Position Of Galactose Research Articles

Highly specific and rapid glycan based amperometric detection of influenza viruses

Influenza is a respiratory disease which infects 0.4–0.6 billion children and 0.2–5.0 billion adults worldwide. In addition to seasonal influenza, pandemic strains can infect a large number of people rapidly. For example, the H1N1 2009 “swine” strain pandemic reported in March 2009 in Mexico spread rapidly to over 74 countries in less than two months. Rapid and precise detection of influenza viruses is critical for appropriate interventions. Current methods such as cell culture and PCR are expensive and require well-trained personnel, although they are highly accurate. Antibody based tests are not sensitive. We recently developed an electrochemical assay to detect 19 influenza viruses using a glucose bearing substrate in 1h.1 However, neuraminidase (NA) from bacteria can also cleave this substrate. Additionally, time to results must be less than 15 minutes because patients cannot wait for 1h if this assay is going to be used in a clinical setting. Herein, we have designed and synthesized an influenza specific substrate 4,7di-OMeSα2,3/6Gal (4,7di-OMe N-acetylneuraminic acid attached to the 3 or 6 position of galactose), and developed an assay that uses a portable potentiostat and commercial Accu-Chek strips. We demonstrated that this assay detects the influenza virus with high specificity within 15 minutes. Influenza NA, not bacterial NA, cleaved 4,7di-OMeSα2,3/6Gal to release galactose. In contrast, viral and bacterial NA both cleaved the natural substrate Sα2,3/6Gal (N-acetylneuraminic acid attached to the 3 or 6 position of galactose). The released galactose was detected amperometrically using a handheld potentiostat and dehydrogenase bearing glucose test strips. The specificity for influenza was confirmed using different influenza strains and respiratory pathogens such as Streptococcus pneumoniae and Haemophilus influenzae; bacteria do not cleave 4,7di-OMeSα2,3/6Gal. The assay was also used to detect co-infections caused by influenza virus and Streptococcus pneumoniae. Viral drug susceptibility and testing with human clinical samples was successful within 15 minutes, indicating that this assay could be used to rapidly detect influenza viruses at primary care or resource poor settings using ubiquitous glucose meters.2 Support or Funding Information NIH-NIAID (R01-AI089450) Workflow and structure of the glycans used for the electrochemical detection This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glycoamino Acid Analogues of the Thomsen-Friedenreich Tumor-Associated Carbohydrate Antigen: Synthesis and Evaluation of Novel Antiproliferative Factor Glycopeptides.

Glycoamino acid analogues of the Thomsen–Friedenreich antigen disaccharide, where the 4′ and 4″ hydroxyl groups were substituted with fluorine or hydrogen, were synthesized and incorporated into the asialylated antiproliferative factor (as-APF), a biologically active form of APF, a glycopeptide found in the urine of patients with interstitial cystitis. Various strategies were employed to incorporate the fluorine atom at the 4-positions of either the galactose or N-acetylgalactosamine unit of the disaccharide antigen, based on stereochemistry and reactivity. These glycopeptides were evaluated in antiproliferative assays on both primary normal bladder epithelial cells and T24 bladder carcinoma cells. Unlike many previously published substitutions to APF, mono-4′-fluorination of the GalNAc residue did not affect the activity, whereas fluoro-derivatives of the galactose 4″-position or both 4′ and 4″ hydroxyls showed a reduced potency relative to the monosubstituted GalNAc derivative. A fourth compound where the 4″ position of galactose was deoxygenated showed a lower potency than the parent and monosubstituted compounds. These results suggest that specific substitutions in the sugar moieties in the APF can be tolerated, and the glycomimetic design of APF analogues can include fluorine in the GalNAc sugar of the disaccharide.

Phase-Variable Changes in the Position of O-Methyl Phosphoramidate Modifications on the Polysaccharide Capsule of Campylobacter jejuni Modulate Serum Resistance.

