrients once depolymerized by glycosidases. Therefore, it is reasonable to hypothesise that the pneumococcus would rely on these glycan-derived sugars to grow. Here, we identified the sugar-specific catabolic pathways used by S. pneumoniae during growth on mucin. Transcriptome analysis of cells grown on mucin showed specific upregulation of genes likely to be involved in deglycosylation, transport and catabolism of galactose, mannose and N acetylglucosamine. In contrast to growth on mannose and N-acetylglucosamine, S. pneumoniae grown on galactose re-route their metabolic pathway from homolactic fermentation to a truly mixed acid fermentation regime. By measuring intracellular metabolites, enzymatic activities and mutant analysis, we provide an accurate map of the biochemical pathways for galactose, mannose and N-acetylglucosamine catabolism in S. pneumoniae. Intranasal mouse infection models of pneumococcal colonisation and disease showed that only mutants in galactose catabolic genes were attenuated. Our data pinpoint galactose as a key nutrient for growth in the respiratory tract and highlights the importance of central carbon metabolism for pneumococcal pathogenesis. PMID:25826206

Cytochemical analysis of alkaline phosphatase and esterase activities and of lectin-binding and anionic sites in rat and mouse Peyer's patch M cells.

PubMed

Owen, R L; Bhalla, D K

1983-10-01

M cells in Peyer's patch follicle epithelium endocytose and transport luminal materials to intraepithelial lymphocytes. We examined (1) enzymatic characteristics of the epithelium covering mouse and rat Peyer's patches by using cytochemical techniques, (2) distribution of lectin-binding sites by peroxidase-labeled lectins, and (3) anionic site distribution by using cationized ferritin to develop a profile of M cell surface properties. Alkaline phosphatase activity resulted in deposits of dense reaction product over follicle surfaces but was markedly reduced over M cells, unlike esterase which formed equivalent or greater product over M cells. Concanavalin A, ricinus communis agglutinin, wheat germ agglutinin and peanut agglutinin reacted equally with M cells and with surrounding enterocytes over follicle surfaces. Cationized ferritin distributed in a random fashion along microvillus membranes of both M cells and enterocytes, indicating equivalent anionic site distribution. Staining for alkaline phosphatase activity provides a new approach for distinguishing M cells from enterocytes at the light microscopic level. Identical binding of lectins indicates that M cells and enterocytes share common glycoconjugates even though molecular groupings may differ. Lectin binding and anionic charge similarities of M cells and enterocytes may facilitate antigen sampling by M cells of particles and compounds that adhere to intestinal surfaces in non-Peyer's patch areas.

Trypanosoma cruzi TcSMUG L-surface mucins promote development and infectivity in the triatomine vector Rhodnius prolixus.

PubMed

Gonzalez, Marcelo S; Souza, Marcela S; Garcia, Eloi S; Nogueira, Nadir F S; Mello, Cícero B; Cánepa, Gaspar E; Bertotti, Santiago; Durante, Ignacio M; Azambuja, Patrícia; Buscaglia, Carlos A

2013-11-01

TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector. Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy. Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.

Leishmania infantum: Lipophosphoglycan intraspecific variation and interaction with vertebrate and invertebrate hosts.

PubMed

Coelho-Finamore, J M; Freitas, V C; Assis, R R; Melo, M N; Novozhilova, N; Secundino, N F; Pimenta, P F; Turco, S J; Soares, R P

2011-03-01

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly-Leishmania interaction. This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO(4) backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one β-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG. Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Preserved antibody levels and loss of memory B cells against pneumococcus and tetanus after splenectomy: tailoring better vaccination strategies.

PubMed

Rosado, M Manuela; Gesualdo, Francesco; Marcellini, Valentina; Di Sabatino, Antonio; Corazza, Gino Roberto; Smacchia, Maria Paola; Nobili, Bruno; Baronci, Carlo; Russo, Lidia; Rossi, Francesca; Vito, Rita De; Nicolosi, Luciana; Inserra, Alessandro; Locatelli, Franco; Tozzi, Alberto E; Carsetti, Rita

2013-10-01

Splenectomized patients are exposed to an increased risk of septicemia caused by encapsulated bacteria. Defense against infection is ensured by preformed serum antibodies produced by long-lived plasma cells and by memory B cells that secrete immunoglobulin in response to specific antigenic stimuli. Studying a group of asplenic individuals (57 adults and 21 children) without additional immunologic defects, we found that spleen removal does not alter serum anti-pneumococcal polysaccharide (PnPS) IgG concentration, but reduces the number of PnPS-specific memory B cells, of both IgM and IgG isotypes. The number of specific memory B cells was low in splenectomized adults and children that had received the PnPS vaccine either before or after splenectomy. Seven children were given the 13-valent pneumococcal conjugated vaccine after splenectomy. In this group, the number of PnPS-specific IgG memory B cells was similar to that of eusplenic children, suggesting that pneumococcal conjugated vaccine administered after splenectomy is able to restore the pool of anti-PnPS IgG memory B cells. Our data further elucidate the crucial role of the spleen in the immunological response to infections caused by encapsulated bacteria and suggest that glycoconjugated vaccines may be the most suitable choice to generate IgG-mediated protection in these patients. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multi-specificity of a Psathyrella velutina mushroom lectin: heparin/pectin binding occurs at a site different from the N-acetylglucosamine/N-acetylneuraminic acid-specific site.

PubMed

Ueda, H; Saitoh, T; Kojima, K; Ogawa, H

1999-09-01

An N-acetylglucosamine (GlcNAc)/N-acetylneuraminic acid-specific lectin from the fruiting body of Psathyrella velutina (PVL) is a useful probe for the detection and fractionation of specific carbohydrates. In this study, PVL was found to exhibit multispecificity to acidic polysaccharides and sulfatides. Purified PVL and a counterpart lectin to PVL in the mycelium interact with heparin neoproteoglycans, as detected by both membrane analysis and solid phase assay. The pH-dependencies of the binding to heparin and GlcNAc5-6 differ. The heparin binding of PVL is inhibited best by pectin, polygalacturonic acid, and highly sulfated polysaccharides, but not by GlcNAc, colominic acid, or other glycosaminoglycans. Sandwich affinity chromatography indicated that PVL can simultaneously interact with heparin- and GlcNAc-containing macromolecules. Extensive biotinylation was found to suppress the binding activity to heparin while the GlcNAc binding activity is retained. On the other hand, biotinyl PVL binds to sulfatide and the binding is not inhibited by GlcNAc, N-acetylneuraminic acid, or heparin. These results indicate that PVL is a multi-ligand adhesive lectin that can interact with various glycoconjugates. This multispecificity needs to be recognized when using PVL as a sugar-specific probe to avoid misleading information about the nature of glycoforms.

C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia

PubMed Central

Yamasaki, Sho; Matsumoto, Makoto; Takeuchi, Osamu; Matsuzawa, Tetsuhiro; Ishikawa, Eri; Sakuma, Machie; Tateno, Hiroaki; Uno, Jun; Hirabayashi, Jun; Mikami, Yuzuru; Takeda, Kiyoshi; Akira, Shizuo; Saito, Takashi

2009-01-01

Mincle (also called as Clec4e and Clecsf9) is a C-type lectin receptor expressed in activated phagocytes. Recently, we have demonstrated that Mincle is an FcRγ-associated activating receptor that senses damaged cells. To search an exogenous ligand(s), we screened pathogenic fungi using cell line expressing Mincle, FcRγ, and NFAT-GFP reporter. We found that Mincle specifically recognizes the Malassezia species among 50 different fungal species tested. Malassezia is a pathogenic fungus that causes skin diseases, such as tinea versicolor and atopic dermatitis, and fatal sepsis. However, the specific receptor on host cells has not been identified. Mutation of the putative mannose-binding motif within C-type lectin domain of Mincle abrogated Malassezia recognition. Analyses of glycoconjugate microarray revealed that Mincle selectively binds to α-mannose but not mannan. Thus, Mincle may recognize specific geometry of α-mannosyl residues on Malassezia species and use this to distinguish them from other fungi. Malassezia activated macrophages to produce inflammatory cytokines/chemokines. To elucidate the physiological function of Mincle, Mincle-deficient mice were established. Malassezia-induced cytokine/chemokine production by macrophages from Mincle−/− mice was significantly impaired. In vivo inflammatory responses against Malassezia was also impaired in Mincle−/− mice. These results indicate that Mincle is the first specific receptor for Malassezia species to be reported and plays a crucial role in immune responses to this fungus. PMID:19171887

Patterns of binding of aluminum-containing adjuvants to Haemophilus influenzae type b and meningococcal group C conjugate vaccines and components.

PubMed

Otto, Robert B D; Burkin, Karena; Amir, Saba Erum; Crane, Dennis T; Bolgiano, Barbara

2015-09-01

The basis of Haemophilus influenzae type b (Hib) and Neisseria meningitidis serogroup C (MenC) glycoconjugates binding to aluminum-containing adjuvants was studied. By measuring the amount of polysaccharide and protein in the non-adsorbed supernatant, the adjuvant, aluminum phosphate, AlPO4, was found to be less efficient than aluminum hydroxide, Al(OH)3 at binding to the conjugates, at concentrations relevant to licensed vaccine formulations and when equimolar. At neutral pH, binding of TT conjugates to AlPO4 was facilitated through the carrier protein, with only weak binding of AlPO4 to CRM197 being observed. There was slightly higher binding of either adjuvant to tetanus toxoid conjugates, than to CRM197 conjugates. This was verified in AlPO4 formulations containing DTwP-Hib, where the adsorption of TT-conjugated Hib was higher than CRM197-conjugated Hib. At neutral pH, the anionic Hib and MenC polysaccharides did not appreciably bind to AlPO4, but did bind to Al(OH)3, due to electrostatic interactions. Phosphate ions reduced the binding of the conjugates to the adjuvants. These patterns of adjuvant adsorption can form the basis for future formulation studies with individual and combination vaccines containing saccharide-protein conjugates. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

A mouse model for fucosidosis recapitulates storage pathology and neurological features of the milder form of the human disease

PubMed Central

Wolf, Heike; Stroobants, Stijn; D'Hooge, Rudi; Hermans-Borgmeyer, Irm; Lüllmann-Rauch, Renate; Dierks, Thomas; Lübke, Torben

2016-01-01

ABSTRACT Fucosidosis is a rare lysosomal storage disorder caused by the inherited deficiency of the lysosomal hydrolase α-L-fucosidase, which leads to an impaired degradation of fucosylated glycoconjugates. Here, we report the generation of a fucosidosis mouse model, in which the gene for lysosomal α-L-fucosidase (Fuca1) was disrupted by gene targeting. Homozygous knockout mice completely lack α-L-fucosidase activity in all tested organs leading to highly elevated amounts of the core-fucosylated glycoasparagine Fuc(α1,6)-GlcNAc(β1-N)-Asn and, to a lesser extent, other fucosylated glycoasparagines, which all were also partially excreted in urine. Lysosomal storage pathology was observed in many visceral organs, such as in the liver, kidney, spleen and bladder, as well as in the central nervous system (CNS). On the cellular level, storage was characterized by membrane-limited cytoplasmic vacuoles primarily containing water-soluble storage material. In the CNS, cellular alterations included enlargement of the lysosomal compartment in various cell types, accumulation of secondary storage material and neuroinflammation, as well as a progressive loss of Purkinje cells combined with astrogliosis leading to psychomotor and memory deficits. Our results demonstrate that this new fucosidosis mouse model resembles the human disease and thus will help to unravel underlying pathological processes. Moreover, this model could be utilized to establish diagnostic and therapeutic strategies for fucosidosis. PMID:27491075

Ketone isosteres of 2-N-acetamidosugars as substrates for metabolic cell surface engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hang, Howard C.; Bertozzi, Carolyn R.

2000-08-22

Novel chemical reactivity can be engendered on cell surfaces by the metabolic incorporation of unnatural sugars into cell surface glycoconjuagtes. 2-N-Acetamido sugars such as GalNAc and GlcNAc are abundant components of cell surface glycoconjugates, and hence attractive targets for metabolic cell surface engineering. Here we report (1) the synthesis of isosteric analogs bearing a ketone group in place of the N-acetamido group, and (2) evaluation of their metabolic incorporation into mammalian cell surface glycans. A ketone isostere of GalNAc was metabolized by CHO cells through the salvage pathway and delivered to O-linked glycoproteins on the cell surface. Its residence atmore » the core position of O-linked glycans is suggested by studies with a-benzyl GalNAc, an inhibitor of O-linked oligosaccharide extension. A mutant CHO cell line lacking endogenous UDP-GalNAc demonstrated enhanced metabolism of the GalNAc analog, suggesting that competition with native intermediates might limits enzymatic transformation in mammalian cells. A ketone isostere of GlcNAc could not be detected on CHO or human cell surfaces after incubation. Thus, the enzymes in the GlcNAc salvage pathway might be less permissive of unnatural substrates than those comprising the GalNAc salvage pathway. Alternatively, high levels of endogenous GlcNAc derivatives might compete with the ketone isostere and prevent its incorporation into oligosaccharides.« less

Profiles of Human Milk Oligosaccharides and Production of Some Human Milk Oligosaccharides in Transgenic Animals12

PubMed Central

Prieto, Pedro Antonio

2012-01-01

During the decade of the 1990s and the first years of the current century, our group embarked on a project to study and synthesize human milk oligosaccharides. This report describes 2 unexpected collateral observations from that endeavor. The first observation was the detection and confirmation of 2 rare neutral human milk oligosaccharides profiles that were uncovered while assessing oligosaccharide content in hundreds of samples of human milk. One of these lacked fucosylated structures altogether, and the other lacked the oligosaccharide 3-fucosyllactose [Galβ1–4(Fucα1–3)Glc]. We used glycoconjugate probes to determine whether the unusual profiles were mirrored by fucosylation of milk glycoproteins. The results show that the lack of fucosylated oligosaccharides in these samples corresponds to the absence of equivalent fucosylated motifs in milk glycoproteins. The second finding was a shortened and distinct lactation process in transgenic rabbits expressing the human fucosyltransferase 1. During the first day of lactation, these animals expressed milk that contained both lactose and 2′-fucosylactose, but on the second day, the production of milk was severely diminished, and by the fourth day, no lactose was detected in their milk. Meanwhile, the concentration of fucosylated glycoproteins increased from the onset of lactation through its premature termination. These 2 findings may shed light on the glycobiology of milk and perhaps on mammary gland differentiation. PMID:22585925

Leishmania UDP-sugar pyrophosphorylase: the missing link in galactose salvage?

PubMed

Damerow, Sebastian; Lamerz, Anne-Christin; Haselhorst, Thomas; Führing, Jana; Zarnovican, Patricia; von Itzstein, Mark; Routier, Françoise H

2010-01-08

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.

Helicobacter pylori and Complex Gangliosides

PubMed Central

Roche, Niamh; Ångström, Jonas; Hurtig, Marina; Larsson, Thomas; Borén, Thomas; Teneberg, Susann

2004-01-01

Recognition of sialic acid-containing glycoconjugates by the human gastric pathogen Helicobacter pylori has been repeatedly demonstrated. To investigate the structural requirements for H. pylori binding to complex gangliosides, a large number of gangliosides were isolated and characterized by mass spectrometry and proton nuclear magnetic resonance. Ganglioside binding of sialic acid-recognizing H. pylori strains (strains J99 and CCUG 17874) and knockout mutant strains with the sialic acid binding adhesin SabA or the NeuAcα3Galβ4GlcNAcβ3Galβ4GlcNAcβ-binding neutrophil-activating protein HPNAP deleted was investigated using the thin-layer chromatogram binding assay. The wild-type bacteria bound to N-acetyllactosamine-based gangliosides with terminal α3-linked NeuAc, while gangliosides with terminal NeuGcα3, NeuAcα6, or NeuAcα8NeuAcα3 were not recognized. The factors affecting binding affinity were identified as (i) the length of the N-acetyllactosamine carbohydrate chain, (ii) the branches of the carbohydrate chain, and (iii) fucose substitution of the N-acetyllactosamine core chain. While the J99/NAP− mutant strain displayed a ganglioside binding pattern identical to that of the parent J99 wild-type strain, no ganglioside binding was obtained with the J99/SabA− mutant strain, demonstrating that the SabA adhesin is the sole factor responsible for the binding of H. pylori bacterial cells to gangliosides. PMID:14977958

Galectins in Intestinal Inflammation: Galectin-1 Expression Delineates Response to Treatment in Celiac Disease Patients

PubMed Central

Sundblad, Victoria; Quintar, Amado A.; Morosi, Luciano G.; Niveloni, Sonia I.; Cabanne, Ana; Smecuol, Edgardo; Mauriño, Eduardo; Mariño, Karina V.; Bai, Julio C.; Maldonado, Cristina A.; Rabinovich, Gabriel A.

2018-01-01

Galectins, a family of animal lectins characterized by their affinity for N-acetyllactosamine-enriched glycoconjugates, modulate several immune cell processes shaping the course of innate and adaptive immune responses. Through interaction with a wide range of glycosylated receptors bearing complex branched N-glycans and core 2-O-glycans, these endogenous lectins trigger distinct signaling programs thereby controling immune cell activation, differentiation, recruitment and survival. Given the unique features of mucosal inflammation and the differential expression of galectins throughout the gastrointestinal tract, we discuss here key findings on the role of galectins in intestinal inflammation, particularly Crohn’s disease, ulcerative colitis, and celiac disease (CeD) patients, as well as in murine models resembling these inflammatory conditions. In addition, we present new data highlighting the regulated expression of galectin-1 (Gal-1), a proto-type member of the galectin family, during intestinal inflammation in untreated and treated CeD patients. Our results unveil a substantial upregulation of Gal-1 accompanying the anti-inflammatory and tolerogenic response associated with gluten-free diet in CeD patients, suggesting a major role of this lectin in favoring resolution of inflammation and restoration of mucosal homeostasis. Thus, a coordinated network of galectins and their glycosylated ligands, exerting either anti-inflammatory or proinflammatory responses, may influence the interplay between intestinal epithelial cells and the highly specialized gut immune system in physiologic and pathologic settings. PMID:29545799

Galectins in Intestinal Inflammation: Galectin-1 Expression Delineates Response to Treatment in Celiac Disease Patients.

PubMed

Sundblad, Victoria; Quintar, Amado A; Morosi, Luciano G; Niveloni, Sonia I; Cabanne, Ana; Smecuol, Edgardo; Mauriño, Eduardo; Mariño, Karina V; Bai, Julio C; Maldonado, Cristina A; Rabinovich, Gabriel A

2018-01-01

Galectins, a family of animal lectins characterized by their affinity for N-acetyllactosamine-enriched glycoconjugates, modulate several immune cell processes shaping the course of innate and adaptive immune responses. Through interaction with a wide range of glycosylated receptors bearing complex branched N-glycans and core 2-O-glycans, these endogenous lectins trigger distinct signaling programs thereby controling immune cell activation, differentiation, recruitment and survival. Given the unique features of mucosal inflammation and the differential expression of galectins throughout the gastrointestinal tract, we discuss here key findings on the role of galectins in intestinal inflammation, particularly Crohn's disease, ulcerative colitis, and celiac disease (CeD) patients, as well as in murine models resembling these inflammatory conditions. In addition, we present new data highlighting the regulated expression of galectin-1 (Gal-1), a proto-type member of the galectin family, during intestinal inflammation in untreated and treated CeD patients. Our results unveil a substantial upregulation of Gal-1 accompanying the anti-inflammatory and tolerogenic response associated with gluten-free diet in CeD patients, suggesting a major role of this lectin in favoring resolution of inflammation and restoration of mucosal homeostasis. Thus, a coordinated network of galectins and their glycosylated ligands, exerting either anti-inflammatory or proinflammatory responses, may influence the interplay between intestinal epithelial cells and the highly specialized gut immune system in physiologic and pathologic settings.

Monoclonal antibody DS6 detects a tumor-associated sialoglycotope expressed on human serous ovarian carcinomas.

PubMed

Kearse, K P; Smith, N L; Semer, D A; Eagles, L; Finley, J L; Kazmierczak, S; Kovacs, C J; Rodriguez, A A; Kellogg-Wennerberg, A E

2000-12-15

A newly developed murine monoclonal antibody, DS6, immunohistochemically reacts with an antigen, CA6, that is expressed by human serous ovarian carcinomas but not by normal ovarian surface epithelium or mesothelium. CA6 has a limited distribution in normal adult tissues and is most characteristically detected in fallopian tube epithelium, inner urothelium and type 2 pneumocytes. Pre-treatment of tissue sections with either periodic acid or neuraminidase from Vibrio cholerae abolishes immunoreactivity with DS6, indicating that CA6 is a neuraminidase-sensitive and periodic acid-sensitive sialic acid glycoconjugate ("sialoglycotope"). SDS-PAGE of OVCAR5 cell lysates has revealed that the CA6 epitope is expressed on an 80 kDa non-disulfide-linked glycoprotein containing N-linked oligosaccharides. Two-dimensional non-equilibrium pH gradient gel electrophoresis indicates an isoelectric point of approximately 6.2 to 6.5. Comparison of the immunohistochemical distribution of CA6 in human serous ovarian adenocarcinomas has revealed similarities to that of CA125; however, distinct differences and some complementarity of antigen expression were revealed by double-label, 2-color immunohistochemical studies. The DS6-detected CA6 antigen appears to be distinct from other well-characterized tumor-associated antigens, including MUC1, CA125 and the histo-blood group-related antigens sLea, sLex and sTn. Copyright 2000 Wiley-Liss, Inc.

Structural analysis of O-polysaccharide chains extracted from different Salmonella Typhimurium strains.

PubMed

Micoli, Francesca; Ravenscroft, Neil; Cescutti, Paola; Stefanetti, Giuseppe; Londero, Silvia; Rondini, Simona; Maclennan, Calman A

2014-02-19

Salmonella Typhimurium is the major cause of invasive nontyphoidal Salmonella disease in Africa, with high mortality among children and HIV-infected individuals. Currently, no vaccine is available for use in humans. Antibodies directed against the O-polysaccharide of the lipopolysaccharide molecule of Salmonella mediate bacterial killing and are protective, and conjugation of the O-polysaccharide to a carrier protein represents a possible strategy for vaccine development. Here we have purified the O-polysaccharide from six different strains of S. Typhimurium and fully characterized them using analytical methods including HPLC-SEC, HPAEC-PAD, GC, GC-MS, 1D and 2D NMR spectroscopy. All the O-polysaccharide samples showed a similar bimodal molecular mass distribution, but differed with respect to the amount and position of O-acetylation and glucosylation. For some strains, O-acetyl groups were found not only on C-2 of abequose (factor 5 specificity), but also on C-2 and C-3 of rhamnose; glucose was found to be linked 1→4 or 1→6 to galactose in different amounts according to the strain of origin. This structural variability could have an impact on the immunogenicity of corresponding glycoconjugate vaccines and different strains need to be evaluated in order to identify the appropriate source of O-polysaccharide to use for the development of a candidate conjugate vaccine with broad coverage against S. Typhimurium. Copyright © 2013 Elsevier Ltd. All rights reserved.

Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells.

PubMed

Anastasia, Luigi; Holguera, Javier; Bianchi, Anna; D'Avila, Francesca; Papini, Nadia; Tringali, Cristina; Monti, Eugenio; Villar, Enrique; Venerando, Bruno; Muñoz-Barroso, Isabel; Tettamanti, Guido

2008-03-01

The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.

Towards defining the role of glycans as hardware in information storage and transfer: basic principles, experimental approaches and recent progress.

PubMed

Solís, D; Jiménez-Barbero, J; Kaltner, H; Romero, A; Siebert, H C; von der Lieth, C W; Gabius, H J

2001-01-01

The term 'code' in biological information transfer appears to be tightly and hitherto exclusively connected with the genetic code based on nucleotides and translated into functional activities via proteins. However, the recent appreciation of the enormous coding capacity of oligosaccharide chains of natural glycoconjugates has spurred to give heed to a new concept: versatile glycan assembly by the genetically encoded glycosyltransferases endows cells with a probably not yet fully catalogued array of meaningful messages. Enciphered by sugar receptors such as endogenous lectins the information of code words established by a series of covalently linked monosaccharides as letters for example guides correct intra- and intercellular routing of glycoproteins, modulates cell proliferation or migration and mediates cell adhesion. Evidently, the elucidation of the structural frameworks and the recognition strategies within the operation of the sugar code poses a fascinating conundrum. The far-reaching impact of this recognition mode on the level of cells, tissues and organs has fueled vigorous investigations to probe the subtleties of protein-carbohydrate interactions. This review presents information on the necessarily concerted approach using X-ray crystallography, molecular modeling, nuclear magnetic resonance spectroscopy, thermodynamic analysis and engineered ligands and receptors. This part of the treatise is flanked by exemplarily chosen insights made possible by these techniques. Copyright 2001 S. Karger AG, Basel

Carrier priming or suppression: understanding carrier priming enhancement of anti-polysaccharide antibody response to conjugate vaccines.

PubMed

Pobre, Karl; Tashani, Mohamed; Ridda, Iman; Rashid, Harunor; Wong, Melanie; Booy, Robert

2014-03-14

With the availability of newer conjugate vaccines, immunization schedules have become increasingly complex due to the potential for unpredictable immunologic interference such as 'carrier priming' and 'carrier induced epitopic suppression'. Carrier priming refers to an augmented antibody response to a carbohydrate portion of a glycoconjugate vaccine in an individual previously primed with the carrier protein. This review aims to provide a critical evaluation of the available data on carrier priming (and suppression) and conceptualize ways by which this phenomenon can be utilized to strengthen vaccination schedules. We conducted this literature review by searching well-known databases to date to identify relevant studies, then extracted and synthesized the data on carrier priming of widely used conjugate polysaccharide vaccines, such as, pneumococcal conjugate vaccine (PCV), meningococcal conjugate vaccine (MenCV) and Haemophilus influenzae type b conjugate vaccines (HibV). We found evidence of carrier priming with some conjugate vaccines, particularly HibV and PCV, in both animal and human models but controversy surrounds MenCV. This has implications for the immunogenicity of conjugate polysaccharide vaccines following the administration of tetanus-toxoid or diphtheria-toxoid containing vaccine (such as DTP). Available evidence supports a promising role for carrier priming in terms of maximizing the immunogenicity of conjugate vaccines and enhancing immunization schedule by making it more efficient and cost effective. Copyright © 2014 Elsevier Ltd. All rights reserved.

The disruption of GDP-fucose de novo biosynthesis suggests the presence of a novel fucose-containing glycoconjugate in Plasmodium asexual blood stages

PubMed Central

Sanz, Sílvia; López-Gutiérrez, Borja; Bandini, Giulia; Damerow, Sebastian; Absalon, Sabrina; Dinglasan, Rhoel R.; Samuelson, John; Izquierdo, Luis

2016-01-01

Glycosylation is an important posttranslational protein modification in all eukaryotes. Besides glycosylphosphatidylinositol (GPI) anchors and N-glycosylation, O-fucosylation has been recently reported in key sporozoite proteins of the malaria parasite. Previous analyses showed the presence of GDP-fucose (GDP-Fuc), the precursor for all fucosylation reactions, in the blood stages of Plasmodium falciparum. The GDP-Fuc de novo pathway, which requires the action of GDP-mannose 4,6-dehydratase (GMD) and GDP-L-fucose synthase (FS), is conserved in the parasite genome, but the importance of fucose metabolism for the parasite is unknown. To functionally characterize the pathway we generated a PfGMD mutant and analyzed its phenotype. Although the labelling by the fucose-binding Ulex europaeus agglutinin I (UEA-I) was completely abrogated, GDP-Fuc was still detected in the mutant. This unexpected result suggests the presence of an alternative mechanism for maintaining GDP-Fuc in the parasite. Furthermore, PfGMD null mutant exhibited normal growth and invasion rates, revealing that the GDP-Fuc de novo metabolic pathway is not essential for the development in culture of the malaria parasite during the asexual blood stages. Nonetheless, the function of this metabolic route and the GDP-Fuc pool that is generated during this stage may be important for gametocytogenesis and sporogonic development in the mosquito. PMID:27849032

ASGR1 and ASGR2, the Genes that Encode the Asialoglycoprotein Receptor (Ashwell Receptor), Are Expressed in Peripheral Blood Monocytes and Show Interindividual Differences in Transcript Profile

PubMed Central

Harris, Rebecca Louise; van den Berg, Carmen Wilma; Bowen, Derrick John

2012-01-01

Background. The asialoglycoprotein receptor (ASGPR) is a hepatic receptor that mediates removal of potentially hazardous glycoconjugates from blood in health and disease. The receptor comprises two proteins, asialoglycoprotein receptor 1 and 2 (ASGR1 and ASGR2), encoded by the genes ASGR1 and ASGR2. Design and Methods. Using reverse transcription amplification (RT-PCR), expression of ASGR1 and ASGR2 was investigated in human peripheral blood monocytes. Results. Monocytes were found to express ASGR1 and ASGR2 transcripts. Correctly spliced transcript variants encoding different isoforms of ASGR1 and ASGR2 were present in monocytes. The profile of transcript variants from both ASGR1 and ASGR2 differed among individuals. Transcript expression levels were compared with the hepatocyte cell line HepG2 which produces high levels of ASGPR. Monocyte transcripts were 4 to 6 orders of magnitude less than in HepG2 but nonetheless readily detectable using standard RT-PCR. The monocyte cell line THP1 gave similar results to monocytes harvested from peripheral blood, indicating it may provide a suitable model system for studying ASGPR function in this cell type. Conclusions. Monocytes transcribe and correctly process transcripts encoding the constituent proteins of the ASGPR. Monocytes may therefore represent a mobile pool of the receptor, capable of reaching sites remote from the liver. PMID:22919488

Comparison of some characteristics of aerobic granules and sludge flocs from sequencing batch reactors.

PubMed

Li, J; Garny, K; Neu, T; He, M; Lindenblatt, C; Horn, H

2007-01-01

Physical, chemical and biological characteristics were investigated for aerobic granules and sludge flocs from three laboratory-scale sequencing batch reactors (SBRs). One reactor was operated as normal SBR (N-SBR) and two reactors were operated as granular SBRs (G-SBR1 and G-SBR2). G-SBR1 was inoculated with activated sludge and G-SBR2 with granules from the municipal wastewater plant in Garching (Germany). The following major parameters and functions were measured and compared between the three reactors: morphology, settling velocity, specific gravity (SG), sludge volume index (SVI), specific oxygen uptake rate (SOUR), distribution of the volume fraction of extracellular polymeric substances (EPS) and bacteria, organic carbon and nitrogen removal. Compared with sludge flocs, granular sludge had excellent settling properties, good solid-liquid separation, high biomass concentration, simultaneous nitrification and denitrification. Aerobic granular sludge does not have a higher microbial activity and there are some problems including higher effluent suspended solids, lower ratio of VSS/SS and no nitrification at the beginning of cultivation. Measurement with CLSM and additional image analysis showed that EPS glycoconjugates build one main fraction inside the granules. The aerobic granules from G-SBR1 prove to be heavier, smaller and have a higher microbial activity compared with G-SBR2. Furthermore, the granules were more compact, with lower SVI and less filamentous bacteria.

Effect of OPC-12759 on EGF receptor activation, p44/p42 MAPK activity, and secretion in conjunctival goblet cells.

PubMed

Ríos, J David; Shatos, Marie A; Urashima, Hiroki; Dartt, Darlene A

2008-04-01

The purpose of the study was to determine if OPC-12759 stimulates secretion from conjunctival goblet cells in culture and if it activates the EGF receptor (EGFR) and p44/p42 mitogen-activated protein kinase (MAPK) to cause mucin secretion. Conjunctival goblet cells were cultured from pieces of male rat conjunctiva. OPC-12759 was added at increasing concentrations and for varying times to the cultured cells. The cholinergic agonist carbachol was used as a positive control. In selected experiments an inhibitor of the EGFR, AG1478, or an inhibitor of the kinase that activates MAPK, U0126, were added before OPC-12759. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay using the lectin UEA-1. Activation of the EGFR and MAPK were determined with Western blotting analysis using antibodies specific to the phosphorylated and the total amounts of these proteins. We found that OPC-12759 induced goblet cell secretion in a time- and concentration-dependent manner. Inhibition of the EGFR with AG1478 blocked secretion stimulated by OPC-12759. Inhibition of MAPK with U0126 also blocked secretion stimulated by OPC-12759. OPC-12759 increased the phosphorylation of the EGFR and MAPK in a time-dependent manner. We concluded that OPC-12759 stimulates secretion from cultured conjunctival goblet cells by activating the EGFR, which then induces MAPK activity.

The Macrophage Galactose-Type Lectin-1 (MGL1) Recognizes Taenia crassiceps Antigens, Triggers Intracellular Signaling, and Is Critical for Resistance to This Infection

PubMed Central

Montero-Barrera, Daniel; Valderrama-Carvajal, Héctor; Terrazas, César A.; Rojas-Hernández, Saúl; Ledesma-Soto, Yadira; Vera-Arias, Laura; Carrasco-Yépez, Maricela; Gómez-García, Lorena; Martínez-Saucedo, Diana; Becerra-Díaz, Mireya; Terrazas, Luis I.

2015-01-01

C-type lectins are multifunctional sugar-binding molecules expressed on dendritic cells (DCs) and macrophages that internalize antigens for processing and presentation. Macrophage galactose-type lectin 1 (MGL1) recognizes glycoconjugates expressing Lewis X structures which contain galactose residues, and it is selectively expressed on immature DCs and macrophages. Helminth parasites contain large amounts of glycosylated components, which play a role in the immune regulation induced by such infections. Macrophages from MGL1−/− mice showed less binding ability toward parasite antigens than their wild-type (WT) counterparts. Exposure of WT macrophages to T. crassiceps antigens triggered tyrosine phosphorylation signaling activity, which was diminished in MGL1−/− macrophages. Following T. crassiceps infection, MGL1−/− mice failed to produce significant levels of inflammatory cytokines early in the infection compared to WT mice. In contrast, MGL1−/− mice developed a Th2-dominant immune response that was associated with significantly higher parasite loads, whereas WT mice were resistant. Flow cytometry and RT-PCR analyses showed overexpression of the mannose receptors, IL-4Rα, PDL2, arginase-1, Ym1, and RELM-α on MGL1−/− macrophages. These studies indicate that MGL1 is involved in T. crassiceps recognition and subsequent innate immune activation and resistance. PMID:25664320

Membrane-associated mucins in normal human conjunctiva.

PubMed

Berry, M; Ellingham, R B; Corfield, A P

2000-02-01

To examine the presence of specific membrane-associated mucins in normal human conjunctiva. Glycoconjugates were extracted from membranes with two detergents: octylglucoside and Triton X114. Mucins were separated by cesium chloride density gradient centrifugation. Size was assessed by gel filtration on Sepharose CL2B and charge by ion-exchange chromatography on MonoQ. Cross reaction with antibodies against mucin gene products was assessed in blots of electrophoresis gels. Extraction of total tissue membranes yielded material with a buoyant density typical of mucins. Gel filtration showed material reacting with antimucin antibodies in a range of molecular sizes. Agarose electrophoresis confirmed the presence of MUC1 and MUC4 and the absence of MUC2 or MUC5AC. Isolation of membrane mucins by sequential, exhaustive extraction with octylglucoside followed by Triton X114 suggested the existence of mucins in different membrane environments. Reagents to carbohydrate epitopes revealed high mobility material, comigrating with MUC1 and MUC4. Low mobility membrane-bound mucins did not cross-react with any antibodies to mucin genes known to be expressed in human conjunctiva. Membrane-associated mucins are distinct from secreted mucins in normal human conjunctiva. MUC1 and MUC4 mature products decorate the membranes of conjunctival epithelial cells. Their segregation between octyl glucoside and the detergent and aqueous phases of Triton X114 suggests a variety of membrane anchoring modes.

Neutralization of B. anthracis toxins during ex vivo phagocytosis.

PubMed

Tarasenko, Olga; Scott, Ashley; Jones, April; Soderberg, Lee; Alusta, Pierre

2013-07-01

Glycoconjugates (GCs) are recognized as stimulation and signaling agents, affecting cell adhesion, activation, and growth of living organisms. Among GC targets, macrophages are considered ideal since they play a central role in inflammation and immune responses against foreign agents. In this context, we studied the effects of highly selective GCs in neutralizing toxin factors produced by B. anthracis during phagocytosis using murine macrophages. The effects of GCs were studied under three conditions: A) prior to, B) during, and C) following exposure of macrophages to B. anthracis individual toxin (protective antigen [PA], edema factor [EF], lethal factor [LF] or toxin complexes (PA-EF-LF, PA-EF, and PA-LF). We employed ex vivo phagocytosis and post-phagocytosis analysis including direct microscopic observation of macrophage viability, and macrophage activation. Our results demonstrated that macrophages are more prone to adhere to GC-altered PA-EF-LF, PA-EF, and PA-LF toxin complexes. This adhesion results in a higher phagocytosis rate and toxin complex neutralization during phagocytosis. In addition, GCs enhance macrophage viability, activate macrophages, and stimulate nitric oxide (NO) production. The present study may be helpful in identifying GC ligands with toxin-neutralizing and/or immunomodulating properties. In addition, our study could suggest GCs as new targets for existing vaccines and the prospective development of vaccines and immunomodulators used to combat the effects of B. anthracis.

Neutral glycoconjugated amide-based calix[4]arenes: complexation of alkali metal cations in water.

PubMed

Cindro, Nikola; Požar, Josip; Barišić, Dajana; Bregović, Nikola; Pičuljan, Katarina; Tomaš, Renato; Frkanec, Leo; Tomišić, Vladislav

2018-02-07

Cation complexation in water presents a unique challenge in calixarene chemistry, mostly due to the fact that a vast majority of calixarene-based cation receptors is not soluble in water or their solubility has been achieved by introducing functionalities capable of (de)protonation. Such an approach inevitably involves the presence of counterions which compete with target cations for the calixarene binding site, and also rather often requires the use of ion-containing buffer solutions in order to control the pH. Herein we devised a new strategy towards the solution of this problem, based on introducing carbohydrate units at the lower or upper rim of calix[4]arenes which comprise efficient cation binding sites. In this context, we prepared neutral, water-soluble receptors with secondary or tertiary amide coordinating groups, and studied their complexation with alkali metal cations in aqueous and methanol (for the comparison purpose) solutions. Complexation thermodynamics was quantitatively characterized by UV spectrometry and isothermal titration calorimetry, revealing that one of the prepared tertiary amide derivatives is capable of remarkably efficient (log K ≈ 5) and selective binding of sodium cations among alkali metal cations in water. Given the ease of the synthetic procedure used, and thus the variety of accessible analogues, this study can serve as a platform for the development of reagents for diverse purposes in aqueous media.

Fimbrial phase variation and systemic E. coli infection studied in the mouse peritonitis model.

PubMed

Nowicki, B; Vuopio-Varkila, J; Viljanen, P; Korhonen, T K; Mäkelä, P H

1986-08-01

Mouse peritonitis induced by intraperitoneal injection of a virulent (LD50 4 x 10(5) E. coli 018:K1:H7 strain isolated from neonatal meningitis was studied. These bacteria are capable of producing both type 1 and S fimbriae, binding to mannose or sialic acid containing glycoconjugates, respectively; the production of both fimbrial types is subject to phase variation. A broth culture of the bacteria was fractionated into subpopulations containing either type 1 or S fimbriae or neither (nonfimbriated cells), and each fraction, grown in broth to logarithmic growth phase, was used to infect groups of mice. The type 1 fraction was associated with decreased virulence as the fraction was eliminated rapidly without causing a progressive infection even at 10(6) bacteria/mouse, whereas both S and nonfimbriated cells started rapid multiplication in the peritoneal cavity and spread to the blood. In nonfibriated cells, however, S fimbriae production was induced at the same time so that at 1 h after injection, 60-70% of the bacteria in the peritoneal cavity and in the blood of the mice had S fimbriae. The injected S-fimbriated fraction remained completely S-fimbriated. Rapid induction of S fimbriae also took place in vitro when the nonfimbriated bacteria were grown in mouse serum or peritoneal fluid. Anti-S serum protected the mice from a lethal dose of S-fimbriated bacteria.

A toolbox of lectins for translating the sugar code: the galectin network in phylogenesis and tumors.

PubMed

Kaltner, H; Gabius, H-J

2012-04-01

Lectin histochemistry has revealed cell-type-selective glycosylation. It is under dynamic and spatially controlled regulation. Since their chemical properties allow carbohydrates to reach unsurpassed structural diversity in oligomers, they are ideal for high density information coding. Consequently, the concept of the sugar code assigns a functional dimension to the glycans of cellular glycoconjugates. Indeed, multifarious cell processes depend on specific recognition of glycans by their receptors (lectins), which translate the sugar-encoded information into effects. Duplication of ancestral genes and the following divergence of sequences account for the evolutionary dynamics in lectin families. Differences in gene number can even appear among closely related species. The adhesion/growth-regulatory galectins are selected as an instructive example to trace the phylogenetic diversification in several animals, most of them popular models in developmental and tumor biology. Chicken galectins are identified as a low-level-complexity set, thus singled out for further detailed analysis. The various operative means for establishing protein diversity among the chicken galectins are delineated, and individual characteristics in expression profiles discerned. To apply this galectin-fingerprinting approach in histopathology has potential for refining differential diagnosis and for obtaining prognostic assessments. On the grounds of in vitro work with tumor cells a strategically orchestrated co-regulation of galectin expression with presentation of cognate glycans is detected. This coordination epitomizes the far-reaching physiological significance of sugar coding.

The disruption of GDP-fucose de novo biosynthesis suggests the presence of a novel fucose-containing glycoconjugate in Plasmodium asexual blood stages.

PubMed

Sanz, Sílvia; López-Gutiérrez, Borja; Bandini, Giulia; Damerow, Sebastian; Absalon, Sabrina; Dinglasan, Rhoel R; Samuelson, John; Izquierdo, Luis

2016-11-16

Glycosylation is an important posttranslational protein modification in all eukaryotes. Besides glycosylphosphatidylinositol (GPI) anchors and N-glycosylation, O-fucosylation has been recently reported in key sporozoite proteins of the malaria parasite. Previous analyses showed the presence of GDP-fucose (GDP-Fuc), the precursor for all fucosylation reactions, in the blood stages of Plasmodium falciparum. The GDP-Fuc de novo pathway, which requires the action of GDP-mannose 4,6-dehydratase (GMD) and GDP-L-fucose synthase (FS), is conserved in the parasite genome, but the importance of fucose metabolism for the parasite is unknown. To functionally characterize the pathway we generated a PfGMD mutant and analyzed its phenotype. Although the labelling by the fucose-binding Ulex europaeus agglutinin I (UEA-I) was completely abrogated, GDP-Fuc was still detected in the mutant. This unexpected result suggests the presence of an alternative mechanism for maintaining GDP-Fuc in the parasite. Furthermore, PfGMD null mutant exhibited normal growth and invasion rates, revealing that the GDP-Fuc de novo metabolic pathway is not essential for the development in culture of the malaria parasite during the asexual blood stages. Nonetheless, the function of this metabolic route and the GDP-Fuc pool that is generated during this stage may be important for gametocytogenesis and sporogonic development in the mosquito.

Structural Analysis of N- and O-glycans Using ZIC-HILIC/Dialysis Coupled to NMR Detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qu, Yi; Feng, Ju; Deng, Shuang

2014-11-19

Protein glycosylation, an important and complex post-translational modification (PTM), is involved in various biological processes including the receptor-ligand and cell-cell interaction, and plays a crucial role in many biological functions. However, little is known about the glycan structures of important biological complex samples, and the conventional glycan enrichment strategy (i.e., size-exclusion column [SEC] separation,) prior to nuclear magnetic resonance (NMR) detection is time-consuming and tedious. In this study, we employed SEC, Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), and ZIC-HILIC coupled with dialysis strategies to enrich the glycopeptides from the pronase E digests of RNase B, followed by NMR analysis ofmore » the glycoconjugate. Our results suggest that the ZIC-HILIC enrichment coupled with dialysis is the most efficient, which was thus applied to the analysis of biological complex sample, the pronase E digest of the secreted proteins from the fungi Aspergillus niger. The NMR spectra revealed that the secreted proteins from A. niger contain both N-linked glycans with a high-mannose core and O-linked glycans bearing mannose and glucose with 1->3 and 1->6 linkages. In all, our study provides compelling evidence that ZIC-HILIC separation coupled to dialysis is superior to the commonly used SEC separation to prepare glycopeptides for the downstream NMR analysis, which could greatly facilitate the future NMR-based glycoproteomics research.« less

Role of IGF-1R in ameliorating apoptosis of GNE deficient cells.

PubMed

Singh, Reema; Chaudhary, Priyanka; Arya, Ranjana

2018-05-09

Sialic acids (SAs) are nine carbon acidic amino sugars, found at the outermost termini of glycoconjugates performing various physiological and pathological functions. SA synthesis is regulated by UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) that catalyzes rate limiting steps. Mutations in GNE result in rare genetic disorders, GNE myopathy and Sialuria. Recent studies indicate an alternate role of GNE in cell apoptosis and adhesion, besides SA biosynthesis. In the present study, using a HEK cell-based model for GNE myopathy, the role of Insulin-like Growth Factor Receptor (IGF-1R) as cell survival receptor protein was studied to counter the apoptotic effect of non-functional GNE. In the absence of functional GNE, IGF-1R was hyposialylated and transduced a downstream signal upon IGF-1 (IGF-1R ligand) treatment. IGF-1 induced activation of IGF-1R led to AKT (Protein Kinase B) phosphorylation that may phosphorylate BAD (BCL2 Associated Death Promoter) and its dissociation from BCL2 to prevent apoptosis. However, reduced ERK (Extracellular signal-regulated kinases) phosphorylation in GNE deficient cells after IGF-1 treatment suggests downregulation of the ERK pathway. A balance between the ERK and AKT pathways may determine the cell fate towards survival or apoptosis. Our study suggests that IGF-1R activation may rescue apoptotic cell death of GNE deficient cell lines and has potential as therapeutic target.

Histochemical study of lymphocystis disease in skin of gilthead seabream, Sparus aurata L.

PubMed

Sarasquete, C; González de Canales, M L; Arellano, J; Pérez-Prieto, S; García-Rosado, E; Borrego, J J

1998-01-01

A battery of horseradish peroxidase-conjugated lectins (Con A, WGA and DBA), as well as conventional histochemical techniques (PAS, saponification, Alcian Blue pH 0.1, 1, 2.5, chlorhydric hydrolisis, sialidase, Bromophenol blue, Tioglycollate reduction and Ferric-ferricyanide-FeIII) were used to study the content and distribution of carbohydrates, proteins and glycoconjugate sugar residues on the skin and on the lymphocystis-infected cells of gilthead seabream, Sparus aurata. Variable amounts of glycoproteins containing sialic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, mannose and/or glucose residues were observed in the cuticle and mucous cells of the corporal skin, tails and fins. Germinative and epithelial cells of the epidermis contained glycogen, proteins, carboxylated groups, as well as glycoproteins with mannose and/or glucose and N-acetyl-D-galactosamine residues. Hyaline capsule of the mature lymphocystis-infected cells was strongly stained with PAS, Alcian Blue (pH 0.5 and 2.5) and weakly positive with Alcian Blue (pH 1). Con A reacted with the granular cytoplasm, specially around hyaline capsule, and with the basophilic intracytoplasmic inclusions developed in mature lymphocystis-infected cells of Sparus aurata skin. These sugar residues (mannose and/or glucose), as well as N-acetyl-D-glucosamine and/or sialic acid and N-acetyl-D-galactosamine were not detected in the hyaline capsule of the lymphocystis disease.