Campylobacter jejuni polysaccharide capsules (CPS) are characterized by the presence of nonstoichiometric O-methyl phosphoramidate (MeOPN) modifications. The lack of stoichiometry is due to phase variation at homopolymeric tracts within the MeOPN transferase genes. C. jejuni strain 81-176 contains two MeOPN transferase genes and has been shown previously to contain MeOPN modifications at the 2 and 6 positions of the galactose (Gal) moiety in the CPS. We demonstrate here that one of the two MeOPN transferases, encoded by CJJ81176_1435, is bifunctional and is responsible for the addition of MeOPN to both the 2 and the 6 positions of Gal. A new MeOPN at the 4 position of Gal was observed in a mutant lacking the CJJ81176_1435 transferase and this was encoded by the CJJ81176_1420 transferase. During routine growth of 81-176, the CJJ81176_1420 transferase was predominantly in an off configuration, while the CJJ81176_1435 transferase was primarily on. However, exposure to normal human serum selected for cells expressing the CJJ81176_1420 transferase. MeOPN modifications appear to block binding of naturally occurring antibodies to the 81-176 CPS. The absence of MeOPN-4-Gal resulted in enhanced sensitivity to serum killing, whereas the loss of MeOPN-2-Gal and MeOPN-6-Gal resulted in enhanced resistance to serum killing, perhaps by allowing more MeOPN to be put onto the 4 position of Gal.IMPORTANCECampylobacter jejuni undergoes phase variation in genes encoding surface antigens, leading to the concept that a strain of this organism consists of multiple genotypes that are selected for fitness in various environments. Methyl phosphoramidate modifications on the capsule of C. jejuni block access of preexisting antibodies in normal human sera to the polysaccharide chain, thus preventing activation of the classical arm of the complement cascade. We show that the capsule of strain 81-176 contains more sites of MeOPN modifications than previously recognized and that one site, on the 4 position of galactose, is more critical to complement resistance than the others. Exposure to normal human serum selects for variants in the population expressing this MeOPN modification.

Theoretical investigation on the binding specificity of fluorinated sialyldisaccharides Neu5Acα(2–3)Gal and Neu5Acα(2–6)Gal with influenza hemagglutinin H1 – A Molecular Dynamics Study

ABSTRACTIn the present study, the hydroxyl groups at the C4 and C7 positions of sialic acid and C6 position of galactose in Neu5Acα(2–3)Gal (N23G) and the hydroxyl groups at the C8 position of sialic acid and C3 and C4 positions of galactose in Neu5Acα(2–6)Gal (N26G) were substituted with fluorine atoms, respectively. Molecular dynamics simulations of 100 ns duration were carried out to investigate the structural and dynamical behavior of H1 bound with the tri-fluorinated N23G and N26G (FN23G and FN26G). Based on energy analysis, it was concluded that FN26G should be a better binder for hemagglutinin (H1) than FN23G and it might act as an inhibitor for influenza.

Highly specific and rapid glycan based amperometric detection of influenza viruses.

Rapid and precise detection of influenza viruses in a point of care setting is critical for applying appropriate countermeasures. Current methods such as nucleic acid or antibody based techniques are expensive or suffer from low sensitivity, respectively. We have developed an assay that uses glucose test strips and a handheld potentiostat to detect the influenza virus with high specificity. Influenza surface glycoprotein neuraminidase (NA), but not bacterial NA, cleaved galactose bearing substrates, 4,7di-OMe N-acetylneuraminic acid attached to the 3 or 6 position of galactose, to release galactose. In contrast, viral and bacterial NA cleaved the natural substrate, N-acetylneuraminic acid attached to the 3 or 6 position of galactose. The released galactose was detected amperometrically using a handheld potentiostat and dehydrogenase bearing glucose test strips. The specificity for influenza was confirmed using influenza strains and different respiratory pathogens that include Streptococcus pneumoniae and Haemophilus influenzae; bacteria do not cleave these molecules. The assay was also used to detect co-infections caused by influenza and bacterial NA. Viral drug susceptibility and testing with human clinical samples was successful in 15 minutes, indicating that this assay could be used to rapidly detect influenza viruses at primary care or resource poor settings using ubiquitous glucose meters.

Galactose 6-O-Sulfotransferases Are Not Required for the Generation of Siglec-F Ligands in Leukocytes or Lung Tissue