Leishmania infantum Lipophosphoglycan-Deficient Mutants: A Tool to Study Host Cell-Parasite Interplay

PubMed Central

Lázaro-Souza, Milena; Matte, Christine; Lima, Jonilson B.; Arango Duque, Guillermo; Quintela-Carvalho, Graziele; de Carvalho Vivarini, Áislan; Moura-Pontes, Sara; Figueira, Cláudio P.; Jesus-Santos, Flávio H.; Gazos Lopes, Ulisses; Farias, Leonardo P.; Araújo-Santos, Théo; Descoteaux, Albert; Borges, Valéria M.

2018-01-01

Lipophosphoglycan (LPG) is the major surface glycoconjugate of metacyclic Leishmania promastigotes and is associated with virulence in various species of this parasite. Here, we generated a LPG-deficient mutant of Leishmania infantum, the foremost etiologic agent of visceral leishmaniasis in Brazil. The L. infantum LPG-deficient mutant (Δlpg1) was obtained by homologous recombination and complemented via episomal expression of LPG1 (Δlpg1 + LPG1). Deletion of LPG1 had no observable effect on parasite morphology or on the presence of subcellular organelles, such as lipid droplets. While both wild-type and add-back parasites reached late phase in axenic cultures, the growth of Δlpg1 parasites was delayed. Additionally, the deletion of LPG1 impaired the outcome of infection in murine bone marrow-derived macrophages. Although no significant differences were observed in parasite load after 4 h of infection, survival of Δlpg1 parasites was significantly reduced at 72 h post-infection. Interestingly, L. infantum LPG-deficient mutants induced a strong NF-κB-dependent activation of the inducible nitric oxide synthase (iNOS) promoter compared to wild type and Δlpg1 + LPG1 parasites. In conclusion, the L. infantum Δlpg1 mutant constitutes a powerful tool to investigate the role(s) played by LPG in host cell-parasite interactions. PMID:29675001

New glycoside derivatives of carnosine and analogs resistant to carnosinase hydrolysis: synthesis and characterization of their copper(II) complexes.

PubMed

Lanza, Valeria; Bellia, Francesco; D'Agata, Roberta; Grasso, Giuseppe; Rizzarelli, Enrico; Vecchio, Graziella

2011-02-01

Carnosine (β-alanyl-L-histidine) is an endogenous dipeptide widely and abundantly distributed in muscle and nervous tissues of several animal species. Many functions have been proposed for this compound, such as antioxidant and metal ion-chelator properties. However, the main limitation on therapeutic use of carnosine on pathologies related to increased oxidative stress and/or metal ion dishomeostasis is associated with the hydrolysis by the specific dipeptidase carnosinase. Several attempts have been made to overcome this limitation. On this basis, we functionalized carnosine and its amide derivative with small sugars such as glucose and lactose. The resistance of these derivatives to the carnosinase hydrolysis was tested and compared with that of carnosine. We found that the glycoconjugation protects the dipeptide moiety from carnosinase hydrolysis, thus potentially improving the availability of carnosine. The copper(II) binding properties of all the new synthesized compounds were investigated by spectroscopic (UV-Visible and circular dichroism) and ESI-MS studies. Particularly, the new family of amide derivatives that are not significantly hydrolyzed by carnosinase is a very promising class of carnosine derivatives. The sugar moiety can act as a recognition element. These new derivatives are potentially able to act as chelating agents in the development of clinical approaches for the regulation of metal homeostasis in the field of medicinal inorganic chemistry. Copyright © 2010 Elsevier Inc. All rights reserved.

Carbohydrate-based heteronuclear complexes as topoisomerase Iα inhibitor: approach toward anticancer chemotherapeutics.

PubMed

Afzal, Mohd; Al-Lohedan, Hamad A; Usman, Mohammad; Tabassum, Sartaj

2018-04-18

Due to the critical role of cellular enzymes necessary for cell proliferation by deciphering topological hurdles in the process of DNA replication, topoisomerases have been one of the major targets in the anticancer drug development area. A need, therefore, arises for new metallodrugs that specifically recognizes DNA and inhibits the activity of topoisomerase enzymes, herein, we report the synthesis and characterization of new metal-based glycoconjugate entities containing heterobimetallic core Cu II -Sn IV (1) and Ni II -Sn IV (2) derived from N-glycoside ligand (L). The optimized structure of complex 1 and other significant vibrational modes have been explained using dispersion corrected B3LYP/DFT calculations. In vitro DNA binding profile of the L and both the complexes 1 and 2 were done by various biophysical studies. Complex 1 breaks pBR322 DNA via a hydrolytic means which was validated by T4 DNA enzymatic assay. To get a mechanistic insight of mode of action topoisomerase I (Topo I) inhibition assay was carried out. Also, we have taken the help of molecular modeling studies in accordance with experimental findings. In vitro cytotoxicity of the complex 1 was evaluated against a panel of cancer cells which exhibited remarkably good anticancer activity (GI 50 values <10 μg/ml). Moreover, intracellular localization of the complex 1 was visualized by confocal microscopy against HeLa cells.

Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection

PubMed Central

Nogueira, Paula M.; Assis, Rafael R.; Torrecilhas, Ana C.; Saraiva, Elvira M.; Pessoa, Natália L.; Campos, Marco A.; Marialva, Eric F.; Ríos-Velasquez, Cláudia M.; Pessoa, Felipe A.; Secundino, Nágila F.; Rugani, Jerônimo N.; Nieves, Elsa; Turco, Salvatore J.; Melo, Maria N.

2016-01-01

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly. PMID:27508930

Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection.

PubMed

Nogueira, Paula M; Assis, Rafael R; Torrecilhas, Ana C; Saraiva, Elvira M; Pessoa, Natália L; Campos, Marco A; Marialva, Eric F; Ríos-Velasquez, Cláudia M; Pessoa, Felipe A; Secundino, Nágila F; Rugani, Jerônimo N; Nieves, Elsa; Turco, Salvatore J; Melo, Maria N; Soares, Rodrigo P

2016-08-01

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.

Identification and Characterization of C-type Lectins in Ostrinia furnacalis (Lepidoptera: Pyralidae)

PubMed Central

Shen, Dongxu; Wang, Lei; Ji, Jiayue; Liu, Qizhi; An, Chunju

2018-01-01

Abstract C-type lectins (CTLs) are a large family of calcium-dependent carbohydrate-binding proteins. They function primarily in cell adhesion and immunity by recognizing various glycoconjugates. We identified 14 transcripts encoding proteins with one or two CTL domains from the transcriptome from Asian corn borer, Ostrinia furnacalis (Guenée; Lepidoptera: Pyralidae). Among them, five (OfCTL-S1 through S5) only contain one CTL domain, the remaining nine (OfIML-1 through 9) have two tandem CTL domains. Five CTL-Ss and six OfIMLs have a signal peptide are likely extracellular while another two OfIMLs might be cytoplasmic. Phylogenetic analysis indicated that OfCTL-Ss had 1:1 orthologs in Lepidoptera, Diptera, Coleoptera and Hymenoptera species, but OfIMLs only clustered with immulectins (IMLs) from Lepidopteran. Structural modeling revealed that the 22 CTL domains adopt a similar double-loop fold consisting of β-sheets and α-helices. The key residues for calcium-dependent or independent binding of specific carbohydrates by CTL domains were predicted with homology modeling. Expression profiles assay showed distinct expression pattern of 14 CTLs: the expression and induction were related to the developmental stages and infected microorganisms. Overall, our work including the gene identification, sequence alignment, phylogenetic analysis, structural modeling, and expression profile assay would provide a valuable basis for the further functional studies of O. furnacalis CTLs. PMID:29718486

Synthesis and Immunological Properties of N-Modified GM3 Antigens as Therapeutic Cancer Vaccines

PubMed Central

Pan, Yanbin; Chefalo, Peter; Nagy, Nancy; Harding, Clifford; Guo, Zhongwu

2011-01-01

The problem of immunotolerance to GM3, an important tumor-associated trisaccharide antigen, seriously hinders its usage in cancer vaccine development. To solve this problem, the keyhole limpet hemocyanin (KLH) conjugates of a series of GM3 derivatives were synthesized and screened as therapeutic cancer vaccines. First, the β-linked anomeric azides of differently N-acylated GM3 analogs were prepared by a highly convergent procedure. Next, a pentenoyl group was linked to the reducing end of the carbohydrate antigens following selective reduction of the azido group. The linker was thereafter ozonolyzed to give an aldehyde functionality permitting the conjugation of the antigens to KLH via reductive amination. Finally, the immunological properties of the resultant glycoconjugates were studied in C57BL/6 mice by assessing the titers of specific antibodies induced by the GM3 analogs. While KLH-GM3 elicited low levels of immune response, the KLH conjugates of N-propionyl, N-butanoyl, N-iso-butanoyl and N-phenylacetyl GM3’s induced robust immune reactions with antibodies of multiple isotypes, indicating significantly improved and T-cell dependent immune responses that lead to isotype switching, affinity maturation and the induction of immunological ‘memory’. It was suggested that GM3PhAc-KLH is a promising vaccine candidate for glycoengineered immunotherapy of cancer with GM3 as the primary target. PMID:15689172

Safety and immunogenicity of one dose of MenACWY-CRM, an investigational quadrivalent meningococcal glycoconjugate vaccine, when administered to adolescents concomitantly or sequentially with Tdap and HPV vaccines.

PubMed

Arguedas, A; Soley, C; Loaiza, C; Rincon, G; Guevara, S; Perez, A; Porras, W; Alvarado, O; Aguilar, L; Abdelnour, A; Grunwald, U; Bedell, L; Anemona, A; Dull, P M

2010-04-19

This Phase III study evaluates an investigational quadrivalent meningococcal CRM(197) conjugate vaccine, MenACWY-CRM (Novartis Vaccines), when administered concomitantly or sequentially with two other recommended adolescent vaccines; combined tetanus, reduced diphtheria and acellular pertussis (Tdap), and human papillomavirus (HPV) vaccine. In this single-centre study, 1620 subjects 11-18 years of age, were randomized to three groups (1:1:1) to receive MenACWY-CRM concomitantly or sequentially with Tdap and HPV. Meningococcal serogroup-specific serum bactericidal assay using human complement (hSBA), and antibodies to Tdap antigens and HPV virus-like particles were determined before and 1 month after study vaccinations. Proportions of subjects with hSBA titres > or =1:8 for all four meningococcal serogroups (A, C, W-135, Y) were non-inferior for both concomitant and sequential administration. Immune responses to Tdap and HPV antigens were comparable when these vaccines were given alone or concomitantly with MenACWY-CRM. All vaccines were well tolerated; concomitant or sequential administration did not increase reactogenicity. MenACWY-CRM was well tolerated and immunogenic in subjects 11-18 years of age, with comparable immune responses to the four serogroups when given alone or concomitantly with Tdap or HPV antigens. This is the first demonstration that these currently recommended adolescent vaccines could be administered concomitantly without causing increased reactogenicity. Copyright 2010 Elsevier Ltd. All rights reserved.

Biofilm formation and interspecies interactions in mixed cultures of thermo-acidophilic archaea Acidianus spp. and Sulfolobus metallicus.

PubMed

Castro, Camila; Zhang, Ruiyong; Liu, Jing; Bellenberg, Sören; Neu, Thomas R; Donati, Edgardo; Sand, Wolfgang; Vera, Mario

2016-09-01

The understanding of biofilm formation by bioleaching microorganisms is of great importance for influencing mineral dissolution rates and to prevent acid mine drainage (AMD). Thermo-acidophilic archaea such as Acidianus, Sulfolobus and Metallosphaera are of special interest due to their ability to perform leaching at high temperatures, thereby enhancing leaching rates. In this work, leaching experiments and visualization by microscopy of cell attachment and biofilm formation patterns of the crenarchaeotes Sulfolobus metallicus DSM 6482(T) and the Acidianus isolates DSM 29038 and DSM 29099 in pure and mixed cultures on sulfur or pyrite were studied. Confocal laser scanning microscopy (CLSM) combined with fluorescent dyes as well as fluorescently labeled lectins were used to visualize different components (e.g. DNA, proteins or glycoconjugates) of the aforementioned species. The data indicate that cell attachment and the subsequently formed biofilms were species- and substrate-dependent. Pyrite leaching experiments coupled with pre-colonization and further inoculation with a second species suggest that both species may negatively influence each other during pyrite leaching with respect to initial attachment and pyrite dissolution rates. In addition, the investigation of binary biofilms on pyrite showed that both species were heterogeneously distributed on pyrite surfaces in the form of individual cells or microcolonies. Physical contact between the two species seems to occur, as revealed by specific lectins able to specifically bind single species within mixed cultures. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism.

PubMed

Laitinen, Teresa; Morreel, Kris; Delhomme, Nicolas; Gauthier, Adrien; Schiffthaler, Bastian; Nickolov, Kaloian; Brader, Günter; Lim, Kean-Jin; Teeri, Teemu H; Street, Nathaniel R; Boerjan, Wout; Kärkönen, Anna

2017-07-01

Apoplastic events such as monolignol oxidation and lignin polymerization are difficult to study in intact trees. To investigate the role of apoplastic hydrogen peroxide (H 2 O 2 ) in gymnosperm phenolic metabolism, an extracellular lignin-forming cell culture of Norway spruce ( Picea abies ) was used as a research model. Scavenging of apoplastic H 2 O 2 by potassium iodide repressed lignin formation, in line with peroxidases activating monolignols for lignin polymerization. Time-course analyses coupled to candidate substrate-product pair network propagation revealed differential accumulation of low-molecular-weight phenolics, including (glycosylated) oligolignols, (glycosylated) flavonoids, and proanthocyanidins, in lignin-forming and H 2 O 2 -scavenging cultures and supported that monolignols are oxidatively coupled not only in the cell wall but also in the cytoplasm, where they are coupled to other monolignols and proanthocyanidins. Dilignol glycoconjugates with reduced structures were found in the culture medium, suggesting that cells are able to transport glycosylated dilignols to the apoplast. Transcriptomic analyses revealed that scavenging of apoplastic H 2 O 2 resulted in remodulation of the transcriptome, with reduced carbon flux into the shikimate pathway propagating down to monolignol biosynthesis. Aggregated coexpression network analysis identified candidate enzymes and transcription factors for monolignol oxidation and apoplastic H 2 O 2 production in addition to potential H 2 O 2 receptors. The results presented indicate that the redox state of the apoplast has a profound influence on cellular metabolism. © 2017 American Society of Plant Biologists. All Rights Reserved.

Trypanosoma cruzi TcSMUG L-surface Mucins Promote Development and Infectivity in the Triatomine Vector Rhodnius prolixus

PubMed Central

Gonzalez, Marcelo S.; Souza, Marcela S.; Garcia, Eloi S.; Nogueira, Nadir F. S.; Mello, Cícero B.; Cánepa, Gaspar E.; Bertotti, Santiago; Durante, Ignacio M.; Azambuja, Patrícia; Buscaglia, Carlos A.

2013-01-01

Background TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector. Methodology and Principal Findings Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy. Conclusion and Significance Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus. PMID:24244781

High-Throughput Analysis and Automation for Glycomics Studies.

PubMed

Shubhakar, Archana; Reiding, Karli R; Gardner, Richard A; Spencer, Daniel I R; Fernandes, Daryl L; Wuhrer, Manfred

This review covers advances in analytical technologies for high-throughput (HTP) glycomics. Our focus is on structural studies of glycoprotein glycosylation to support biopharmaceutical realization and the discovery of glycan biomarkers for human disease. For biopharmaceuticals, there is increasing use of glycomics in Quality by Design studies to help optimize glycan profiles of drugs with a view to improving their clinical performance. Glycomics is also used in comparability studies to ensure consistency of glycosylation both throughout product development and between biosimilars and innovator drugs. In clinical studies there is as well an expanding interest in the use of glycomics-for example in Genome Wide Association Studies-to follow changes in glycosylation patterns of biological tissues and fluids with the progress of certain diseases. These include cancers, neurodegenerative disorders and inflammatory conditions. Despite rising activity in this field, there are significant challenges in performing large scale glycomics studies. The requirement is accurate identification and quantitation of individual glycan structures. However, glycoconjugate samples are often very complex and heterogeneous and contain many diverse branched glycan structures. In this article we cover HTP sample preparation and derivatization methods, sample purification, robotization, optimized glycan profiling by UHPLC, MS and multiplexed CE, as well as hyphenated techniques and automated data analysis tools. Throughout, we summarize the advantages and challenges with each of these technologies. The issues considered include reliability of the methods for glycan identification and quantitation, sample throughput, labor intensity, and affordability for large sample numbers.

Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5).

PubMed

Aspeborg, Henrik; Coutinho, Pedro M; Wang, Yang; Brumer, Harry; Henrissat, Bernard

2012-09-20

The large Glycoside Hydrolase family 5 (GH5) groups together a wide range of enzymes acting on β-linked oligo- and polysaccharides, and glycoconjugates from a large spectrum of organisms. The long and complex evolution of this family of enzymes and its broad sequence diversity limits functional prediction. With the objective of improving the differentiation of enzyme specificities in a knowledge-based context, and to obtain new evolutionary insights, we present here a new, robust subfamily classification of family GH5. About 80% of the current sequences were assigned into 51 subfamilies in a global analysis of all publicly available GH5 sequences and associated biochemical data. Examination of subfamilies with catalytically-active members revealed that one third are monospecific (containing a single enzyme activity), although new functions may be discovered with biochemical characterization in the future. Furthermore, twenty subfamilies presently have no characterization whatsoever and many others have only limited structural and biochemical data. Mapping of functional knowledge onto the GH5 phylogenetic tree revealed that the sequence space of this historical and industrially important family is far from well dispersed, highlighting targets in need of further study. The analysis also uncovered a number of GH5 proteins which have lost their catalytic machinery, indicating evolution towards novel functions. Overall, the subfamily division of GH5 provides an actively curated resource for large-scale protein sequence annotation for glycogenomics; the subfamily assignments are openly accessible via the Carbohydrate-Active Enzyme database at http://www.cazy.org/GH5.html.

Transition state-based ST6Gal I inhibitors: Mimicking the phosphodiester linkage with a triazole or carbamate through an enthalpy-entropy compensation.

PubMed

Montgomery, Andrew P; Skropeta, Danielle; Yu, Haibo

2017-10-31

Human β-galactoside α-2,6-sialyltransferase I (ST6Gal I) catalyses the synthesis of sialylated glycoconjugates. Overexpression of ST6Gal I is observed in many cancers, where it promotes metastasis through altered cell surface sialylation. A wide range of sialyltransferase inhibitors have been developed, with analogues structurally similar to the transition state exhibiting the highest inhibitory activity. To improve synthetic accessibility and pharmacokinetics of previously reported inhibitors, the replacement of the charged phosphodiester linker with a potential neutral isostere such as a carbamate or a 1,2,3-triazole has been investigated. Extensive molecular dynamics simulations have demonstrated that compounds with the alternate linkers could maintain key interactions with the human ST6Gal I active site, demonstrating the potential of a carbamate or a 1,2,3-triazole as a phosphodiester isostere. Free energy perturbation calculations provided energetic evidence suggesting that the carbamate and 1,2,3-triazole were slightly more favourable than the phosphodiester. Further exploration with free energy component, quasi-harmonic and cluster analysis suggested that there is an enthalpy-entropy compensation accounting for the replacement of the flexible charged phosphodiester with a neutral and rigid isostere. Overall, these simulations provide a strong rationale for the use of a carbamate or 1,2,3-triazole as a phosphodiester isostere in the development of novel inhibitors of human ST6Gal I.

Expression of human β-N-acetylhexosaminidase B in yeast eases the search for selective inhibitors.

PubMed

Krejzová, Jana; Kulik, Natallia; Slámová, Kristýna; Křen, Vladimír

2016-07-01

Human lysosomal β-N-acetylhexosaminidases from the family 20 of glycoside hydrolases are dimeric enzymes catalysing the cleavage of terminal β-N-acetylglucosamine and β-N-acetylgalactosamine residues from a broad spectrum of glycoconjugates. Here, we present a facile, robust, and cost-effective extracellular expression of human β-N-acetylhexosaminidase B in Pichia pastoris KM71H strain. The prepared Hex B was purified in a single step with 33% yield obtaining 10mg of the pure enzyme per 1L of the culture media. The enzyme was used in the inhibition assays with the known mechanism-based inhibitor NAG-thiazoline and a wide variety of its derivatives in the search for specific inhibitors of the human GH20 β-N-acetylhexosaminidases over the human GH84 β-N-acetylglucosaminidase, which was expressed, purified and used in the inhibition experiments as well. Moreover, enzyme-inhibitor complexes were analysed employing computational tools in order to reveal the structural basis of the results of the inhibition assays, showing the importance of water-mediated interactions between the enzyme and respective ligands. The presented method for the heterologous expression of human Hex B is robust, it significantly reduces the costs and equipment demands in comparison to the expression in mammalian cell lines. This will enhance accessibility of this human enzyme to the broad scientific community and may speed up the research of specific inhibitors of this physiologically important glycosidase family. Copyright © 2016 Elsevier Inc. All rights reserved.

A Key Role for Apoplastic H2O2 in Norway Spruce Phenolic Metabolism1[OPEN

PubMed Central

Laitinen, Teresa

2017-01-01

Apoplastic events such as monolignol oxidation and lignin polymerization are difficult to study in intact trees. To investigate the role of apoplastic hydrogen peroxide (H2O2) in gymnosperm phenolic metabolism, an extracellular lignin-forming cell culture of Norway spruce (Picea abies) was used as a research model. Scavenging of apoplastic H2O2 by potassium iodide repressed lignin formation, in line with peroxidases activating monolignols for lignin polymerization. Time-course analyses coupled to candidate substrate-product pair network propagation revealed differential accumulation of low-molecular-weight phenolics, including (glycosylated) oligolignols, (glycosylated) flavonoids, and proanthocyanidins, in lignin-forming and H2O2-scavenging cultures and supported that monolignols are oxidatively coupled not only in the cell wall but also in the cytoplasm, where they are coupled to other monolignols and proanthocyanidins. Dilignol glycoconjugates with reduced structures were found in the culture medium, suggesting that cells are able to transport glycosylated dilignols to the apoplast. Transcriptomic analyses revealed that scavenging of apoplastic H2O2 resulted in remodulation of the transcriptome, with reduced carbon flux into the shikimate pathway propagating down to monolignol biosynthesis. Aggregated coexpression network analysis identified candidate enzymes and transcription factors for monolignol oxidation and apoplastic H2O2 production in addition to potential H2O2 receptors. The results presented indicate that the redox state of the apoplast has a profound influence on cellular metabolism. PMID:28522458

Glycans: bioactive signals decoded by lectins.