Eosinophil accumulation is a characteristic feature of the immune response to parasitic worms and allergens. The cell surface carbohydrate-binding receptor Siglec-F is highly expressed on eosinophils and negatively regulates their accumulation during inflammation. Although endogenous ligands for Siglec-F have yet to be biochemically defined, binding studies using glycan arrays have implicated galactose 6-O-sulfate (Gal6S) as a partial recognition determinant for this receptor. Only two sulfotransferases are known to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase 1 (C6ST-1). Here we use mice deficient in both KSGal6ST and C6ST-1 to determine whether these sulfotransferases are required for the generation of endogenous Siglec-F ligands. First, we characterize ligand expression on leukocyte populations and find that ligands are predominantly expressed on cell types also expressing Siglec-F, namely eosinophils, neutrophils, and alveolar macrophages. We also detect Siglec-F ligand activity in bronchoalveolar lavage fluid fractions containing polymeric secreted mucins, including MUC5B. Consistent with these observations, ligands in the lung increase dramatically during infection with the parasitic nematode, Nippostrongylus brasiliensis, which is known to induce eosinophil accumulation and mucus production. Surprisingly, Gal6S is undetectable in sialylated glycans from eosinophils and BAL fluid analyzed by mass spectrometry. Furthermore, none of the ligands we describe are diminished in mice lacking KSGal6ST and C6ST-1, indicating that neither of the known galactose 6-O-sulfotransferases is required for ligand synthesis. These results establish that ligands for Siglec-F are present on several cell types that are relevant during allergic lung inflammation and argue against the widely held view that Gal6S is critical for glycan recognition by this receptor.

Functional and structural characterization of potent Th1 biasing 6′-derivatised α-GalCer iNKT cell agonists, and their superior role in tumor protection. (156.4)

Abstract Invariant natural killer T cells are known to have marked immunomodulatory capacity due to their ability to produce copious amounts of effector cytokines. In this study, we report the first crystal structures of a novel class of strong Th1 biasing structural analogues of α-galactosylceramide by addition of aromatic structures on the 6-OH position of galactose. They are characterized by marked Th1 polarized cytokine patterns that are highly conserved between mice and men, and marked tumour protection in vivo. The strength of the Th1 response correlates well with enhanced lipid binding to CD1d as a result of an induced fit mechanism that binds the aromatic substitution as a third anchor, in addition to the two lipid chains. This induced fit is in contrast to another Th1 biasing glycolipid, α-C-GalCer, whose CD1d binding follows a conventional key-lock principle. These findings highlight the previously unexploited flexibility of CD1d in accommodating galactose-modified glycolipids and broaden the range of glycolipids that can stimulate iNKT cells. We speculate that glycolipids can be designed that induce a similar fit, thereby leading to superior and more sustained iNKT cell responses in vivo.

Specific modification of xyloglucan from Hymenaea courbaril seeds

In this work, xyloglucan extracted from the seeds of Hymenaea courbaril (jatobá) collected in Rancho Alegre, Paraná, Brazil (JRAXG), was oxidized at the C-6 position of galactose or glucose units, using 2,3,6,6-tetramethylpiperidine-1-oxyl, to form uronic acid-containning polysaccharides (JRAOXGs), with different degrees of oxidation (10, 23 and 34%) and high yield (~ 95%). The properties of the news polyelectrolyte were studied by intrinsic viscosity, Gel Permeation Chromatography (GPC), Infrared spectroscopy (FTIR), 13C-NMR ( 13C-Nuclear Magnetic Resonance), and by Atomic Force Microscopy (AFM). The FTIR and 13C-NMR spectra confirmed the oxidation process. In comparison to JRA XG the JRAOXGs derivatives showed by GPC a homogeneous profile, but with hydrodynamic volume and molecular weight (Mw) reduction. Furthermore, AFM images on mica, after dewetting hydrodynamic volume, show different states of aggregation accordingly to the degree of oxidation when compared to the native state. The native XG presents a rather compact and quite homogeneous topography with a few holes and absence of aggregates, while the oxidation products present spherical-like conformation whose diameters is in the range of 8–30 nm. The morphological changes observed by AFM to JRAOXG34 derivative, can be very important to guide to drug encapsulation, for example.

Recognition Mechanism of Galectin-4 for Cholesterol 3-Sulfate

Galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, and it co-localizes on the cell surface of human colonic adenocarcinoma cells with glycosphingolipids carrying SO(-)(3)-->3Galbeta1-->3(GalNAc) residues (Ideo, H., Seko, A., and Yamashita, K. (2005) J. Biol. Chem. 280, 4730-4737). In the present study, it was found that galectin-4 also binds to cholesterol 3-sulfate, which has no beta-galactoside moiety. This characteristic of galectin-4 is unique within the galectin family. The site-directed mutated galectin-4-R45A had diminished binding ability toward cholesterol 3-sulfate, suggesting that Arg(45) of galectin-4 is indispensable for cholesterol 3-sulfate recognition. Gel filtration and chemical cross-linking experiments revealed that some galectin-4 exists as dimers, and this multivalency seemed to enhance its avidity for cholesterol 3-sulfate binding. Cholesterol 3-sulfate and sulfatide co-existed with galectin-4 in detergent-insoluble fractions of porcine esophagus and intestine, respectively. These results suggested that not only sulfated glycosphingolipids but also cholesterol 3-sulfate are endogenous ligands for galectin-4 in vivo.