PubMed

Gabius, Hans-Joachim

2008-12-01

The glycan part of cellular glycoconjugates affords a versatile means to build biochemical signals. These oligosaccharides have an exceptional talent in this respect. They surpass any other class of biomolecule in coding capacity within an oligomer (code word). Four structural factors account for this property: the potential for variability of linkage points, anomeric position and ring size as well as the aptitude for branching (first and second dimensions of the sugar code). Specific intermolecular recognition is favoured by abundant potential for hydrogen/co-ordination bonds and for C-H/pi-interactions. Fittingly, an array of protein folds has developed in evolution with the ability to select certain glycans from the natural diversity. The thermodynamics of this reaction profits from the occurrence of these ligands in only a few energetically favoured conformers, comparing favourably with highly flexible peptides (third dimension of the sugar code). Sequence, shape and local aspects of glycan presentation (e.g. multivalency) are key factors to regulate the avidity of lectin binding. At the level of cells, distinct glycan determinants, a result of enzymatic synthesis and dynamic remodelling, are being defined as biomarkers. Their presence gains a functional perspective by co-regulation of the cognate lectin as effector, for example in growth regulation. The way to tie sugar signal and lectin together is illustrated herein for two tumour model systems. In this sense, orchestration of glycan and lectin expression is an efficient means, with far-reaching relevance, to exploit the coding potential of oligosaccharides physiologically and medically.

Analysis of Bufo arenarum oviductal secretion during the sexual cycle.

PubMed

Crespo, Claudia A; Ramos, Inés; Medina, Marcela F; Fernández, Silvia N

2009-11-01

SummaryBufo arenarum oocytes are oviposited surrounded by jelly coats, one component of the extracellular matrix required for fertilization. The secretion, released to the oviductal lumen, was analysed by SDS-PAGE. The coomassie blue staining evidenced an electrophoretic pattern with molecules ranging between 300 and 19 kDa that showed variations in their secretion profiles during the sexual cycle. In the preovulatory period the densitometric analysis showed the presence of nine peaks with marked predominance of the 74 kDa molecule. Once ovulation has occurred, the jelly coats become arranged around the oocytes during their transit throughout the oviductal pars convoluta (PC), revealing the addition of three proteins only observed during this period, which suggests a differential secretion. Some of these proteins could not diffuse under any extraction treatment, indicating for them a structural or in situ function. Proteins of low molecular mass diffused totally while others showed a partial diffusing capacity. After ovulation a marked decrease in the relative amount of all the proteins released to the lumen, especially the 74 kDa protein, could be detected. During this period, unlike the other stages of the sexual cycle, a differential secretion pattern was observed along the PC. The histochemical analysis performed during the ovulatory period showed the presence of glycoconjugates including both acidic and neutral groups. The present results are in agreement with previous ultrastructural and histochemical studies that describe the role of Bufo arenarum jelly coats in fertilization.

Activity-Based Profiling of a Physiologic Aglycone Library Reveals Sugar Acceptor Promiscuity of Family 1 UDP-Glucosyltransferases from Grape1[W

PubMed Central

Bönisch, Friedericke; Frotscher, Johanna; Stanitzek, Sarah; Rühl, Ernst; Wüst, Matthias; Bitz, Oliver; Schwab, Wilfried

2014-01-01

Monoterpenols serve various biological functions and accumulate in grape (Vitis vinifera), where a major fraction occurs as nonvolatile glycosides. We have screened the grape genome for sequences with similarity to terpene URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASES (UGTs) from Arabidopsis (Arabidopsis thaliana). A ripening-related expression pattern was shown for three candidates by spatial and temporal expression analyses in five grape cultivars. Transcript accumulation correlated with the production of monoterpenyl β-d-glucosides in grape exocarp during ripening and was low in vegetative tissue. Targeted functional screening of the recombinant UGTs for their biological substrates was performed by activity-based metabolite profiling (ABMP) employing a physiologic library of aglycones built from glycosides isolated from grape. This approach led to the identification of two UDP-glucose:monoterpenol β-d-glucosyltransferases. Whereas VvGT14a glucosylated geraniol, R,S-citronellol, and nerol with similar efficiency, the three allelic forms VvGT15a, VvGT15b, and VvGT15c preferred geraniol over nerol. Kinetic resolution of R,S-citronellol and R,S-linalool was shown for VvGT15a and VvGT14a, respectively. ABMP revealed geraniol as the major biological substrate but also disclosed that these UGTs may add to the production of further glycoconjugates in planta. ABMP of aglycone libraries provides a versatile tool to uncover novel biologically relevant substrates of small-molecule glycosyltransferases that often show broad sugar acceptor promiscuity. PMID:25073706

Silencing of sterol glycosyltransferases modulates the withanolide biosynthesis and leads to compromised basal immunity of Withania somnifera

PubMed Central

Singh, Gaurav; Tiwari, Manish; Singh, Surendra Pratap; Singh, Surendra; Trivedi, Prabodh Kumar; Misra, Pratibha

2016-01-01

Sterol glycosyltransferases (SGTs) catalyse transfer of glycon moiety to sterols and their related compounds to produce diverse glyco-conjugates or steryl glycosides with different biological and pharmacological activities. Functional studies of SGTs from Withania somnifera indicated their role in abiotic stresses but details about role under biotic stress are still unknown. Here, we have elucidated the function of SGTs by silencing SGTL1, SGTL2 and SGTL4 in Withania somnifera. Down-regulation of SGTs by artificial miRNAs led to the enhanced accumulation of withanolide A, withaferin A, sitosterol, stigmasterol and decreased content of withanoside V in Virus Induced Gene Silencing (VIGS) lines. This was further correlated with increased expression of WsHMGR, WsDXR, WsFPPS, WsCYP710A1, WsSTE1 and WsDWF5 genes, involved in withanolide biosynthesis. These variations of withanolide concentrations in silenced lines resulted in pathogen susceptibility as compared to control plants. The infection of Alternaria alternata causes increased salicylic acid, callose deposition, superoxide dismutase and H2O2 in aMIR-VIGS lines. The expression of biotic stress related genes, namely, WsPR1, WsDFS, WsSPI and WsPR10 were also enhanced in aMIR-VIGS lines in time dependent manner. Taken together, our observations revealed that a positive feedback regulation of withanolide biosynthesis occurred by silencing of SGTLs which resulted in reduced biotic tolerance. PMID:27146059

Decreased sialidase activity in alveolar macrophages of guinea pigs exposed to coal mine dust.

PubMed Central

Terzidis-Trabelsi, H; Lefèvre, J P; Bignon, J; Lambré, C R

1992-01-01

The origin of immune dysfunctions that are observed in pneumoconiotic miners still remains unknown. There is evidence that the carbohydrate moiety of membrane glycoconjugates is of primary importance in many functions of immunocompetent cells. The glycosylation, and especially the sialylation level of membrane components of various lymphocyte and macrophage subsets, vary depending on the state of cellular differentiation and activation. Sialidases, which may regulate the amount of sialic acids exposed on the cell membrane, can thus be considered as immunoregulatory enzymes. In this report, the sialidase activity has been measured in alveolar macrophages (AM) and in cell-free bronchoalveolar lavage fluid (BALF) from guinea pigs exposed for 4 months to coal mine dust at a concentration of 300 mg/m3. The samples were collected by bronchoalveolar lavage 2 months after cessation of exposure. The sialidase activity in the cell-free fluid and in the purified alveolar macrophages showed a 10-fold decrease (p less than 0.001). Kinetic parameters of the enzyme such as Km and optimum pH did not change. This changed activity was specific for sialidase, as two other lysosomal glycosidases, beta-galactosidase and N-acetylglucosaminidase, showed unchanged activities. These results suggest the possibility that, by inducing a decreased sialidase activity, exposure to coal mine dust may lead to a modified expression of AM membrane-associated sialic acids giving rise to altered immune functions (i. e., phagocytosis, antigen processing, response to cytokines, etc.). PMID:1396442

Receptors and aging: structural selectivity of the rhamnose-receptor on fibroblasts as shown by Ca(2+)-mobilization and gene-expression profiles.

PubMed

Faury, G; Molinari, J; Rusova, E; Mariko, B; Raveaud, S; Huber, P; Velebny, V; Robert, A M; Robert, L

2011-01-01

Qualitative and quantitative modifications of receptors were shown to play a key role in cell and tissue aging. We recently described the properties of a rhamnose-recognizing receptor on fibroblasts involved in the mediation of age-dependent functions of these cells. Using Ca(2+)-mobilization and DNA-microarrays we could show in the presence of rhamnose-rich oligo- and polysaccharides (RROPs) Ca(2+)-mobilization and changes in gene regulation. Here, we compared the effects of several RROPs, differing in their carbohydrate sequence and molecular weights, in normal human dermal fibroblasts (NHDFs). It appeared that different structural features were required for maximal effects on Ca(2+)-mobilization and gene-expression profiles. Maximal effect on Ca(2+) influx and intracellular free calcium regulation was exhibited by RROP-1, a 50 kDa average molecular weight polysaccharide, and RROP-3, a 5 kDa average molecular weight oligosaccharide with a different carbohydrate sequence. Maximal effect on gene-expression profiles was obtained with RROP-3. These results suggest the possibility of several different transmission pathways from the rhamnose-receptor to intracellular targets, differentially affecting these two intracellular functions, with potential consequences on aging. Although of only relative specificity, this receptor site exhibits a high affinity for rhamnose, absent from vertebrate glycoconjugates. The rhamnose-receptor might well represent an evolutionary conserved conformation of a prokaryote lectin. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Studies on the Selectivity Between Nickel-Catalyzed 1,2-Cis-2-Amino Glycosylation of Hydroxyl Groups of Thioglycoside Acceptors with C(2)-Substituted Benzylidene N-Phenyl Trifluoroacetimidates and Intermolecular Aglycon Transfer of the Sulfide Group

PubMed Central

Yu, Fei; Nguyen, Hien M.

2012-01-01

The stereoselective synthesis of saccharide thioglycosides containing 1,2-cis-2-amino glycosidic linkages is challenging. In addition to the difficulties associated with achieving high α-selectivity in the formation of 1,2-cis-2-amino glycosidic bonds, the glycosylation reaction is hampered by undesired transfer of the anomeric sulfide group from the glycosyl acceptor to the glycosyl donor. Overcoming these obstacles will pave the way for the preparation of oligosaccharides and glycoconjugates bearing the 1,2-cis-2-amino glycosidic linkages because the saccharide thioglycosides obtained can serve as donors for another coupling iteration. This approach streamlines selective deprotection and anomeric derivatization steps prior to the subsequent coupling event. We have developed an efficient approach for the synthesis of highly yielding and α-selective saccharide thioglycosides containing 1,2-cis-2-amino glycosidic bonds, via cationic nickel-catalyzed glycosylation of thioglycoside acceptors bearing the 2-trifluoromethylphenyl aglycon with N-phenyl trifluoroacetimidate donors. The 2-trifluoromethylphenyl group effectively blocks transfer of the anomeric sulfide group from the glycosyl acceptor to the C(2)-benzylidene donor and can be easily installed and activated. The current method also highlights the efficacy of the nickel catalyst selectively activating the C(2)-benzylidene imidate group in the presence of the anomeric sulfide group on the glycosyl acceptors. PMID:22838405

Alternative vaccine administration by powder injection: Needle-free dermal delivery of the glycoconjugate meningococcal group Y vaccine

PubMed Central

Schiffter, Heiko A.; Carlisle, Robert C.; Rollier, Christine S.; Prud’homme, Robert K.; Pollard, Andrew J.

2017-01-01

Powder-injectors use gas propulsion to deposit lyophilised drug or vaccine particles in the epidermal and sub epidermal layers of the skin. We prepared dry-powder (Tg = 45.2 ± 0.5°C) microparticles (58.1 μm) of a MenY-CRM197 glyconjugate vaccine (0.5% wt.) for intradermal needle-free powder injection (NFPI). SFD used ultrasound atomisation of the liquid vaccine-containing excipient feed, followed by lyophilisation above the glass transition temperature (Tg’ = − 29.9 ± 0.3°C). This resulted in robust particles (density~ 0.53 ±0.09 g/cm3) with a narrow volume size distribution (mean diameter 58.1 μm, and span = 1.2), and an impact parameter (ρvr ~ 11.5 kg/m·s) sufficient to breach the Stratum corneum (sc). The trehalose, manitol, dextran (10 kDa), dextran (150 kDa) formulation, or TMDD (3:3:3:1), protected the MenY-CRM197 glyconjugate during SFD with minimal loss, no detectable chemical degradation or physical aggregation. In a capsular group Y Neisseria meningitidis serum bactericidal assay (SBA) with human serum complement, the needle free vaccine, which contained no alum adjuvant, induced functional protective antibody responses in vivo of similar magnitude to the conventional vaccine injected by hypodermic needle and syringe and containing alum adjuvant. These results demonstrate that needle-free vaccination is both technically and immunologically valid, and could be considered for vaccines in development. PMID:28837693

First characterization of immunogenic conjugates of Vi negative Salmonella Typhi O-specific polysaccharides with rEPA protein for vaccine development.

PubMed

Salman, M; St Michael, F; Ali, A; Jabbar, A; Cairns, C; Hayes, A C; Rahman, M; Iqbal, M; Haque, A; Cox, A D

2017-11-01

Efficacious typhoid vaccines for young children will significantly reduce the disease burden in developing world. The Vi polysaccharide based conjugate vaccines (Vi-rEPA) against Salmonella Typhi Vi positive strains has shown high efficacy but may be ineffective against Vi negative S. Typhi. In this study, for the first time, we report the synthesis and evaluation of polysaccharide-protein conjugates of Vi negative S. Typhi as potential vaccine candidates. Four different conjugates were synthesized using recombinant exoprotein A of Pseudomonas aeruginosa (rEPA) and human serum albumin (HSA) as the carrier proteins, using either direct reductive amination or an intermediate linker molecule, adipic acid dihydrazide (ADH). Upon injection into mice, a significantly higher antibody titer was observed in mice administrated with conjugate-1 (OSP-HSA) (P=0.0001) and conjugate 2 (OSP-rEPA) (P≤0.0001) as compared to OSP alone. In contrast, the antibody titer elicited by conjugate 3 (OSP ADH -HSA) and conjugate 4 (OSP ADH -rEPA) were insignificant (P=0.1684 and P=0.3794, respectively). We conclude that reductive amination is the superior method to prepare the S. Typhi OSP glycoconjugate. Moreover, rEPA was a better carrier protein than HSA. Thus OSP-rEPA conjugate seems to be efficacious typhoid vaccines candidate, it may be evaluated further and recommended for the clinical trials. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Precocious development of lectin (Ulex europaeus agglutinin I) receptors in dome epithelium of gut-associated lymphoid tissues.

PubMed

Roy, M J

1987-06-01

Dome epithelium (DE), the tissue covering lymphoid domes of gut-associated lymphoid tissues, was examined in both adult and neonatal rabbit appendix or sacculus rotundus to determine if dome epithelial cells matured earlier than epithelial cells covering adjacent villi. The localization of well-differentiated epithelial cells in rabbit gut-associated lymphoid tissues (GALT) was accomplished histochemically by use of molecular probes: fluorescein isothiocyanate or horseradish peroxidase conjugates of Ulex europaeus agglutinin I (UEA), a lectin specific for terminal L-fucose molecules on certain glycoconjugates. The villus epithelial cells of newborn and 2-, 5-, or 10-day-old rabbits did not bind UEA, but between the twelfth and fifteenth days of postnatal life, UEA receptors were expressed by well-differentiated villus epithelial cells. In contrast to villus epithelium, DE in appendix and sacculus rotundus of neonatal rabbits expressed UEA receptors two days after birth, a feature that distinguished the DE of neonatal GALT for the next two weeks. In adult rabbits, UEA receptors were associated with dome epithelial cells extending from the mouths of glandular crypts to the upper domes; in contrast to the domes, UEA receptors were only present on well-differentiated epithelial cells at the villus tips. Results suggested that in neonatal rabbits most dome epithelial cells developed UEA receptors shortly after birth, reflecting precocious development of DE as compared to villus epithelium. In adult rabbit dome epithelium UEA receptors appeared on dome epithelial cells as they left the glandular crypts, representing accelerated epithelial maturation.

New GlcNAc/GalNAc-specific lectin from the ascidian Didemnum ternatanum.

PubMed

Molchanova, Valentina; Chikalovets, Irina; Li, Wei; Kobelev, Stanislav; Kozyrevskaya, Svetlana; Bogdanovich, Raisa; Howard, Eric; Belogortseva, Natalia

2005-05-25

Previously we isolated GlcNAc-specific lectin (DTL) from the ascidian Didemnum ternatanum by affinity chromatography on cross-linked ovalbumin. Here we report the purification and characterization of new D-GlcNAc/D-GalNAc-specific lectin DTL-A from the same ascidian. This lectin was isolated from non-bound cross-linked ovalbumin fraction and further was purified by gel filtration on Sepharose CL-4B, affinity chromatography on GlcNAc-agarose and gel filtration on Superdex 200. SDS-polyacrylamide gel electrophoresis and gel filtration of purified lectin on Sepharose CL-4B indicates that it exists as large aggregates in the native state. Investigations of the carbohydrate specificity of DTL-A by enzyme-linked lectin assay suggest the multi-specificity of this lectin. DTL-A binds BSM, asialo-BSM as well as heparin and dextran sulfate. The binding of DTL-A to BSM was inhibited by monosaccharides D-GlcNAc and D-GalNAc, their alpha- but not beta-anomers. Among polysaccharides and glycoconjugates, DTL-A binding to BSM was effectively inhibited by BSM, asialo-BSM, pronase-treated BSM and synthetic alpha-D-GalNAc-PAA. Fetuin and asialofetuin showed a much lower inhibitory potency, heparin and dextran sulfate were noninhibitory. On the other hand, DTL-A binding to heparin was effectively inhibited by dextran sulfate, fucoidan, whereas BSM showed insignificantly inhibitory effect. DTL-A binding to heparin was not inhibited by D-GlcNAc and D-GalNAc.

Abnormal accumulation and recycling of glycoproteins visualized in Niemann–Pick type C cells using the chemical reporter strategy

PubMed Central

Mbua, Ngalle Eric; Flanagan-Steet, Heather; Johnson, Steven; Wolfert, Margreet A.; Boons, Geert-Jan; Steet, Richard

2013-01-01

Niemann–Pick type C (NPC) disease is characterized by impaired cholesterol efflux from late endosomes and lysosomes and secondary accumulation of lipids. Although impaired trafficking of individual glycoproteins and glycolipids has been noted in NPC cells and other storage disorders, there is currently no effective way to monitor their localization and movement en masse. Using a chemical reporter strategy in combination with pharmacologic treatments, we demonstrate a disease-specific and previously unrecognized accumulation of a diverse set of glycoconjugates in NPC1-null and NPC2-deficient fibroblasts within endocytic compartments. These labeled vesicles do not colocalize with the cholesterol-laden compartments of NPC cells. Experiments using the endocytic uptake marker dextran show that the endosomal accumulation of sialylated molecules can be largely attributed to impaired recycling as opposed to altered fusion of vesicles. Treatment of either NPC1-null or NPC2-deficient cells with cyclodextrin was effective in reducing cholesterol storage as well as the endocytic accumulation of sialoglycoproteins, demonstrating a direct link between cholesterol storage and abnormal recycling. Our data further demonstrate that this accumulation is largely glycoproteins, given that inhibitors of O-glycan initiation or N-glycan processing led to a significant reduction in staining intensity. Taken together, our results provide a unique perspective on the trafficking defects in NPC cells, and highlight the utility of this methodology in analyzing cells with altered recycling and turnover of glycoproteins. PMID:23733943

Pneumococcal Neuraminidase A (NanA) Promotes Biofilm Formation and Synergizes with Influenza A Virus in Nasal Colonization and Middle Ear Infection

PubMed Central

Wren, John T.; Blevins, Lance K.; Pang, Bing; Basu Roy, Ankita; Oliver, Melissa B.; Reimche, Jennifer L.; Wozniak, Jessie E.; Alexander-Miller, Martha A.

2017-01-01

ABSTRACT Even in the vaccine era, Streptococcus pneumoniae (the pneumococcus) remains a leading cause of otitis media, a significant public health burden, in large part because of the high prevalence of nasal colonization with the pneumococcus in children. The primary pneumococcal neuraminidase, NanA, which is a sialidase that catalyzes the cleavage of terminal sialic acids from host glycoconjugates, is involved in both of these processes. Coinfection with influenza A virus, which also expresses a neuraminidase, exacerbates nasal colonization and disease by S. pneumoniae, in part via the synergistic contributions of the viral neuraminidase. The specific role of its pneumococcal counterpart, NanA, in this interaction, however, is less well understood. We demonstrate in a mouse model that NanA-deficient pneumococci are impaired in their ability to cause both nasal colonization and middle ear infection. Coinfection with neuraminidase-expressing influenza virus and S. pneumoniae potentiates both colonization and infection but not to wild-type levels, suggesting an intrinsic role of NanA. Using in vitro models, we show that while NanA contributes to both epithelial adherence and biofilm viability, its effect on the latter is actually independent of its sialidase activity. These data indicate that NanA contributes both enzymatically and nonenzymatically to pneumococcal pathogenesis and, as such, suggest that it is not a redundant bystander during coinfection with influenza A virus. Rather, its expression is required for the full synergism between these two pathogens. PMID:28096183

Functional and Evolutionary Characterization of a UDP-Xylose Synthase Gene from the Plant Pathogen Xylella fastidiosa, Involved in the Synthesis of Bacterial Lipopolysaccharide.

PubMed

Alencar, Valquíria Campos; Jabes, Daniela Leite; Menegidio, Fabiano Bezerra; Sassaki, Guilherme Lanzi; de Souza, Lucas Rodrigo; Puzer, Luciano; Meneghetti, Maria Cecília Zorél; Lima, Marcelo Andrade; Tersariol, Ivarne Luis Dos Santos; de Oliveira, Regina Costa; Nunes, Luiz R

2017-02-07

Xylella fastidiosa is a plant-infecting bacillus, responsible for many important crop diseases, such as Pierce's disease of vineyards, citrus variegated chlorosis, and coffee leaf scorch (CLS), among others. Recent genomic comparisons involving two CLS-related strains, belonging to X. fastidiosa subsp. pauca, revealed that one of them carries a frameshift mutation that inactivates a gene encoding an oxidoreductase of the short-chain dehydrogenase/reductase (SDR) superfamily, which may play important roles in determining structural variations in bacterial glycans and glycoconjugates. However, the exact nature of this SDR has been a matter of controversy, as different annotations of X. fastidiosa genomes have implicated it in distinct reactions. To confirm the nature of this mutated SDR, a comparative analysis was initially performed, suggesting that it belongs to a subgroup of SDR decarboxylases, representing a UDP-xylose synthase (Uxs). Functional assays, using a recombinant derivative of this enzyme, confirmed its nature as XfUxs, and carbohydrate composition analyses, performed with lipopolysaccharide (LPS) molecules obtained from different strains, indicate that inactivation of the X. fastidiosa uxs gene affects the LPS structure among CLS-related X. fastidiosa strains. Finally, a comparative sequence analysis suggests that this mutation is likely to result in a morphological and evolutionary hallmark that differentiates two subgroups of CLS-related strains, which may influence interactions between these bacteria and their plant and/or insect hosts.

Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from Streptomyces Species

PubMed Central

Matsunaga, Emiko; Higuchi, Yujiro; Mori, Kazuki; Yairo, Nao; Oka, Takuji; Shinozuka, Saki; Tashiro, Kosuke; Izumi, Minoru; Kuhara, Satoru; Takegawa, Kaoru

2015-01-01

β-D-galactofuranose (Galf) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Galf-ase) gene has been identified in any organism. To search for a Galf-ase gene we screened soil samples and discovered a strain, identified as a Streptomyces species by the 16S ribosomal RNA gene analysis, that exhibits Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Galf-ases. Using recombinant proteins expressed in Escherichia coli, we found that three out of four candidates displayed the activity of not only Galf-ase but also α-L-arabinofuranosidase (Araf-ase), whereas the other one showed only the Galf-ase activity. This novel Galf-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate pNP-β-D-Galf. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus Aspergillus fumigatus, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Galf-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Galf-specific Galf-ases widely exist in microorganisms. PMID:26340350

Mycoplasma infection of cell lines can simulate the expression of Fc receptors by binding of the carbohydrate moiety of antibodies.

PubMed

Lemke, H; Krausse, R; Lorenzen, J; Havsteen, B

1985-05-01

During the production of Fc receptor (FcR)-bearing hybridomas it was observed with a particular monoclonal anti-sheep red blood cell antibody (anti-SRBC 1/5, IgG1) that the contamination with Mycoplasma arginini of in vitro cultured cell lines leads to an apparent FcR activity. This property did not correspond with the serological typing since other antibodies of the same isotype could not support FcR rosette formation. Another mycoplasma strain M. orale lacked this property. Analysis of the binding reaction revealed that M. arginini contains a lectin which binds the carbohydrate moiety of the anti-SRBC 1/5 antibody, i.e. anti-SRBC 1/5 synthesized under the influence of tunicamycin or deglycosylated by NaIO4 oxidation did not support rosette formation. These data suggest that binding of antibodies to certain mycoplasma strains may be a pathogenic factor during mycoplasma infections by masking the microorganisms with the host's own defense molecules. The experiments with M. arginini-infected cell lines gain immunological importance since we obtained identical results with staphylococcal protein A, as another bacteriological FcR, and cell lines expressing intrinsic membrane FcR. Although it is an open question whether the glycoconjugates are directly bound by the FcR or else by influencing the three-dimensional structure of the antibodies, it seems possible that FcR in general may be lectins.

Molecular characterization of the receptor binding structure-activity relationships of influenza B virus hemagglutinin.

PubMed

Carbone, V; Kim, H; Huang, J X; Baker, M A; Ong, C; Cooper, M A; Li, J; Rockman, S; Velkov, T

2013-01-01

Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.

Structure and binding analysis of Polyporus squamosus lectin in complex with the Neu5Acα2-6Galβ1-4GlcNAc human-type influenza receptor

PubMed Central

Kadirvelraj, Renuka; Grant, Oliver C; Goldstein, Irwin J; Winter, Harry C; Tateno, Hiroaki; Fadda, Elisa; Woods, Robert J

2011-01-01

Glycan chains that terminate in sialic acid (Neu5Ac) are frequently the receptors targeted by pathogens for initial adhesion. Carbohydrate-binding proteins (lectins) with specificity for Neu5Ac are particularly useful in the detection and isolation of sialylated glycoconjugates, such as those associated with pathogen adhesion as well as those characteristic of several diseases including cancer. Structural studies of lectins are essential in order to understand the origin of their specificity, which is particularly important when employing such reagents as diagnostic tools. Here, we report a crystallographic and molecular dynamics (MD) analysis of a lectin from Polyporus squamosus (PSL) that is specific for glycans terminating with the sequence Neu5Acα2-6Galβ. Because of its importance as a histological reagent, the PSL structure was solved (to 1.7 Å) in complex with a trisaccharide, whose sequence (Neu5Acα2-6Galβ1-4GlcNAc) is exploited by influenza A hemagglutinin for viral adhesion to human tissue. The structural data illuminate the origin of the high specificity of PSL for the Neu5Acα2-6Gal sequence. Theoretical binding free energies derived from the MD data confirm the key interactions identified crystallographically and provide additional insight into the relative contributions from each amino acid, as well as estimates of the importance of entropic and enthalpic contributions to binding. PMID:21436237

Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control

PubMed Central

Williams, Danielle M.; Ovchinnikova, Olga G.; Koizumi, Akihiko; Mainprize, Iain L.; Kimber, Matthew S.; Lowary, Todd L.

2017-01-01

Lipopolysaccharides (LPS) are essential outer membrane glycolipids in most gram-negative bacteria. Biosynthesis of the O-antigenic polysaccharide (OPS) component of LPS follows one of three widely distributed strategies, and similar processes are used to assemble other bacterial surface glycoconjugates. This study focuses on the ATP-binding cassette (ABC) transporter-dependent pathway, where glycans are completed on undecaprenyl diphosphate carriers at the cytosol:membrane interface, before export by the ABC transporter. We describe Raoultella terrigena WbbB, a prototype for a family of proteins that, remarkably, integrates several key activities in polysaccharide biosynthesis into a single polypeptide. WbbB contains three glycosyltransferase (GT) modules. Each of the GT102 and GT103 modules characterized here represents a previously unrecognized GT family. They form a polymerase, generating a polysaccharide of [4)-α-Rhap-(1→3)-β-GlcpNAc-(1→] repeat units. The polymer chain is terminated by a β-linked Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) residue added by a third GT module belonging to the recently discovered GT99 family. The polymerase GT modules are separated from the GT99 chain terminator by a coiled-coil structure that forms a molecular ruler to determine product length. Different GT modules in the polymerase domains of other family members produce diversified OPS structures. These findings offer insight into glycan assembly mechanisms and the generation of antigenic diversity as well as potential tools for glycoengineering. PMID:28137848

Lactobacillus johnsonii glycolipids, their structure and immunoreactivity with sera from inflammatory bowel disease patients.

PubMed

Paściak, Mariola; Górska, Sabina; Jawiarczyk, Natalia; Gamian, Andrzej

2017-03-01

Structural studies of the major glycolipids produced by two Lactobacillus johnsonii (LJ) strains, LJ 151 isolated from intestinal tract of healthy mice and LJ 142 isolated from mice with experimentally induced inflammatory bowel disease (IBD), were performed. Two major glycolipids, GL1 and GL2, were present in lipid extracts from L. johnsonii 142 and 151 strains. Glycolipid GL1 has been identified as β-D-Glcp-(1→6)-α-D-Galp-(1→2)-α-D-Glcp-diglyceride and GL2 as α-D-Galp-(1→2)-α-D-Glcp-diglyceride. The main fatty acid residues identified by gas-liquid chromatography-mass spectrometry were palmitic, stearic and lactobacillic acids. Besides structural elucidation of the major glycolipids, the aim of this study was to determine the immunochemical properties of these glycolipids and to compare their immunoreactivity to that of polysaccharides obtained from the same strains. Sera from rabbits immunized with bacterial cells possessed much higher serological reactivity with polysaccharides than with glycolipids. Inversely, reactivity of the glycolipids with human sera from patients with IBD was much higher than that determined for the polysaccharides, while reactivity of glycolipids with human sera from healthy individuals was much lower than one measured for the polysaccharides. Results indicate that glycoconjugates from Lactobacillus cell wall act as antigens and may represent new IBD diagnostic biomarkers. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

An enzyme-linked immunosorbent assay (ELISA) for quantification of human collectin 11 (CL-11, CL-K1)

PubMed Central

Selman, L.; Henriksen, M.L.; Brandt, J.; Palarasah, Y.; Waters, A.; Beales, P.L.; Holmskov, U.; Jørgensen, T.J.D.; Nielsen, C.; Skjodt, K.; Hansen, S.

2012-01-01

Collectin 11 (CL-11), also referred to as collectin kidney 1 (CL-K1), is a pattern recognition molecule that belongs to the collectin group of proteins involved in innate immunity. It interacts with glycoconjugates on pathogen surfaces and has been found in complex with mannose-binding lectin-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies. The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7–104%). The working range is 0.15–34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined to be below 2.1 ng/ml. We measured the mean serum CL-11 concentration to 284 ng/ml in 100 Danish blood donors, with a 95% confidence interval of 269–299 ng/ml. There was no significant difference in the CL-11 concentration measured in matched serum and plasma samples. Storage of samples and repeated freezing and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes. PMID:22301270

Bivalent O-glycoside mimetics with S/disulfide/Se substitutions and aromatic core: Synthesis, molecular modeling and inhibitory activity on biomedically relevant lectins in assays of increasing physiological relevance.

PubMed

Kaltner, Herbert; Szabó, Tamás; Fehér, Krisztina; André, Sabine; Balla, Sára; Manning, Joachim C; Szilágyi, László; Gabius, Hans-Joachim

2017-06-15

The emerging significance of recognition of cellular glycans by lectins for diverse aspects of pathophysiology is a strong incentive for considering development of bioactive and non-hydrolyzable glycoside derivatives, for example by introducing S/Se atoms and the disulfide group instead of oxygen into the glycosidic linkage. We report the synthesis of 12 bivalent thio-, disulfido- and selenoglycosides attached to benzene/naphthalene cores. They present galactose, for blocking a plant toxin, or lactose, the canonical ligand of adhesion/growth-regulatory galectins. Modeling reveals unrestrained flexibility and inter-headgroup distances too small to bridge two sites in the same lectin. Inhibitory activity was first detected by solid-phase assays using a surface-presented glycoprotein, with relative activity enhancements per sugar unit relative to free cognate sugar up to nearly 10fold. Inhibitory activity was also seen on lectin binding to surfaces of human carcinoma cells. In order to proceed to characterize this capacity in the tissue context monitoring of lectin binding in the presence of inhibitors was extended to sections of three types of murine organs as models. This procedure proved to be well-suited to determine relative activity levels of the glycocompounds to block binding of the toxin and different human galectins to natural glycoconjugates at different sites in sections. The results on most effective inhibition by two naphthalene-based disulfides and a selenide raise the perspective for broad applicability of the histochemical assay in testing glycoclusters that target biomedically relevant lectins. Copyright © 2017 Elsevier Ltd. All rights reserved.

Wholly Rickettsia! Reconstructed Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells

PubMed Central

Driscoll, Timothy P.; Verhoeve, Victoria I.; Guillotte, Mark L.; Lehman, Stephanie S.; Rennoll, Sherri A.; Beier-Sexton, Magda; Rahman, M. Sayeedur; Azad, Abdu F.

2017-01-01

ABSTRACT Reductive genome evolution has purged many metabolic pathways from obligate intracellular Rickettsia (Alphaproteobacteria; Rickettsiaceae). While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenic Rickettsia culture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed the Rickettsia metabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized) needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycoconjugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycerophospholipid pathways also initiate from host precursors, and import of both isoprenes and terpenoids is required for the synthesis of ubiquinone and the lipid carrier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accentuating their parasitic nature. Six biosynthesis pathways contain holes (missing enzymes); similar patterns in taxonomically diverse bacteria suggest alternative enzymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host metabolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell. PMID:28951473

Aspartic protease inhibitors as potential anti-Candida albicans drugs: impacts on fungal biology, virulence and pathogenesis.

PubMed

Braga-Silva, L A; Santos, A L S

2011-01-01

Mycoses are still one of the most problematic illnesses worldwide, especially affecting immunocompromised individuals. The development of novel antifungal drugs is becoming more demanding every day, since existing drugs either have too many side effects or they tend to lose effectiveness due to the resistant fungal strains. In this scenario, Candida albicans is still the main fungal pathogen isolated in hospitals. Pathogenicity results essentially from modifications of the host defense mechanisms that secondarily initiate transformations in the fungal behavior. The pathogenesis of C. albicans is multifactorial and different virulence attributes are important during the various stages of infection. Some virulence factors, like the secreted aspartic proteases (Saps), play a role in several infection stages and the inhibition of one of the many stages may contribute to the containment of the pathogen and thus should help in the treatment of disease. Therefore, Saps are potential targets for the development of novel anti-C. albicans drugs. Herein, we review the beneficial properties of pepstatin A and aspartic-type protease inhibitors used in the anti-human immunodeficiency virus chemotherapy on C. albicans, with particular emphasis in the effects on Sap activity, proliferation, morphogenesis (yeasts into mycelia transformation), ultrastructural architecture, adhesion to mammalian cells and abiotic materials, modulation of unrelated virulence factors (e.g., surface glycoconjugates, lipases and sterol), experimental candidiasis infection as well as synergistic properties when conjugated with classical antifungals. Collectively, these positive findings have stimulated the search for novel natural and/or synthetic pharmacological compounds with anti-aspartic protease properties against the human opportunistic fungus C. albicans.

INTEGRATION OF SYSTEMS GLYCOBIOLOGY WITH BIOINFORMATICS TOOLBOXES, GLYCOINFORMATICS RESOURCES AND GLYCOPROTEOMICS DATA

PubMed Central

Liu, Gang; Neelamegham, Sriram

2015-01-01

The glycome constitutes the entire complement of free carbohydrates and glycoconjugates expressed on whole cells or tissues. ‘Systems Glycobiology’ is an emerging discipline that aims to quantitatively describe and analyse the glycome. Here, instead of developing a detailed understanding of single biochemical processes, a combination of computational and experimental tools are used to seek an integrated or ‘systems-level’ view. This can explain how multiple biochemical reactions and transport processes interact with each other to control glycome biosynthesis and function. Computational methods in this field commonly build in silico reaction network models to describe experimental data derived from structural studies that measure cell-surface glycan distribution. While considerable progress has been made, several challenges remain due to the complex and heterogeneous nature of this post-translational modification. First, for the in silico models to be standardized and shared among laboratories, it is necessary to integrate glycan structure information and glycosylation-related enzyme definitions into the mathematical models. Second, as glycoinformatics resources grow, it would be attractive to utilize ‘Big Data’ stored in these repositories for model construction and validation. Third, while the technology for profiling the glycome at the whole-cell level has been standardized, there is a need to integrate mass spectrometry derived site-specific glycosylation data into the models. The current review discusses progress that is being made to resolve the above bottlenecks. The focus is on how computational models can bridge the gap between ‘data’ generated in wet-laboratory studies with ‘knowledge’ that can enhance our understanding of the glycome. PMID:25871730

A recombinant fungal lectin for labeling truncated glycans on human cancer cells.

PubMed

Audfray, Aymeric; Beldjoudi, Mona; Breiman, Adrien; Hurbin, Amandine; Boos, Irene; Unverzagt, Carlo; Bouras, Mourad; Lantuejoul, Sylvie; Coll, Jean-Luc; Varrot, Annabelle; Le Pendu, Jacques; Busser, Benoit; Imberty, Anne

2015-01-01

Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases.

A Recombinant Fungal Lectin for Labeling Truncated Glycans on Human Cancer Cells

PubMed Central

Hurbin, Amandine; Boos, Irene; Unverzagt, Carlo; Bouras, Mourad; Lantuejoul, Sylvie; Coll, Jean-Luc; Varrot, Annabelle; Le Pendu, Jacques; Busser, Benoit; Imberty, Anne

2015-01-01

Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases. PMID:26042789

Quality, immunogenicity and stability of meningococcal serogroup ACWY-CRM197, DT and TT glycoconjugate vaccines.

PubMed

Beresford, Nicola J; Martino, Angela; Feavers, Ian M; Corbel, Michael J; Bai, Xilian; Borrow, Ray; Bolgiano, Barbara

2017-06-16

A physicochemical and immunological study of the stability of three different meningococcal (Men) ACWY conjugate vaccines was performed to evaluate any patterns of serogroup oligo- or polysaccharide-specific or carrier protein-specific stability that would affect immunogenicity. Critical quality and stability-indicating characteristics were measured, with the study supporting the suitability of both HPLC-SEC and HPAEC-PAD methods to detect changes following inappropriate vaccine storage. All three final products, ACWY-CRM 197 , -DT and -TT conjugate vaccines had expected quality indicator values and similar immunogenicity in a mouse model (anti-PS IgG and rSBA) when stored at +2-8°C. When stored at ≥+37°C, all conjugated carrier proteins and serogroup saccharides were affected. Direct correlations were observed between the depolymerization of the MenA saccharide as evidenced by a size-reduction in the MenA conjugates (CRM 197 , DT and TT) and their immunogenicity. MenA was the most labile serogroup, followed by MenC; then MenW and Y, which were similar. At high temperatures, the conjugated carrier proteins were prone to unfolding and/or aggregation. The anti-MenC IgG responses of the multivalent conjugate vaccines in mice were equivalent to those observed in monovalent MenC conjugate vaccines, and were independent of the carrier protein. For any newly developing MenACWY saccharide-protein conjugate vaccines, a key recommendation would be to consider the lyophilization of final product to prevent deleterious degradation that would affect immunogenicity. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Effects of Anethum graveolens L. (dill) on Oocyte and Fertility of Adult Female Rats

PubMed Central

Monsefi, Malihezaman; Ghasemi, Aazam; Alaee, Sanaz; Aliabadi, Elham

2015-01-01

Background Our previous studies revealed Anethum graveolens L. caused some changes in female reproductive system that induced infertility. Therefore, in this study, oocyte changes as one of probable reasons of infertility were investigated. Methods In this study, 59 adult female rats were divided into 3 groups of control, low dose (0.5 g/kg) and high dose (5 g/kg) of dill seed aqueous extract (LDE and HDE) treated groups that were gavaged with 1 ml of each dose for 10 days (2 estrous cycles). Vaginal smears were prepared daily. Oocytes of superovulated animals were extracted and their morphometrical changes were measured (n = 5). Oocyte cell membrane glycoconjugates were stained with UEA, PNA, and DBA-FITC lectins (n = 5). Ultrastructural studies of oocytes were performed using TEM (n = 5). The number, weight, and crown-rump length of newborns were examined in three groups after mating with untreated males (n = 5). Data were analyzed using SPSS software. Results Results demonstrated that the duration of the estrous cycle, the diestrus phase and progesterone concentration in the experimental groups increased significantly compared to the control group (p < 0.05). Granulosa cells of corpus luteum in HDE-treated group were larger and clearer. The intensity reactions of galactose/Nacetylgalactoseamine terminal sugar of oocyte decreased insignificantly in experimental groups compared to the control group p > 0.05. Duration of mating to pregnancy increased and the weight and crown-rump length of newborns decreased in experimental groups significantly (p < 0.05). Conclusion Dill seed aqueous extract can induce infertility without any effect on oocyte structure. PMID:25717430

Alterations in protein glycosylation in PMA-differentiated U-937 cells exposed to mineral particles.

PubMed Central

Trabelsi, N; Greffard, A; Pairon, J C; Bignon, J; Zanetti, G; Fubini, B; Pilatte, Y

1997-01-01

Carbohydrate moieties of cell glycoconjugates play a pivotal role in molecular recognition phenomena involved in the regulation of most biological systems and the changes observed in cell surface carbohydrates during cell activation or differentiation frequently modulate certain cell functions. Consequently, some aspects of macrophage response to particle exposure might conceivably result from alterations in glycosylation. Therefore, the effect of mineral particles on protein glycosylation was investigated in phorbol myristate acetate (PMA)-differentiated U-937. Jacalin, a lectin specific for O-glycosylated structures, showed a global increase in O-glycosylation in particle-treated cells. In contrast, no significant modifications were observed with concanavalin A, a lectin that recognizes certain N-glycosylated structures. The sialic acid-specific lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin and the galactose-specific lectin Ricinus communis agglutinin revealed a complex pattern of alterations in glycoprotein glycosylation after crystalline silica or manganese dioxide treatments. Expression of sialyl Lewis(x), a glycosylated structure implicated in leukocyte trafficking, could not be detected in control or treated cells. This finding was consistent with the decrease in sialyl Lewis(x) expression observed during PMA-induced differentiation. In conclusion, various treatments used in this study induced quantitative as well as qualitative changes in protein glycosylation. Whether these changes are due to glycosidase release or to an alteration in glycosyltransferase expression remains to be determined. The potential functional implications of these changes are currently under investigation. Images Figure 1. A Figure 1. B Figure 2. A Figure 2. B Figure 3. A Figure 3. B Figure 3. C Figure 4. PMID:9400716

In search of a solution to the sphinx-like riddle of GM1.

PubMed

Ledeen, Robert W; Wu, Gusheng

2010-12-01

Among the many glycoconjugates contributing to the sugar code, gangliosides have drawn special attention owing to their predominance as the major sialoglycoconjugate category within the nervous system. However, their occurrence, albeit at lower levels, appears ubiquitous in vertebrate cells and even some invertebrate tissues. Now that over 100 gangliosides have been structurally characterized, their diverse physiological functions constitute a remaining enigma. This has been especially true of GM1, for which a surprising array of functions has already been revealed. Our current research has focused on two areas of GM1 function: (a) signaling induced in neural and immune cells by cross-linking of GM1 in the plasma membrane that leads to activation of TRPC5 (transient receptor potiential, canonical form 5) channels, a process important in neuritogenesis and autoimmune suppression; (b) activation by GM1 of a sodium-calcium exchanger (NCX) in the inner membrane of the nuclear envelope (NE) with resulting modulation of nuclear and cellular calcium. The latter has a role in maintaining neuronal viability, loss of which renders neurons vulnerable to Ca(2+) overload. Pathological manifestations in mutant mice and their cultured neurons lacking GM1 have shown dramatic rescue with a membrane permeable derivative of GM1 that enters the nucleus and restores NCX activity. Nuclear function of GM1 is related to the presence of neuraminidase in the NE, an enzyme that generates GM1 through hydrolysis of GD1a. A different isoform of this enzyme was found in each of the two membranes of the NE.

The Effect of Fucoidan from the Brown Alga Fucus evanescence on the Activity of α-N-Acetylgalactosaminidase of Human Colon Carcinoma Cells.

PubMed

Bakunina, Irina; Chadova, Oksana; Malyarenko, Olesya; Ermakova, Svetlana

2018-05-10

α- N -acetylgalactosaminidase (EC 3.2.1.49) (alpha-NaGalase) catalyzes the hydrolysis of N -acetamido-2-deoxy-α-d-galactoside residues from non-reducing ends of various complex carbohydrates and glycoconjugates. It is known that human cancer cells express an alpha-NaGalase, which accumulates in the blood plasma of patients. The enzyme deglycosylates the Gc protein-derived macrophage activating factor (GcMAF) and inhibits macrophage activity acting as an immunosuppressor. The high specific activity 0.033 ± 0.002 μmol mg −1 min −1 of the enzyme was found in human colon carcinoma cells DLD-1. The alpha-NaGalase of DLD-1 cells was isolated and biochemical characterized. The enzyme exhibits maximum activity at pH 5.2 and temperature 55 °C. The K m is 2.15 mM, V max ⁻0.021 μmol min −1 mL −1 , k cat ⁻1.55 min −1 and k cat / K m ⁻0.72 min −1 mM −1 at 37 °C, pH 5.2. The effects of fucoidan from the brown alga Fucus evanescence on the activity of alpha-NaGalase in human colon carcinoma DLD-1 cells and on the biosynthesis of this enzyme were investigated. It was shown that fucoidan did not inhibit free alpha-NaGalase, however, it reduced the expression of the enzyme in the DLD-1 cells at IC 50 73 ± 4 μg mL −1 .

Effect of structural modifications of ganglioside GM2 on intra-molecular carbohydrate-to-carbohydrate interaction and enzymatic susceptibility.

PubMed

Li, Yu-Teh; Li, Su-Chen; Kiso, Makoto; Ishida, Hideharu; Mauri, Laura; Raimondi, Laura; Bernardi, Anna; Sonnino, Sandro

2008-03-01

The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcbeta1-->4(Neu5Acalpha2-->3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to beta-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcbeta1-->4Gal linkage in GM2 was converted to the GalNAcbeta1-->6Gal, both the GalNAc and the Neu5Ac became susceptible to beta-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates.

Carbohydrate recognition by the rhamnose-binding lectin SUL-I with a novel three-domain structure isolated from the venom of globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus.

PubMed

Hatakeyama, Tomomitsu; Ichise, Ayaka; Unno, Hideaki; Goda, Shuichiro; Oda, Tatsuya; Tateno, Hiroaki; Hirabayashi, Jun; Sakai, Hitomi; Nakagawa, Hideyuki

2017-08-01

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure. © 2017 The Protein Society.

An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in Cells.

PubMed

Li, Jing; Wang, Jiajia; Wen, Liuqing; Zhu, He; Li, Shanshan; Huang, Kenneth; Jiang, Kuan; Li, Xu; Ma, Cheng; Qu, Jingyao; Parameswaran, Aishwarya; Song, Jing; Zhao, Wei; Wang, Peng George

2016-11-18

O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac 3 4dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac 3 4dGlcNAz allowed the selective visualization of O-GlcNAc in cells and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.

Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

USGS Publications Warehouse

Cardenas, W.; Dankert, J.R.; Jenkins, J.A.

2004-01-01

Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P < 0.05). Furthermore, addition of trypsin inhibitor to the LPS treatments caused noticeable delays in cell size and viability changes. These patterns of cellular activation by LPS formulations indicated that crayfish haemocytes react differently to the polysaccharide and lipid A moieties of LPS, where lipid A is cytotoxic and the polysaccharide portion is stimulatory. These effects concur with the general pattern of mammalian cell activation by LPS, thereby indicting commone innate immune recognition mechanisms to bacterial antigens between cells from mammals and invertebrates. These definitive molecular approaches used to verify and identify mechanisms of invertbrate haemocyte responses to LPS could be applied with other glycoconjugates, soluble mediators, or xenobiotic compounds.

Histochemical Characterization of Oocytes in the Pink Cuskeel (Genypterus blacodes).

PubMed

Cohen, Stefanía; Petcoff, Gladys; Freijo, Roberto O; Portiansky, Enrique L; Barbeito, Claudio G; Macchi, Gustavo J; Díaz, Alcira O

2015-08-01

In the present study we histochemically and lectinhistochemically characterized the growing oocytes of the pink cuskeel (Genypterus blacodes). We used histochemical methods for the localization and characterization of glycoconjugates (GCs) and lectin histochemical techniques for the identification of specific sugar residues. We analyzed presence and distribution of GCs in the different structures of the growing follicles (cortical alveoli, globules, yolk granules and zona radiata). During the initial stage of vitellogenesis, the oocytes presented small yolk granules composed of GCs that gradually increased during exogenous vitellogenesis. These GCs contained moderate quantities of α-D-mannose, D-glucose, N-acetylglucosamine and N-acetyl-neuraminic acid. The cortical alveoli contained both neutral and carboxylated GCs, and lectin techniques detected N-acetylgalactosamine, galactose and L-fucose. The zona radiata showed a strong positive reaction to PAS and it reacted weakly with more specific techniques, such as KOH/PA*S and PA/Bh/KOH/PAS. This structure showed GCs with oxidizable vicinal diols, O-acyl sugars and sialic acid residues with different substitution types and presented N-acetylgalactosamine and L-fucose specific residues. The oocytes follicular envelope evidenced neutral and acidic non-sulfated GCs and high concentrations of α-D-mannose, D-glucose, galactose and N-acetylgalactosamine. The intergranular cytoplasmic GCs were mainly rich in α-D-mannose, D-glucose, N-acetylgalactosamine, N-acetylglucosamine and N-acetyl-neuraminic acid. These results enhance the comprehension of the structure and functionality of the pink cuskeel ovarian follicles, and provide a useful tool for the study of this tissue in other teleost species.

Immunity to adult cestodes: basic knowledge and vaccination problems. A review.

PubMed

Andreassen, J

1991-04-01

Immunity in mammals to intestinal cestodes has been reviewed using the normal final host infected with the tapeworms Hymenolepis diminuta in rats and H. microstoma and H. nana in mice as a model. Primary infections up to a certain level continue to live as long the host, while most worms in infections with larger doses are destrobilated and expelled. It has been argued that concomitant immunity against a superimposed infection exists in rats and mice infected with H. diminuta and H. microstoma, respectively, and suggested that it also takes place in humans infected with Taenia spp. Immunity to secondary infections after expulsion of a primary infection occurs, but immunological memory is rather short-lived, although depression of worm growth occurs for at least two third of the rat's life. Serum antibodies have been shown to produce a direct precipitate on the surface of cestodes in vitro, but a direct effect of antibodies in vivo or the relationship with e.g. host effector cells, like mast cells and eosinophils, is unknown. It has been shown that peritoneal exudate cells from rats are able to kill H. diminuta in vitro. Very little is known about the mechanisms of tapeworms to counteract host immunological responses, but the tegumental glycoconjugates and discoidal secretory bodies are possible candidates. Passive transfer of immunity by mesenteric lymph node cells has only been successful using cells from H. nana egg-infected mice and has shown that only short-lived proliferating cells are responsible for transferring immunity. Vaccination procedures and problems are discussed with special reference to E. granulosus in dogs.

The activity of N-acetyl-β-d-hexosaminidase A and B and β-glucuronidase in nasal polyps and hypertrophic nasal concha.

PubMed

Chojnowska, Sylwia; Minarowska, Alina; Waszkiewicz, Napoleon; Kępka, Alina; Zalewska-Szajda, Beata; Gościk, Elżbieta; Kowal, Krzysztof; Olszewska, Ewa; Konarzewska-Duchnowska, Emilia; Minarowski, Łukasz; Zwierz, Krzysztof; Ładny, Jerzy Robert; Szajda, Sławomir Dariusz

2014-01-01

Nasal polyps and hypertrophic lower nasal conchae are common disorders of nasal cavity. The majority of etiopathogenetic theories indicate inflammatory background of polyps and hypertrophic concha. N-acetyl-β-D-hexosaminidase and β-glucuronidase are lysosomal exoglycosidases revealing accelerated activity in inflammatory processes. The aim of the study was to evaluate the catabolism of glycoconjugates in nasal polyps and hypertrophic nasal concha basing on the activity of N-acetyl-β-D-hexosaminidase (HEX) and β-glucuronidase (GLU). Material consisted of nasal polyps taken from 40 patients during polypectomy in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and hypertrophic lower nasal conchae taken from 20 patients during mucotomy. The activity of HEX, HEX A, HEX B and GLU in supernatant of homogenates of nasal polyps and hypertrophic lower nasal concha tissues has been estimated using colorimetric method. Statistically significant decrease has been observed in concentration of the activity (per 1mg of tissue) of HEX (p<0.05), HEX B (p<0.001) and specific activity (per 1mg of protein) of HEX B (p<0.001) in nasal polyps tissue in comparison to hypertrophic lower nasal conchae tissue. Decrease in the activity and specific activity concentration of the majority of examined lysosomal exoglycosidases (increasing in inflammations) in comparison to hypertrophic lower nasal conchae suggests electrolytes disorders and questions the inflammatory background of nasal polyps. Copyright © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z.o.o. All rights reserved.

Mannose-conjugated platinum complexes reveals effective tumor targeting mediated by glucose transporter 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ran; Li, Hong; Gao, Xiangqian

Despite numerous studies that report the glucose derived glycoconjugates as antitumor candidates, using mannose as sugar motif for specific tumor targeting remains less studied. In this research, two novel mannose-conjugated platinum complexes 4a and 4b that target the Warburg effect were designed, synthesized and evaluated for their antitumor activities in vitro and in vivo. Compared with oxaliplatin, both complexes exhibited substantial enhancement in water solubility as well as excellent or comparative cytotoxicity in six human cancer cell lines. Cytotoxicity assessments on Glucose transporter 1 (GLUT1) down-regulated or overexpressed cells and platinum accumulation study demonstrated that cellular uptake of compound 4a was regulatedmore » by GLUT1. In particular, 4a induced apoptosis in HT29 cells by suppressing expression of Bcl-2 and Bcl-XL, which preliminary explained the mechanism origin of antitumor effect. As indicated by its maximum tolerated dose-finding assay and in vivo anticancer activity, compound 4a exhibits better safety and efficacy profile than oxaliplatin. The findings of this study indicate the possibility of subjecting mannose-conjugated platinum complexes as lead compounds for further preclinical evaluation. - Highlights: • Mannose-conjugated platinum complexes were designed and synthesized to target glucose transporter 1(GLUT1). • Mannose-conjugated platinum complex 4a transport across cancer cells through GLUT1. • Mannose-conjugated platinum complex 4a induce apoptosis in HT29 cells. • Mannose-conjugated platinum complex 4a antitumor activities were more potent than those of oxaliplatin.« less

Prevalence of the F-type lectin domain.

PubMed

Bishnoi, Ritika; Khatri, Indu; Subramanian, Srikrishna; Ramya, T N C

2015-08-01

F-type lectins are fucolectins with characteristic fucose and calcium-binding sequence motifs and a unique lectin fold (the "F-type" fold). F-type lectins are phylogenetically widespread with selective distribution. Several eukaryotic F-type lectins have been biochemically and structurally characterized, and the F-type lectin domain (FLD) has also been studied in the bacterial proteins, Streptococcus mitis lectinolysin and Streptococcus pneumoniae SP2159. However, there is little knowledge about the extent of occurrence of FLDs and their domain organization, especially, in bacteria. We have now mined the extensive genomic sequence information available in the public databases with sensitive sequence search techniques in order to exhaustively survey prokaryotic and eukaryotic FLDs. We report 437 FLD sequence clusters (clustered at 80% sequence identity) from eukaryotic, eubacterial and viral proteins. Domain architectures are diverse but mostly conserved in closely related organisms, and domain organizations of bacterial FLD-containing proteins are very different from their eukaryotic counterparts, suggesting unique specialization of FLDs to suit different requirements. Several atypical phylogenetic associations hint at lateral transfer. Among eukaryotes, we observe an expansion of FLDs in terms of occurrence and domain organization diversity in the taxa Mollusca, Hemichordata and Branchiostomi, perhaps coinciding with greater emphasis on innate immune strategies in these organisms. The naturally occurring FLDs with diverse domain organizations that we have identified here will be useful for future studies aimed at creating designer molecular platforms for directing desired biological activities to fucosylated glycoconjugates in target niches. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Localization of α1-2 Fucose Glycan in the Mouse Olfactory Pathway.

PubMed

Kondoh, Daisuke; Kamikawa, Akihiro; Sasaki, Motoki; Kitamura, Nobuo

2017-01-01

Glycoconjugates in the olfactory system play critical roles in neuronal formation, and α1-2 fucose (α1-2Fuc) glycan mediates neurite outgrowth and synaptic plasticity. Histochemical findings of α1-2Fuc glycan in the mouse olfactory system detected using Ulex europaeus agglutinin-I (UEA-I) vary. This study histochemically assessed the main olfactory and vomeronasal pathways in male and female ICR and C57BL/6J mice aged 3-4 months using UEA-I. Ulex europaeus agglutinin-I reacted with most receptor cells arranged mainly at the basal region of the olfactory epithelium. The olfactory nerve layer and glomerular layer of the main olfactory bulb were speckled with positive UEA-I staining, and positive fibers were scattered from the glomerular to the internal plexiform layer. The lateral olfactory tract and rostral migratory stream were also positive for UEA-I. We identified superficial short-axon cells, interneurons of the external plexiform layer, external, middle and internal tufted cells, mitral cells and granule cells as the origins of the UEA-I-positive fibers in the main olfactory bulb. The anterior olfactory nucleus, anterior piriform cortex and olfactory tubercle were negative for UEA-I. Most receptor cells in the vomeronasal epithelium and most glomeruli of the accessory olfactory bulb were positive for UEA-I. Our findings indicated that α1-2Fuc glycan is located within the primary and secondary, but not the ternary, pathways of the main olfactory system, in local circuits of the main olfactory bulb and within the primary, but not secondary, pathway of the vomeronasal system. © 2016 S. Karger AG, Basel.

Deletion of Aspergillus nidulans GDP-mannose transporters affects hyphal morphometry, cell wall architecture, spore surface character, cell adhesion, and biofilm formation.

PubMed

Kadry, Ashraf A; El-Ganiny, Amira M; Mosbah, Rasha A; Kaminskyj, Susan G W

2018-07-01

Systemic human fungal infections are increasingly common. Aspergillus species cause most of the airborne fungal infections. Life-threatening invasive aspergillosis was formerly found only in immune-suppressed patients, but recently some strains of A. fumigatus have become primary pathogens. Many fungal cell wall components are absent from mammalian systems, so they are potential drug targets. Cell-wall-targeting drugs such as echinocandins are used clinically, although echinocandin-resistant strains were discovered shortly after their introduction. Currently there are no fully effective anti-fungal drugs. Fungal cell wall glycoconjugates modulate human immune responses, as well as fungal cell adhesion, biofilm formation, and drug resistance. Guanosine diphosphate (GDP) mannose transporters (GMTs) transfer GDP-mannose from the cytosol to the Golgi lumen prior to mannosylation. Aspergillus nidulans GMTs are encoded by gmtA and gmtB. Here we elucidate the roles of A. nidulans GMTs. Strains engineered to lack either or both GMTs were assessed for hyphal and colonial morphology, cell wall ultrastructure, antifungal susceptibility, spore hydrophobicity, adherence and biofilm formation. The gmt-deleted strains had smaller colonies with reduced sporulation and with thicker hyphal walls. The gmtA deficient spores had reduced hydrophobicity and were less adherent and less able to form biofilms in vitro. Thus, gmtA not only participates in maintaining the cell wall integrity but also plays an important role in biofilm establishment and adherence of A. nidulans. These findings suggested that GMTs have roles in A. nidulans growth and cell-cell interaction and could be a potential target for new antifungals that target virulence determinants.

Activation of human naïve Th cells increases surface expression of GD3 and induces neoexpression of GD2 that colocalize with TCR clusters.

PubMed

Villanueva-Cabello, Tania M; Mollicone, Rosella; Cruz-Muñoz, Mario E; López-Guerrero, Delia V; Martínez-Duncker, Iván

2015-12-01

CD4+ T helper lymphocytes (Th) orchestrate the immune response after their activation by antigen-presenting cells. Activation of naïve Th cells is reported to generate the reduction in surface epitopes of sialic acid (Sia) in α2,3 and α2,6 linkages. In this work, we report that in spite of this glycophenotype, anti-CD3/anti-CD28-activated purified human naïve Th cells show a significant increase in surface Sia, as assessed by metabolic labeling, compared with resting naïve Th cells, suggesting an increased flux of Sia toward Siaα2,8 glycoconjugates. To understand this increase as a result of ganglioside up-regulation, we observed that very early after activation, human naïve Th cells show an increased expression in surface GD3 and neoexpression of surface GD2 gangliosides, the latter clustering with the T cell receptor (TCR). Also, we report that in contrast to GM2/GD2 synthase null mice, lentiviral vector-mediated silencing of the GM2/GD2 synthase in activated human naïve Th cells reduced efficient TCR clustering and downstream signaling, as assessed by proliferation assays and IL-2 and IL-2R expression, pointing to an important role of this enzyme in activation of human naive Th cells. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Pneumococcal Neuraminidase A (NanA) Promotes Biofilm Formation and Synergizes with Influenza A Virus in Nasal Colonization and Middle Ear Infection.

PubMed

Wren, John T; Blevins, Lance K; Pang, Bing; Basu Roy, Ankita; Oliver, Melissa B; Reimche, Jennifer L; Wozniak, Jessie E; Alexander-Miller, Martha A; Swords, W Edward

2017-04-01

Even in the vaccine era, Streptococcus pneumoniae (the pneumococcus) remains a leading cause of otitis media, a significant public health burden, in large part because of the high prevalence of nasal colonization with the pneumococcus in children. The primary pneumococcal neuraminidase, NanA, which is a sialidase that catalyzes the cleavage of terminal sialic acids from host glycoconjugates, is involved in both of these processes. Coinfection with influenza A virus, which also expresses a neuraminidase, exacerbates nasal colonization and disease by S. pneumoniae , in part via the synergistic contributions of the viral neuraminidase. The specific role of its pneumococcal counterpart, NanA, in this interaction, however, is less well understood. We demonstrate in a mouse model that NanA-deficient pneumococci are impaired in their ability to cause both nasal colonization and middle ear infection. Coinfection with neuraminidase-expressing influenza virus and S. pneumoniae potentiates both colonization and infection but not to wild-type levels, suggesting an intrinsic role of NanA. Using in vitro models, we show that while NanA contributes to both epithelial adherence and biofilm viability, its effect on the latter is actually independent of its sialidase activity. These data indicate that NanA contributes both enzymatically and nonenzymatically to pneumococcal pathogenesis and, as such, suggest that it is not a redundant bystander during coinfection with influenza A virus. Rather, its expression is required for the full synergism between these two pathogens. Copyright © 2017 American Society for Microbiology.

Lactobacillus acidophilus Metabolizes Dietary Plant Glucosides and Externalizes Their Bioactive Phytochemicals

PubMed Central

Theilmann, Mia C.; Nielsen, Kristian Fog; Klaenhammer, Todd R.

2017-01-01

ABSTRACT Therapeutically active glycosylated phytochemicals are ubiquitous in the human diet. The human gut microbiota (HGM) modulates the bioactivities of these compounds, which consequently affect host physiology and microbiota composition. Despite a significant impact on human health, the key players and the underpinning mechanisms of this interplay remain uncharacterized. Here, we demonstrate the growth of Lactobacillus acidophilus on mono- and diglucosyl dietary plant glycosides (PGs) possessing small aromatic aglycones. Transcriptional analysis revealed the upregulation of host interaction genes and identified two loci that encode phosphotransferase system (PTS) transporters and phospho-β-glucosidases, which mediate the uptake and deglucosylation of these compounds, respectively. Inactivating these transport and hydrolysis genes abolished or severely reduced growth on PG, establishing the specificity of the loci to distinct groups of PGs. Following intracellular deglucosylation, the aglycones of PGs are externalized, rendering them available for absorption by the host or for further modification by other microbiota taxa. The PG utilization loci are conserved in L. acidophilus and closely related lactobacilli, in correlation with versatile growth on these compounds. Growth on the tested PG appeared more common among human gut lactobacilli than among counterparts from other ecologic niches. The PGs that supported the growth of L. acidophilus were utilized poorly or not at all by other common HGM strains, underscoring the metabolic specialization of L. acidophilus. These findings highlight the role of human gut L. acidophilus and select lactobacilli in the bioconversion of glycoconjugated phytochemicals, which is likely to have an important impact on the HGM and human host. PMID:29162708

Automation of the anthrone assay for carbohydrate concentration determinations.

PubMed

Turula, Vincent E; Gore, Thomas; Singh, Suddham; Arumugham, Rasappa G

2010-03-01

Reported is the adaptation of a manual polysaccharide assay applicable for glycoconjugate vaccines such as Prevenar to an automated liquid handling system (LHS) for improved performance. The anthrone assay is used for carbohydrate concentration determinations and was scaled to the microtiter plate format with appropriate mixing, dispensing, and measuring operations. Adaptation and development of the LHS platform was performed with both dextran polysaccharides of various sizes and pneumococcal serotype 6A polysaccharide (PnPs 6A). A standard plate configuration was programmed such that the LHS diluted both calibration standards and a test sample multiple times with six replicate preparations per dilution. This extent of replication minimized the effect of any single deviation or delivery error that might have occurred. Analysis of the dextran polymers ranging in size from 214 kDa to 3.755 MDa showed that regardless of polymer chain length the hydrolysis was complete, as evident by uniform concentration measurements. No plate positional absorbance bias was observed; of 12 plates analyzed to examine positional bias the largest deviation observed was 0.02% percent relative standard deviation (%RSD). The high purity dextran also afforded the opportunity to assess LHS accuracy; nine replicate analyses of dextran yielded a mean accuracy of 101% recovery. As for precision, a total of 22 unique analyses were performed on a single lot of PnPs 6A, and the resulting variability was 2.5% RSD. This work demonstrated the capability of a LHS to perform the anthrone assay consistently and a reduced assay cycle time for greater laboratory capacity.

The gastric mucosal barrier.

PubMed

Clamp, J R; Ene, D

1989-01-01

The gastric mucosal barrier is a complex system made up of submucosal, epithelial and mucus elements. The mucus gel layer is a thick tenacious organized layer adherent to the epithelium. Despite these properties it is composed of more than 95% water, the organization being provided by long interacting glycoprotein molecules (mucus glycoprotein or mucin). These molecules are largely made up of carbohydrate which is present in large numbers of relatively small oligosaccharide units packed around the polypeptide core. This structure provides clues to the nature of the protection afforded by the mucus layer. For example, it is relatively resistant to proteolysis in the gastrointestinal tract; it retains water in an unstirred layer; the tangled glycoproteins exclude large molecules and the carbohydrate of the oligosaccharide units mirror that at the surface of the epithelial cell. Few biochemical studies have been carried out on the effect of ulcer-healing drugs on gastric mucus. Normal subjects were, therefore, given two weeks treatment with cimetidine, carbenoxolone or misoprostol and the secretions aspirated from the unstimulated and pentagastrin-stimulated stomach. The volume of secretion and weight and carbohydrate content of non-diffusable glycoconjugates were determined for each specimen, together with the proportion of high molecular mass mucin and qualitative and quantitative analyses of the glycopolypeptide. There were no significant differences between the results for each drug or without drug. This may be because normal subjects were studied who already have an effective mucosal barrier. In addition, it is likely that the process of mucus biosynthesis and secretion in a healthy individual is relatively resistant to the action of ulcer healing drugs.

Characterization and Modeling of the Collision Induced Dissociation Patterns of Deprotonated Glycosphingolipids: Cleavage of the Glycosidic Bond

NASA Astrophysics Data System (ADS)

Rožman, Marko

2016-01-01

Glycosphingolipid fragmentation behavior was investigated by combining results from analysis of a series of negative ion tandem mass spectra and molecular modeling. Fragmentation patterns extracted from 75 tandem mass spectra of mainly acidic glycosphingolipid species (gangliosides) suggest prominent cleavage of the glycosidic bonds with retention of the glycosidic oxygen atom by the species formed from the reducing end (B and Y ion formation). Dominant product ions arise from dissociation of sialic acids glycosidic bonds whereas product ions resulting from cleavage of other glycosidic bonds are less abundant. Potential energy surfaces and unimolecular reaction rates of several low-energy fragmentation pathways leading to cleavage of glycosidic bonds were estimated in order to explain observed dissociation patterns. Glycosidic bond cleavage in both neutral (unsubstituted glycosyl group) and acidic glycosphingolipids was the outcome of the charge-directed intramolecular nucleophilic substitution (SN2) mechanism. According to the suggested mechanism, the nucleophile in a form of carboxylate or oxyanion attacks the carbon at position one of the sugar ring, simultaneously breaking the glycosidic bond and yielding an epoxide. For gangliosides, unimolecular reaction rates suggest that dominant product ions related to the cleavage of sialic acid glycosidic bonds are formed via direct dissociation channels. On the other hand, low abundant product ions related to the dissociation of other glycosidic bonds are more likely to be the result of sequential dissociation. Although results from this study mainly contribute to the understanding of glycosphingolipid fragmentation chemistry, some mechanistic findings regarding cleavage of the glycosidic bond may be applicable to other glycoconjugates.

Oocyte adhesiveness and embryonic development of Astyanax bimaculatus (Linnaeus, 1758) (Pisces: Characidae).

PubMed

Weber, André Alberto; Arantes, Fábio Pereira; Sato, Yoshimi; Rizzo, Elizete; Bazzoli, Nilo

2013-05-01

This study shows for the first time the presence of a jelly coat on oocytes of neotropical Characiformes fish. This structure could be responsible for the adhesiveness of Astyanax bimaculatus oocytes, a species widely distributed in South America including in the São Francisco River basin in Brazil. Adult specimens of A. bimaculatus were submitted to artificial reproduction in order to analyse the egg morphology and embryonic development. The eggs were fertilised and kept in incubators with a water temperature of 24°C so that embryogenesis could be monitored. Ovulated and unfertilised oocytes were also collected and submitted to routine histological techniques. Astyanax bimaculatus oocytes were found to be spherical, yellowish, and covered by a thin jelly coat with a slightly adhesive surface. The mean oocyte diameter was 1.03 ± 0.03 mm, the perivitelline space was 0.21 ± 0.02 mm and the jelly coat's thickness was 0.04 ± 0.01 mm. Positive periodic acid-Schiff (PAS) stain and Alcian blue stain pH 2.5 indicated the presence of neutral glycoproteins, and carboxylated acid glycoconjugates on the jelly coat that formed mucosubstances that may be associated with egg adhesiveness. At a water temperature of 24°C, blastopore closure and hatching occurred at 5 h and 17 h after fertilisation, respectively. The results of this study provide essential information for phylogenetic studies and for a better understanding of the reproductive strategy of A. bimaculatus, currently included in the incertae sedis group of the Characidae family due to the lack of monophyly among the families of the group.

Fucoidan as an inhibitor of thermally induced collagen glycation examined by acetate electrophoresis.

PubMed

Pielesz, Anna; Paluch, Jadwiga

2014-08-01

Non-enzymatic glycation (Maillard reaction) in vitro could be a simple method to obtain glycoconjugates for studying their biological properties. Hence, fucoidan was retained by acetate electrophoresis indicating a strong interaction with the protein. A loss of colour in fucoidan bands was found for samples incubated with collagen as compared with samples of free fucoidan. Also under in vitro conditions at 100°C - simulating a sudden burn incident - fucoidan binds with collagen as a result of the Maillard reaction. In contrast, the colour of the fucoidan bands intensified for samples incubated with collagen, with the addition of glucose. Electrophoretic analyses were carried out after heating the samples to a temperature simulating a burn incident. The bands were found to intensify for samples incubated with collagen during a 30-day-long incubation. Thus, spontaneous in vitro glycation - i.e. without the addition of glucose - was confirmed. This process is highly intensified both by the temperature and time of incubation. For a sample incubated in vitro in a fucoidan solution containing glucose, glycation was confirmed in a preliminary FTIR and acetate electrophoresis examinations, occurring in collagen obtained from chicken skins. In particular, a new band emerging around 1746 cm(-1) was observed for above samples, as was its increasing intensity, as compared with samples without the addition of glucose. In the collagen glycation assay, while glucose reacts with collagen and forms cross-linked aggregates, fucoidan decreases the process of aggregation and recovery of native collagen. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The influence of surface carbohydrates during in vitro infection of mammalian cells by the dermatophyte Trichophyton rubrum.

PubMed

Esquenazi, Daniele; Alviano, Celuta S; de Souza, Wanderley; Rozental, Sonia

2004-04-01

In order to better understand the role played by surface glycoconjugates during host cell adhesion and endocytosis of Trichophyton rubrum, we looked for the presence of carbohydrate-binding adhesins on the microconidia surface and their role on cellular interaction with epithelial and macrophages cells. The interaction of T. rubrum with chinese hamster ovary epithelial cells and their glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, that express mannose and galactose, respectively. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates to the interaction medium, pretreatment with lectins and with sodium periodate decreased the adhesion and endocytic index for all mutants. The ability of the fungus to penetrate into mammalian cells was confirmed in experiments using macrophages treated with cytochalasin D. Flow cytometric analysis showed that this fungus recognizes mannose and galactose. The binding was inhibited by the addition of methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside, and showed higher fluorescence intensity at 37 than at 28 degrees C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature for the microconidia adhesins. The presence of lectin-like molecules in fungus cell could be observed by scanning electron microscopy of the fungus incubated with colloidal-gold labeled neoglycoproteins. Our results suggest that T. rubrum has the ability to invade mammalian cells and expresses carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. These adhesins may play an important role on the adhesion and invasion of the fungus during the infectious process of dermatophytosis.

Genetic characteristics and pathogenic mechanisms of periodontal pathogens.

PubMed

Amano, A; Chen, C; Honma, K; Li, C; Settem, R P; Sharma, A

2014-05-01

Periodontal disease is caused by a group of bacteria that utilize a variety of strategies and molecular mechanisms to evade or overcome host defenses. Recent research has uncovered new evidence illuminating interesting aspects of the virulence of these bacteria and their genomic variability. This paper summarizes some of the strategies utilized by the major species - Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis - implicated in the pathogenesis of periodontal disease. Whole-genome sequencing of 14 diverse A. actinomycetemcomitans strains has revealed variations in their genetic content (ranging between 0.4% and 19.5%) and organization. Strikingly, isolates from human periodontal sites showed no genomic changes during persistent colonization. T. forsythia manipulates the cytokine responses of macrophages and monocytes through its surface glycosylation. Studies have revealed that bacterial surface-expressed O-linked glycans modulate T-cell responses during periodontal inflammation. Periodontal pathogens belonging to the "red complex" consortium express neuraminidases, which enables them to scavenge sialic acid from host glycoconjugates. Analysis of recent data has demonstrated that the cleaved sialic acid acts as an important nutrient for bacterial growth and a molecule for the decoration of bacteria surfaces to help evade the host immune attack. In addition, bacterial entry into host cells is also an important prerequisite for the lifestyle of periodontal pathogens such as P. gingivalis. Studies have shown that, after its entry into the cell, this bacterium uses multiple sorting pathways destined for autophagy, lysosomes, or recycling pathways. In addition, P. gingivalis releases outer membrane vesicles which enter cells via endocytosis and cause cellular functional impairment.

Studies on the defect underlying the lysosomal storage of sialic acid in Salla disease. Lysosomal accumulation of sialic acid formed from N-acetyl-mannosamine or derived from low density lipoprotein in cultured mutant fibroblasts.

PubMed Central

Renlund, M; Kovanen, P T; Raivio, K O; Aula, P; Gahmberg, C G; Ehnholm, C

1986-01-01

Salla disease is a lysosomal storage disorder characterized by mental retardation and disturbed sialic acid metabolism. To study endogenous synthesis and breakdown of sialic acid, fibroblasts were incubated for 5 d in the presence and then in the absence of N-[3H]acetylmannosamine. Labeling of free sialic acid was 5-10 times higher in mutant than in normal cells. Radioactivity decreased in 4 d by 75% in normal but only by 30% in mutant fibroblasts. The labeling pattern was not normalized upon coculture of mutant and normal cells. To study the metabolism of extracellular sialic acid, low-density lipoprotein (LDL) was labeled in the sialic acid moiety (periodate-NaB3H4) or in the protein moiety (125I). Binding, internalization, lysosomal degradation, and exit of products of protein catabolism were similar in normal and mutant fibroblasts. Upon incubation with LDL labeled in the sialic acid moiety, mutant cells accumulated 2-3 times more free sialic acid radioactivity than normal fibroblasts, mostly in the lysosomal fraction. After a 24-h chase incubation, radioactivity in free sialic acid decreased by 70-80% in normal but only by 10-30% in mutant cells. In mutant fibroblasts, 40% of the radioactivity remained in lysosomes, whereas no labeled free sialic acid was detected in lysosomes from normal fibroblasts. We conclude that in Salla disease, fibroblast endogenous synthesis of sialic acid and lysosomal cleavage of exogenous glycoconjugates is normal, but free sialic acid cannot leave the lysosome. These findings suggest that the basic defect in Salla disease is deficient transport of free sialic acid through the lysosomal membrane. PMID:3944269

Versatile On-Resin Synthesis of High Mannose Glycosylated Asparagine with Functional Handles

PubMed Central

Chen, Rui; Pawlicki, Mark A.; Tolbert, Thomas J.

2013-01-01

Here we present a synthetic route for solid phase synthesis of N-linked glycoconjugates containing high mannose oligosaccharides which allows the incorporation of useful functional handles on the N-terminus of asparagine. In this strategy, the C-terminus of an Fmoc protected aspartic acid residue is first attached to a solid phase support. The side chain of aspartic acid is protected by a 2-phenylisopropyl protecting group, which allows selective deprotection for the introduction of glycosylation. By using a convergent on-resin glycosylamine coupling strategy, an N-glycosidic linkage is successfully formed on the free side chain of the resin bound aspartic acid with a large high mannose oligosaccharide, Man8GlcNAc2, to yield N-linked high mannose glycosylated asparagine. The use of on-resin glycosylamine coupling provides excellent glycosylation yield, can be applied to couple other types of oligosaccharides, and also makes it possible to recover excess oligosaccharides conveniently after the on-resin coupling reaction. Useful functional handles including an alkene (p-vinylbenzoic acid), an alkyne (4-pentynoic acid), biotin, and 5-carboxyfluorescein are then conjugated onto the N-terminal amine of asparagine on-resin after the removal of the Fmoc protecting group. In this way, useful functional handles are introduced onto the glycosylated asparagine while maintaining the structural integrity of the reducing end of the oligosaccharide. The asparagine side chain also serves as a linker between the glycan and the functional group and preserves the native presentation of N-linked glycan which may aid in biochemical and structural studies. As an example of a biochemical study using functionalized high mannose glycosylated asparagine, a fluorescence polarization assay has been utilized to study the binding of the lectin Concanavalin A (ConA) using 5-carboxyfluorescein labeled high mannose glycosylated asparagine. PMID:24326091

Effects of chlorate on the sulfation process of Trypanosoma cruzi glycoconjugates. Implication of parasite sulfates in cellular invasion.

PubMed

Ferrero, Maximiliano R; Soprano, Luciana L; Acosta, Diana M; García, Gabriela A; Esteva, Mónica I; Couto, Alicia S; Duschak, Vilma G

2014-09-01

Sulfation, a post-translational modification which plays a key role in various biological processes, is inhibited by competition with chlorate. In Trypanosoma cruzi, the agent of Chagas' disease, sulfated structures have been described as part of glycolipids and we have reported sulfated high-mannose type oligosaccharides in the C-T domain of the cruzipain (Cz) glycoprotein. However, sulfation pathways have not been described yet in this parasite. Herein, we studied the effect of chlorate treatment on T. cruzi with the aim to gain some knowledge about sulfation metabolism and the role of sulfated molecules in this parasite. In chlorate-treated epimastigotes, immunoblotting with anti-sulfates enriched Cz IgGs (AS-enriched IgGs) showed Cz undersulfation. Accordingly, a Cz mobility shift toward higher isoelectric points was observed in 2D-PAGE probed with anti-Cz antibodies. Ultrastructural membrane abnormalities and a significant decrease of dark lipid reservosomes were shown by electron microscopy and a significant decrease in sulfatide levels was confirmed by TLC/UV-MALDI-TOF-MS analysis. Altogether, these results suggest T. cruzi sulfation occurs via PAPS. Sulfated epitopes in trypomastigote and amastigote forms were evidenced using AS-enriched IgGs by immunoblotting. Their presence on trypomastigotes surface was demonstrated by flow cytometry and IF with Cz/dCz specific antibodies. Interestingly, the percentage of infected cardiac HL-1 cells decreased 40% when using chlorate-treated trypomastigotes, suggesting sulfates are involved in the invasion process. The same effect was observed when cells were pre-incubated with dCz, dC-T or an anti-high mannose receptor (HMR) antibody, suggesting Cz sulfates and HMR are also involved in the infection process by T. cruzi. Copyright © 2014 Elsevier B.V. All rights reserved.

Extensive enrichment of N-glycolylneuraminic acid in extracellular sialoglycoproteins abundantly synthesized and secreted by human cancer cells.

PubMed

Inoue, Sadako; Sato, Chihiro; Kitajima, Ken

2010-06-01

N-Glycolylneuraminic acid (Neu5Gc) is the second most populous sialic acid (Sia). The only known biosynthetic pathway of Neu5Gc is the hydroxylation of cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac), catalyzed by CMP-Neu5Ac hydroxylase (CMAH). Neu5Gc is abundantly found in mammals except for human, in which CMAH is inactivated due to mutation in the CMAH gene. Evidence has accumulated to show occurrence of Neu5Gc-containing glycoconjugates in sera of cancer patients, human cancerous tissues and cultured human cell lines. Recently, occurrence of natural antibodies against Neu5Gc was shown in healthy humans and is a serious problem for clinical xenotransplantation and stem cell therapies. Studying human occurrence of Neu5Gc is of importance and interest in a broad area of medical sciences. In this study, using a fluorometric high performance liquid chromatography method, we performed quantitative analyses of Sias both inside and in the external environment of the cell and found that (i) incorporation of Neu5Gc was most prominent in soluble glycoproteins found both in the extracellular space and inside the cell as the major Sia compounds. (ii) Of the total Neu5Gc in the Sia compounds that the cells synthesized, 90% was found in the secreted sialoglycoproteins, whereas for Neu5Ac, 70% was found in the secreted sialoglycoproteins. (iii) The Neu5Gc ratio was higher in the secreted sialoglycoproteins (as high as 40% of total Sias) than in intracellular sialoglycoproteins. (iv) The majority of the secreted sialoglycoproteins was anchored on the culture dishes and solubilized by brief trypsin treatment. Based on these findings, a new idea on the mechanism of accumulation of Neu5Gc in cancer cells was proposed.

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans.

PubMed

Katoh, Toshihiko; Katayama, Takane; Tomabechi, Yusuke; Nishikawa, Yoshihide; Kumada, Jyunichi; Matsuzaki, Yuji; Yamamoto, Kenji

2016-10-28

Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Glycan Reader is improved to recognize most sugar types and chemical modifications in the Protein Data Bank.

PubMed

Park, Sang-Jun; Lee, Jumin; Patel, Dhilon S; Ma, Hongjing; Lee, Hui Sun; Jo, Sunhwan; Im, Wonpil

2017-10-01

Glycans play a central role in many essential biological processes. Glycan Reader was originally developed to simplify the reading of Protein Data Bank (PDB) files containing glycans through the automatic detection and annotation of sugars and glycosidic linkages between sugar units and to proteins, all based on atomic coordinates and connectivity information. Carbohydrates can have various chemical modifications at different positions, making their chemical space much diverse. Unfortunately, current PDB files do not provide exact annotations for most carbohydrate derivatives and more than 50% of PDB glycan chains have at least one carbohydrate derivative that could not be correctly recognized by the original Glycan Reader. Glycan Reader has been improved and now identifies most sugar types and chemical modifications (including various glycolipids) in the PDB, and both PDB and PDBx/mmCIF formats are supported. CHARMM-GUI Glycan Reader is updated to generate the simulation system and input of various glycoconjugates with most sugar types and chemical modifications. It also offers a new functionality to edit the glycan structures through addition/deletion/modification of glycosylation types, sugar types, chemical modifications, glycosidic linkages, and anomeric states. The simulation system and input files can be used for CHARMM, NAMD, GROMACS, AMBER, GENESIS, LAMMPS, Desmond, OpenMM, and CHARMM/OpenMM. Glycan Fragment Database in GlycanStructure.Org is also updated to provide an intuitive glycan sequence search tool for complex glycan structures with various chemical modifications in the PDB. http://www.charmm-gui.org/input/glycan and http://www.glycanstructure.org. wonpil@lehigh.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Heat-stable antigen (CD24) as ligand for mouse P-selectin.

PubMed

Sammar, M; Aigner, S; Hubbe, M; Schirrmacher, V; Schachner, M; Vestweber, D; Altevogt, P

1994-07-01

Heat-stable antigen (HSA)/CD24 is a cell surface molecule expressed by many cell types in the mouse. The molecule has an unusual structure because of its small protein core and extensive glycosylation. In order to study the functional role of the HSA-associated glycoconjugates we have isolated different forms of HSA. Using lectin analysis we provide evidence for extensive heterogeneity in carbohydrate composition and sialic acid linkage. Several HSA forms were recognized by mouse P-selectin-IgG but not E-selectin-IgG in ELISA. As expected, P-selectin-IgG also bound to L2/HNK-1-positive neural glycoproteins (L2-glycoproteins) and sulfatides but not to gangliosides and other control glycoproteins. The binding of P-selectin-IgG to L2-glycoproteins and HSA required bivalent cations. The reactivity to HSA was sensitive to sialidase treatment whereas the binding to L2-glycoproteins was not. Studies with alpha 2-6 sialytransferase indicated that alpha 2-6 linked sialic acid was not involved in the P-selectin binding to HSA. Surprisingly, an L2/HNK-1 specific antibody was found to cross-react with some HSA glycoforms and its binding correlated with P-selectin-IgG reactivity. L2/HNK-1-positive or L2/HNK-1-negative HSA glycoforms were also analyzed after coating to polystyrene beads. Only the L2/HNK-1-positive HSA coated beads were reactive with P-selectin-IgG and could bind to activated bend3 endothelioma cells expressing P-selectin whereas the L2/HNK-1-negative HSA beads did not. It is suggested that in its L2/HNK-1 modified form the HSA molecule on leukocytes could represent a ligand for P-selectin on endothelial cells or platelets.

Effect of nutritional supplements on attentional-deficit hyperactivity disorder.

PubMed

Dykman, K D; Dykman, R A

1998-01-01

This study reports the effects of two nutritional products upon the severity of symptoms in children with confirmed diagnoses of Attention-Deficit Hyperactivity Disorder (ADHD): a glyconutritional product containing saccharides known to be important in healthy functioning and a phytonutritional product containing flash-dried fruits and vegetables. Seventeen ADHD children were recruited from a local parent support group. Parents of five of the subjects did not have their children on methylphenidate. Of the remaining twelve, all on methylphenidate, six were left on prescribed doses (random assignment). The other six had their doses reduced by half after two weeks (study duration was six weeks). The subjects were assessed initially and three subsequent times over a period of six weeks (longitudinal nonrandomized design). The behavior disorder items for ADHD, Oppositional Defiant Disorder (ODD), and Conduct Disorder (CD) as listed in the Diagnostic and Statistical Manual for Mental Disorders (DSM IV) (American Psychiatric Association, 1994) were rated by teachers and parents on a 3-point scale. Also included was a Side Effects Scale described by Barkley (1990). The children received the glyconutritional supplement for the entire six weeks. After three weeks, the phytonutritional supplement was added to the diet to increase the probability of positive results. The glyconutritional supplement decreased the number and severity of ADHD, associated ODD and CD symptoms, and side effects in all groups during the first two weeks of the study. There was little further reduction with the addition of the phytonutritional supplement. The three study groups did not differ statistically in degree of reduction over observations. Present results suggest that symptoms of ADHD may be reduced by the addition to the diet of saccharides used by the body in glycoconjugate synthesis.

Leishmania HASP and SHERP Genes Are Required for In Vivo Differentiation, Parasite Transmission and Virulence Attenuation in the Host

PubMed Central

Doehl, Johannes S. P.; Sádlová, Jovana; Aslan, Hamide; Pružinová, Kateřina; Votýpka, Jan; Kamhawi, Shaden; Volf, Petr

2017-01-01

Differentiation of extracellular Leishmania promastigotes within their sand fly vector, termed metacyclogenesis, is considered to be essential for parasites to regain mammalian host infectivity. Metacyclogenesis is accompanied by changes in the local parasite environment, including secretion of complex glycoconjugates within the promastigote secretory gel and colonization and degradation of the sand fly stomodeal valve. Deletion of the stage-regulated HASP and SHERP genes on chromosome 23 of Leishmania major is known to stall metacyclogenesis in the sand fly but not in in vitro culture. Here, parasite mutants deficient in specific genes within the HASP/SHERP chromosomal region have been used to investigate their role in metacyclogenesis, parasite transmission and establishment of infection. Metacyclogenesis was stalled in HASP/SHERP mutants in vivo and, although still capable of osmotaxis, these mutants failed to secrete promastigote secretory gel, correlating with a lack of parasite accumulation in the thoracic midgut and failure to colonise the stomodeal valve. These defects prevented parasite transmission to a new mammalian host. Sand fly midgut homogenates modulated parasite behaviour in vitro, suggesting a role for molecular interactions between parasite and vector in Leishmania development within the sand fly. For the first time, stage-regulated expression of the small HASPA proteins in Leishmania (Leishmania) has been demonstrated: HASPA2 is expressed only in extracellular promastigotes and HASPA1 only in intracellular amastigotes. Despite its lack of expression in amastigotes, replacement of HASPA2 into the null locus background delays onset of pathology in BALB/c mice. This HASPA2-dependent effect is reversed by HASPA1 gene addition, suggesting that the HASPAs may have a role in host immunomodulation. PMID:28095465

Preferential use of lambda light chains is associated with defective mouse antibody responses to the capsular polysaccharide of Neisseria meningitidis group B.

PubMed

Colino, Jesus; Outschoorn, Ingrid

2004-01-01

The capsular polysaccharide of Neisseria meningitidis group B (CpsB) is a very poor immunogen in mammals; this has been considered to be due to the induction of tolerance to cross-reactive host glycoconjugates. It has hampered the development of an effective vaccine against this meningococcal group for many years. Syngeneic populations have a similar tolerogenic background. Thus, we used the variability in ability to mount CpsB-specific immunoglobulin (Ig) responses of individuals from these populations to reveal underlying mechanisms to tolerance contributing to the poor immunogenicity of CpsB. Here we analyze by ELISA, the individual CpsB-specific Ig response of BALB/c and other syngeneic mice to immunization with intact bacteria, using the distribution of light chains as a direct indicator of the repertoire dynamics of the response. Although approximately 96% of anti-CpsB Ig bear kappa-light chains, BALB/c mouse populations were heterogeneous in the light chain composition of their individual anti-CpsB Ig responses. The proportion of kappa and lambda-light chains used for anti-CpsB Ig was a private characteristic that remained relatively constant, for each individual, through repetitive immunizations regardless of the bacterial stimuli size. Despite the prevalence of individual use of kappa-light chains, 5% of BALB/c mice showed restricted usage of lambda-light chains in their CpsB-specific Ig responses, and an additional 11% use them significantly. The preferential use of lambda-light chains in these mice was strongly associated with defective IgM, and absent or barely detectable IgG anti-CpsB responses even after repetitive bacterial immunization. We conclude that differences in the private repertoire of specific Ig also contribute to mouse unresponsiveness to CpsB.

The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach.

PubMed

Harduin-Lepers, Anne; Mollicone, Rosella; Delannoy, Philippe; Oriol, Rafael

2005-08-01

The animal sialyltransferases are Golgi type II transmembrane glycosyltransferases. Twenty distinct sialyltransferases have been identified in both human and murine genomes. These enzymes catalyze transfer of sialic acid from CMP-Neu5Ac to the glycan moiety of glycoconjugates. Despite low overall identities, they share four conserved peptide motifs [L (large), S (small), motif III, and motif VS (very small)] that are hallmarks for sialyltransferase identification. We have identified 155 new putative genes in 25 animal species, and we have exploited two lines of evidence: (1) sequence comparisons and (2) exon-intron organization of the genes. An ortholog to the ancestor present before the split of ST6Gal I and II subfamilies was detected in arthropods. An ortholog to the ancestor present before the split of ST6GalNAc III, IV, V, and VI subfamilies was detected in sea urchin. An ortholog to the ancestor present before the split of ST3Gal I and II subfamilies was detected in ciona, and an ortholog to the ancestor of all the ST8Sia was detected in amphioxus. Therefore, single examples of the four families (ST3Gal, ST6Gal, ST6GalNAc, and ST8Sia) have appeared in invertebrates, earlier than previously thought, whereas the four families were all detected in bony fishes, amphibians, birds, and mammals. As previously hypothesized, sequence similarities among sialyltransferases suggest a common genetic origin, by successive duplications of an ancestral gene, followed by divergent evolution. Finally, we propose predictions on these invertebrates sialyltransferase-related activities that have not previously been demonstrated and that will ultimately need to be substantiated by protein expression and enzymatic activity assays.

Pea lectin receptor-like kinase promotes high salinity stress tolerance in bacteria and expresses in response to stress in planta.

PubMed

Joshi, Amita; Dang, Hung Quang; Vaid, Neha; Tuteja, Narendra

2010-01-01

The plant lectin receptor-like kinases (LecRLKs) are involved in various signaling pathways but their role in salinity stress tolerance has not heretofore been well described. Salinity stress negatively affects plant growth/productivity and threatens food security worldwide. Based on functional gene-mining assay, we have isolated 34 salinity tolerant genes out of one million Escherichia coli (SOLR) transformants containing pea cDNAs grown in 0.8 M NaCl. Sequence analysis of one of these revealed homology to LecRLK, which possesses N-myristilation and N-glycosylation sites thus corroborating the protein to be a glycoconjugate. The homology based computational modeling of the kinase domain suggested high degree of conservation with the protein already known to be stress responsive in plants. The NaCl tolerance provided by PsLecRLK to the above bacteria was further confirmed in E. coli (DH5alpha). In planta studies showed that the expression of PsLecRLK cDNA was significantly upregulated in response to NaCl as compared to K(+) and Li(+) ions, suggesting the Na(+) ion specific response. Transcript of the PsLecRLK gene accumulates mainly in roots and shoots. The purified 47 kDa recombinant PsLecRLK-KD (kinase domain) protein has been shown to phosphorylate general substrates like MBP and casein. This study not only suggests the conservation of the cellular response to high salinity stress across prokaryotes and plant kingdom but also provides impetus to develop novel concepts for better understanding of mechanism of stress tolerance in bacteria and plants. It also opens up new avenues for studying practical aspects of plant salinity tolerance for enhanced agricultural productivity.

Human Gastric Mucins Differently Regulate Helicobacter pylori Proliferation, Gene Expression and Interactions with Host Cells

PubMed Central

Skoog, Emma C.; Sjöling, Åsa; Navabi, Nazanin; Holgersson, Jan; Lundin, Samuel B.; Lindén, Sara K.

2012-01-01

Helicobacter pylori colonizes the mucus niche of the gastric mucosa and is a risk factor for gastritis, ulcers and cancer. The main components of the mucus layer are heavily glycosylated mucins, to which H. pylori can adhere. Mucin glycosylation differs between individuals and changes during disease. Here we have examined the H. pylori response to purified mucins from a range of tumor and normal human gastric tissue samples. Our results demonstrate that mucins from different individuals differ in how they modulate both proliferation and gene expression of H. pylori. The mucin effect on proliferation varied significantly between samples, and ranged from stimulatory to inhibitory, depending on the type of mucins and the ability of the mucins to bind to H. pylori. Tumor-derived mucins and mucins from the surface mucosa had potential to stimulate proliferation, while gland-derived mucins tended to inhibit proliferation and mucins from healthy uninfected individuals showed little effect. Artificial glycoconjugates containing H. pylori ligands also modulated H. pylori proliferation, albeit to a lesser degree than human mucins. Expression of genes important for the pathogenicity of H. pylori (babA, sabA, cagA, flaA and ureA) appeared co-regulated in response to mucins. The addition of mucins to co-cultures of H. pylori and gastric epithelial cells protected the viability of the cells and modulated the cytokine production in a manner that differed between individuals, was partially dependent of adhesion of H. pylori to the gastric cells, but also revealed that other mucin factors in addition to adhesion are important for H. pylori-induced host signaling. The combined data reveal host-specific effects on proliferation, gene expression and virulence of H. pylori due to the gastric mucin environment, demonstrating a dynamic interplay between the bacterium and its host. PMID:22563496

Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of ganglioside biosynthesis.

PubMed

Harlalka, Gaurav V; Lehman, Anna; Chioza, Barry; Baple, Emma L; Maroofian, Reza; Cross, Harold; Sreekantan-Nair, Ajith; Priestman, David A; Al-Turki, Saeed; McEntagart, Meriel E; Proukakis, Christos; Royle, Louise; Kozak, Radoslaw P; Bastaki, Laila; Patton, Michael; Wagner, Karin; Coblentz, Roselyn; Price, Joy; Mezei, Michelle; Schlade-Bartusiak, Kamilla; Platt, Frances M; Hurles, Matthew E; Crosby, Andrew H

2013-12-01

Glycosphingolipids are ubiquitous constituents of eukaryotic plasma membranes, and their sialylated derivatives, gangliosides, are the major class of glycoconjugates expressed by neurons. Deficiencies in their catabolic pathways give rise to a large and well-studied group of inherited disorders, the lysosomal storage diseases. Although many glycosphingolipid catabolic defects have been defined, only one proven inherited disease arising from a defect in ganglioside biosynthesis is known. This disease, because of defects in the first step of ganglioside biosynthesis (GM3 synthase), results in a severe epileptic disorder found at high frequency amongst the Old Order Amish. Here we investigated an unusual neurodegenerative phenotype, most commonly classified as a complex form of hereditary spastic paraplegia, present in families from Kuwait, Italy and the Old Order Amish. Our genetic studies identified mutations in B4GALNT1 (GM2 synthase), encoding the enzyme that catalyzes the second step in complex ganglioside biosynthesis, as the cause of this neurodegenerative phenotype. Biochemical profiling of glycosphingolipid biosynthesis confirmed a lack of GM2 in affected subjects in association with a predictable increase in levels of its precursor, GM3, a finding that will greatly facilitate diagnosis of this condition. With the description of two neurological human diseases involving defects in two sequentially acting enzymes in ganglioside biosynthesis, there is the real possibility that a previously unidentified family of ganglioside deficiency diseases exist. The study of patients and animal models of these disorders will pave the way for a greater understanding of the role gangliosides play in neuronal structure and function and provide insights into the development of effective treatment therapies.

Effect of structural modifications of ganglioside GM2 on intra-molecular carbohydrate-to-carbohydrate interaction and enzymatic susceptibility

PubMed Central

Li, Yu-Teh; Li, Su-Chen; Kiso, Makoto; Ishida, Hideharu; Mauri, Laura; Raimondi, Laura; Bernardi, Anna; Sonnino, Sandro

2008-01-01

Summary The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcβ1Π4(Neu5Acα2Π3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to β-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcβ1Π4Gal linkage in GM2 was converted to the GalNAcβ1Π6Gal, both the GalNAc and the Neu5Ac became susceptible to β-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates. PMID:17967427

Immunization of breast cancer patients using a synthetic sialyl-Tn glycoconjugate plus Detox adjuvant.

PubMed

MacLean, G D; Reddish, M; Koganty, R R; Wong, T; Gandhi, S; Smolenski, M; Samuel, J; Nabholtz, J M; Longenecker, B M

1993-01-01

We have synthesized various formulations that have potential for active specific immunotherapy (ASI) of human cancers. Sialyl-Tn (STn) is a potentially important target structure for ASI because its expression on mucins is a strong, independent predictor of poor prognosis, suggesting that it may have functional significance in the metastatic process. In this first pilot study of synthetic sialyl-Tn hapten conjugated to keyhole limpet hemocyanin (STn-KLH), with Detox adjuvant, toxicity and humoral immunogenicity were assessed in 12 patients with metastatic breast cancer. Toxicity was minimal, restricted to local cutaneous reactions (apart from transient nausea and vomiting following single low-dose cyclophosphamide treatment). Using STn-conjugated human serum albumin in a solid-phase enzyme-linked immunosorbent assay, it was shown that all patients developed IgM and IgG specific for the synthetic STn hapten. Following immunization, most patients were shown to develop increased titres of complement-mediated cytotoxic antibodies, partially inhibited by synthetic STn hapten, but not by the related TF hapten. We also detected IgM and IgG antibodies reactive with natural STn determinants expressed on ovine submaxillary mucin, the STn specificity of this reactivity being confirmed by hapten inhibition. Evaluation of clinical efficacy in a small pilot study is difficult. Five patients are alive 12 or more months after entry, and another 4 patients are alive 6 or more months after entry into the study. All 3 patients with known widespread bulky disease progressed despite ASI, 2 having died from widespread cancer. Two patients had partial responses, each lasting 6 months. While several patients had disease stability for 3-10 months, 1 patient with pulmonary metastases remains stable 15 months after entry into the program.

Post-metamorphic development of skin glands in a true toad: Parotoids versus dorsal skin.

PubMed

Regueira, Eleonora; Dávila, Camila; Sassone, Alina G; O'Donohoe, María E Ailín; Hermida, Gladys N

2017-05-01

Chemical defenses in amphibians are a common antipredatory and antimicrobial strategy related to the presence of dermal glands that synthesize and store toxic or unpalatable substances. Glands are either distributed throughout the skin or aggregated in multiglandular structures, being the parotoids the most ubiquitous macrogland in toads of Bufonidae. Even though dermal glands begin to develop during late-larval stages, many species, including Rhinella arenarum, have immature glands by the end of metamorphosis, and their post-metamorphic growth is unknown. Herein, we compared the post-metamorphic development of parotoids and dorsal glands by histological and allometric studies in a size series of R. arenarum. Histological and histochemical studies to detect proteins, acidic glycoconjugates, and catecholamines, showed that both, parotoids and dorsal glands, acquire characteristics of adults in individuals larger than 50 mm; that is, a moment in which the cryptic coloration disappears. Parotoid height increased allometrically as a function of body size, whereas the size of small dorsal glands decreased with body size. The number of glands in the dorsum was not linearly related to body size, appearing to be an individual characteristic. Only adult specimens had intraepithelial granular glands in the duct of the largest glands of the parotoids. Since toxic secretions accumulate in the central glands of parotoids, allometric growth of parotoids may translate into greater protection from predators in the largest animals. Conversely, large glands in the dorsum, which produce a proteinaceous secretion of unknown function, grow isometrically to body size. Some characteristics, like intraepithelial glands in the ducts and basophilic glands in the dorsum, are limited to adults. © 2017 Wiley Periodicals, Inc.

Characterization of the Antigenic Heterogeneity of Lipoarabinomannan, the Major Surface Glycolipid of Mycobacterium tuberculosis, and Complexity of Antibody Specificities toward This Antigen

PubMed Central

Choudhary, Alok; Honnen, William; Lai, Zhong; Gennaro, Maria Laura; Garcia-Viveros, Moncerrato; Sahloul, Kamar; Spencer, John S.; Chatterjee, Delphi

2018-01-01

Lipoarabinomannan (LAM), the major antigenic glycolipid of Mycobacterium tuberculosis, is an important immunodiagnostic target for detecting tuberculosis (TB) infection in HIV-1–coinfected patients, and is believed to mediate a number of functions that promote infection and disease development. To probe the human humoral response against LAM during TB infection, several novel LAM-specific human mAbs were molecularly cloned from memory B cells isolated from infected patients and grown in vitro. The fine epitope specificities of these Abs, along with those of a panel of previously described murine and phage-derived LAM-specific mAbs, were mapped using binding assays against LAM Ags from several mycobacterial species and a panel of synthetic glycans and glycoconjugates that represented diverse carbohydrate structures present in LAM. Multiple reactivity patterns were seen that differed in their specificity for LAM from different species, as well as in their dependence on arabinofuranoside branching and nature of capping at the nonreducing termini. Competition studies with mAbs and soluble glycans further defined these epitope specificities and guided the design of highly sensitive immunodetection assays capable of detecting LAM in urine of TB patients, even in the absence of HIV-1 coinfection. These results highlighted the complexity of the antigenic structure of LAM and the diversity of the natural Ab response against this target. The information and novel reagents described in this study will allow further optimization of diagnostic assays for LAM and may facilitate the development of potential immunotherapeutic approaches to inhibit the functional activities of specific structural motifs in LAM. PMID:29610143

Identification of lipopolysaccharide-interacting plasma membrane-type proteins in Arabidopsis thaliana.

PubMed

Vilakazi, Cornelius S; Dubery, Ian A; Piater, Lizelle A

2017-02-01

Lipopolysaccharide (LPS) is an amphiphatic bacterial glycoconjugate found on the external membrane of Gram-negative bacteria. This endotoxin is considered as a microbe-associated molecular pattern (MAMP) molecule and has been shown to elicit defense responses in plants. Here, LPS-interacting proteins from Arabidopsis thaliana plasma membrane (PM)-type fractions were captured and identified in order to investigate those involved in LPS perception and linked to triggering of innate immune responses. A novel proteomics-based affinity-capture strategy coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for the enrichment and identification of LPS-interacting proteins. As such, LPS isolated from Burkholderia cepacia (LPS B.cep. ) was immobilized on three independent and distinct affinity-based matrices to serve as bait for interacting proteins from A. thaliana leaf and callus tissue. These were resolved by 1D electrophoresis and identified by mass spectrometry. Proteins specifically bound to LPS B.cep. have been implicated in membrane structure (e.g. COBRA-like and tubulin proteins), membrane trafficking and/or transport (e.g. soluble NSF attachment protein receptor (SNARE) proteins, patellin, aquaporin, PM instrinsic proteins (PIP) and H + -ATPase), signal transduction (receptor-like kinases and calcium-dependent protein kinases) as well as defense/stress responses (e.g. hypersensitive-induced response (HIR) proteins, jacalin-like lectin domain-containing protein and myrosinase-binding proteins). The novel affinity-capture strategy for the enrichment of LPS-interacting proteins proved to be effective, especially in the binding of proteins involved in plant defense responses, and can thus be used to elucidate LPS-mediated molecular recognition and disease mechanism(s). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Core 1-derived O-glycans are essential E-selectin ligands on neutrophils.

PubMed

Yago, Tadayuki; Fu, Jianxin; McDaniel, J Michael; Miner, Jonathan J; McEver, Rodger P; Xia, Lijun

2010-05-18

Neutrophils roll on E-selectin in inflamed venules through interactions with cell-surface glycoconjugates. The identification of physiologic E-selectin ligands on neutrophils has been elusive. Current evidence suggests that P-selectin glycoprotein ligand-1 (PSGL-1), E-selectin ligand-1 (ESL-1), and CD44 encompass all glycoprotein ligands for E-selectin; that ESL-1 and CD44 use N-glycans to bind to E-selectin; and that neutrophils lacking core 2 O-glycans have partially defective interactions with E-selectin. These data imply that N-glycans on ESL-1 and CD44 and O-glycans on PSGL-1 constitute all E-selectin ligands, with neither glycan subset having a dominant role. The enzyme T-synthase transfers Gal to GalNAcalpha1-Ser/Thr to form the core 1 structure Galbeta1-3GalNAcalpha1-Ser/Thr, a precursor for core 2 and extended core 1 O-glycans that might serve as selectin ligands. Here, using mice lacking T-synthase in endothelial and hematopoietic cells, we found that E-selectin bound to CD44 and ESL-1 in lysates of T-synthase-deficient neutrophils. However, the cells exhibited markedly impaired rolling on E-selectin in vitro and in vivo, failed to activate beta2 integrins while rolling, and did not emigrate into inflamed tissues. These defects were more severe than those of neutrophils lacking PSGL-1, CD44, and the mucin CD43. Our results demonstrate that core 1-derived O-glycans are essential E-selectin ligands; that some of these O-glycans are on protein(s) other than PSGL-1, CD44, and CD43; and that PSGL-1, CD44, and ESL-1 do not constitute all glycoprotein ligands for E-selectin.

Gibberellic Acid, Synthetic Auxins, and Ethylene Differentially Modulate α-l-Arabinofuranosidase Activities in Antisense 1-Aminocyclopropane-1-Carboxylic Acid Synthase Tomato Pericarp Discs1

PubMed Central

Sozzi, Gabriel O.; Greve, L. Carl; Prody, Gerry A.; Labavitch, John M.

2002-01-01

α-l-Arabinofuranosidases (α-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different α-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. α-Af I and II are active throughout fruit ontogeny. α-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. α-Af II activity accounts for over 80% of the total α-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, α-Af III is ethylene dependent and specifically active during ripening. α-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas α-Af II and III acted on Na2CO3-soluble pectins. Different α-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. α-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only α-Af III activity. Results suggest that tomato α-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production. PMID:12114586

Immunogenicity and safety of investigational vaccine formulations against meningococcal serogroups A, B, C, W, and Y in healthy adolescents

PubMed Central

Saez-Llorens, Xavier; Aguilera Vaca, Diana Catalina; Abarca, Katia; Maho, Emmanuelle; Graña, Maria Gabriela; Heijnen, Esther; Smolenov, Igor; Dull, Peter M

2015-01-01

This phase 2 study assessed the immunogenicity, safety, and reactogenicity of investigational formulations of meningococcal ABCWY vaccines, consisting of recombinant proteins (rMenB) and outer membrane vesicle (OMV) components of a licensed serogroup B vaccine, combined with components of a licensed quadrivalent meningococcal glycoconjugate vaccine (MenACWY-CRM). A total of 495 healthy adolescents were randomized to 6 groups to receive 2 doses (Months 0, 2) of one of 4 formulations of rMenB antigens, with or without OMV, combined with MenACWY-CRM, or 2 doses of rMenB alone or one dose of MenACWY-CRM then a placebo. Immunogenicity was assessed by serum bactericidal assay with human complement (hSBA) against serogroups ACWY and serogroup B test strains; solicited reactions and any adverse events (AEs) were assessed. Two MenABCWY vaccinations elicited robust ACWY immune responses, with higher seroresponse rates than one dose of MenACWY-CRM. Bactericidal antibody responses against the rMenB antigens and OMV components were highest in subjects who received 2 doses of OMV-containing MenABCWY formulations, with ≥68% of subjects achieving hSBA titers ≥5 against each of the serogroup B test strains. After the first dose, solicited local reaction rates were higher in the MenABCWY or rMenB groups than the MenACWY-CRM group, but similar across groups after the second dose, consisting mainly of transient injection site pain. Fever (≥38.0°C) was rare and there were no vaccine-related serious AEs. In conclusion, investigational MenABCWY formulations containing OMV components elicited highly immunogenic responses against meningococcal serogroups ACWY, as well as serogroup B test strains, with an acceptable safety profile. [NCT01210885] PMID:25969894

GLUT1-mediated selective tumor targeting with fluorine containing platinum(II) glycoconjugates

PubMed Central

Liu, Ran; Fu, Zheng; Zhao, Meng; Gao, Xiangqian; Li, Hong; Mi, Qian; Liu, Pengxing; Yang, Jinna; Yao, Zhi; Gao, Qingzhi

2017-01-01

Increased glycolysis and overexpression of glucose transporters (GLUTs) are physiological characteristics of human malignancies. Based on the so-called Warburg effect, 18flurodeoxyglucose-positron emission tomography (FDG-PET) has successfully developed as clinical modality for the diagnosis and staging of many cancers. To leverage this glucose transporter mediated metabolic disparity between normal and malignant cells, in the current report, we focus on the fluorine substituted series of glucose, mannose and galactose-conjugated (trans-R,R-cyclohexane-1,2-diamine)-2-flouromalonato-platinum(II) complexes for a comprehensive evaluation on their selective tumor targeting. Besides highly improved water solubility, these sugar-conjugates presented improved cytotoxicity than oxaliplatin in glucose tranporters (GLUTs) overexpressing cancer cell lines and exhibited no cross-resistance to cisplatin. For the highly water soluble glucose-conjugated complex (5a), two novel in vivo assessments were conducted and the results revealed that 5a was more efficacious at a lower equitoxic dose (70% MTD) than oxaliplatin (100% MTD) in HT29 xenograft model, and it was significantly more potent than oxaliplatin in leukemia-bearing DBA/2 mice as well even at equimolar dose levels (18% vs 90% MTD). GLUT inhibitor mediated cell viability analysis, GLUT1 knockdown cell line-based cytotoxicity evaluation, and platinum accumulation study demonstrated that the cellular uptake of the sugar-conjugates was regulated by GLUT1. The higher intrinsic DNA reactivity of the sugar-conjugates was confirmed by kinetic study of platinum(II)-guanosine adduct formation. The mechanistic origin of the antitumor effect of the fluorine complexes was found to be forming the bifunctional Pt-guanine-guanine (Pt-GG) intrastrand cross-links with DNA. The results provide a rationale for Warburg effect targeted anticancer drug design. PMID:28467806

GLUT1-mediated selective tumor targeting with fluorine containing platinum(II) glycoconjugates.

PubMed

Liu, Ran; Fu, Zheng; Zhao, Meng; Gao, Xiangqian; Li, Hong; Mi, Qian; Liu, Pengxing; Yang, Jinna; Yao, Zhi; Gao, Qingzhi

2017-06-13

Increased glycolysis and overexpression of glucose transporters (GLUTs) are physiological characteristics of human malignancies. Based on the so-called Warburg effect, 18flurodeoxyglucose-positron emission tomography (FDG-PET) has successfully developed as clinical modality for the diagnosis and staging of many cancers. To leverage this glucose transporter mediated metabolic disparity between normal and malignant cells, in the current report, we focus on the fluorine substituted series of glucose, mannose and galactose-conjugated (trans-R,R-cyclohexane-1,2-diamine)-2-flouromalonato-platinum(II) complexes for a comprehensive evaluation on their selective tumor targeting. Besides highly improved water solubility, these sugar-conjugates presented improved cytotoxicity than oxaliplatin in glucose tranporters (GLUTs) overexpressing cancer cell lines and exhibited no cross-resistance to cisplatin. For the highly water soluble glucose-conjugated complex (5a), two novel in vivo assessments were conducted and the results revealed that 5a was more efficacious at a lower equitoxic dose (70% MTD) than oxaliplatin (100% MTD) in HT29 xenograft model, and it was significantly more potent than oxaliplatin in leukemia-bearing DBA/2 mice as well even at equimolar dose levels (18% vs 90% MTD). GLUT inhibitor mediated cell viability analysis, GLUT1 knockdown cell line-based cytotoxicity evaluation, and platinum accumulation study demonstrated that the cellular uptake of the sugar-conjugates was regulated by GLUT1. The higher intrinsic DNA reactivity of the sugar-conjugates was confirmed by kinetic study of platinum(II)-guanosine adduct formation. The mechanistic origin of the antitumor effect of the fluorine complexes was found to be forming the bifunctional Pt-guanine-guanine (Pt-GG) intrastrand cross-links with DNA. The results provide a rationale for Warburg effect targeted anticancer drug design.

Development of Subunit Vaccines that Provide High Level Protection and Sterilizing Immunity Against Acute Inhalational Melioidosis.

PubMed

Burtnick, Mary N; Shaffer, Teresa L; Ross, Brittany N; Muruato, Laura A; Sbrana, Elena; DeShazer, David; Torres, Alfredo G; Brett, Paul J

2017-11-06

Burkholderia pseudomallei , the etiologic agent of melioidosis, causes severe disease in humans and animals. Diagnosis and treatment of melioidosis can be challenging and no licensed vaccines currently exist. Several studies have shown that this pathogen expresses a variety of structurally conserved protective antigens that include cell-surface polysaccharides and cell-associated/-secreted proteins. Based on this, such antigens have become important components of the subunit vaccine candidates that we are currently developing. In the present study, the 6-deoxyheptan capsular polysaccharide (CPS) from B. pseudomallei was purified, chemically activated and covalently linked to recombinant CRM197 diphtheria toxin mutant (CRM197) to produce CPS-CRM197. Additionally, tandem nickel-cobalt affinity chromatography was used to prepare highly purified recombinant B. pseudomallei Hcp1 and TssM proteins. Immunization of C57BL/6 mice with CPS-CRM197 produced high-titer IgG and opsonizing antibody responses against the CPS component of the glycoconjugate while immunization with Hcp1 and TssM produced high titer IgG and robust IFN-γ secreting T cell responses against the proteins. Extending upon these studies, we found that when vaccinated with a combination of CPS-CRM197 plus Hcp1, 100% of the mice survived a lethal inhalational challenge of B. pseudomallei Remarkably, 70% of the survivors had no culturable bacteria in their lungs, livers or spleens indicating that the vaccine formulation had generated sterilizing immune responses. Collectively, these studies help to better establish surrogates of antigen-induced immunity against B. pseudomallei as well as provide valuable insights towards the development of a safe, affordable and effective melioidosis vaccine. Copyright © 2017 Burtnick et al.