Carbohydrate-binding properties of human neo-CRP and its relationship to phosphorylcholine-binding site.

Binding characteristics of two types of ligands for human neo-C-reactive protein (neo-CRP), which is a conformationally altered but physiologically relevant form of CRP, were studied fluorometrically by probing CRP immobilized on a polystyrene surface with europium-labeled ligands. Two Eu-ligands used were bovine serum albumin derivatives that contain on average 40 residues of ligand structures, one derivative containing phosphorylcholine (PC) and the other lactosyl residues. The PC-containing ligands required the presence of calcium for binding, whereas galactose-containing derivatives bound in the absence of calcium. The optimal pH for the PC-dependent binding was broad (pH 6-8), whereas the best binding pH for the galactose-dependent binding was around 6. The carbohydrate-mediated binding is rather nonspecific: the binding site prefers galactose configuration, but other hexoses can be accommodated. The two best monosaccharide inhibitors at this site were galactose-6-phosphate and galacturonic acid, suggesting the importance of having a negatively charged group at C-6 position of galactose. In fact, the phosphate-binding site is common to both PC and sugar phosphates, and the choline- and the sugar-binding sites are probably located on either side of the phosphate-binding site. Binding characteristics of Eu-labeled PC-BSA to neo-CRP are quite similar to that found for native CRP in solution phase [Lee et al. (2002) J. Biol. Chem., 277, 225-232], whereas binding of sugar phosphates by neo-CRP shows considerably less stringent requirements compared to native CRP. For instance, galactose-alpha1-phosphate was not inhibitory at all in the native CRP binding assay, whereas it was a good inhibitor in the neo-CRP assay.

Specific modifications of galactomannans

A sample of galactomannan extracted from the seeds of Leucaema leucocephala is modified on a specific position using the reactant 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). This method allows oxidation at the C-6 position of galactose and free mannose (not connected to a side group). The kinetics of oxidation is followed under well-defined conditions. Different methods are proposed for the characterization of the modified polysaccharides and NMR spectroscopy is shown to give useful information. New polyelectrolytes are produced with different degrees of oxidation. Successive alkylations on the C-6 sites are investigated. The degree of alkylation obtained remains low but it can be increased easily to reinforce the amphiphilic character.

Identification and distribution of oviposition stimulants for monarch butterflies in hosts and nonhosts

Flavonol glycosides that act as oviposition stimulants for monarch butterflies were surveyed from a range of asclepiad hosts and some nonhosts. Major stimulants also were identified as quercetin-3-O-(2″-O-β-xylosyl)-β-D-galactoside and quercetin-3-O-β-D-galactoside from Asclepias syriaca and A. incarnata, respectively. The flavonol glycosides in A. curassavica, A. tuberosa, A. incarnata, A. syriaca, A. humistrata, A. albicans, A. eriocarpa, Calotropis procera, Cynanchum acutum, Vincetoxicum (Cynanchum) nigrum and in nonhosts Hoya australis and Nerium oleander were compared and characterized by HPLC and spectral studies. There was great variation in quercetin glycoside content. On the basis of the sugar moieties attached to quercetin, the asclepiad glycosides were classified broadly as those containing: (1) galactose, glucose, and rhamnose; (2) galactose, glucose, and xylose; and (3) galactose, glucose, xylose, and rhamnose. In most cases, galactose was attached to the 3-O-position (1″) of quercetin and other sugars were attached either to the 2″ or 6″ position of galactose. The sugars of triglycosides were attached at both 2″ and 6″ positions. A geographical pattern of flavonol distribution that may have affected the evolution of host recognition by the butterflies is suggested.

Synthesis of Gal-beta-(1-->4)-GlcNac-beta-(1-->6)-[Gal-beta-(1-->3]-GalNAc-alpha- OBn oligosaccharides bearing O-methyl or O-sulfo groups at C-3 of the Gal residue: specific acceptors for Gal: 3-O-sulfotransferases.

Our recent studies have revealed the existence of two distinct Gal: 3-O-sulfotransferases capable of acting on the C-3 position of galactose in a Core 2 branched structure, e.g., Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNacalph a1-->OBenzyl as acceptor to give 3-O-sulfoGalbeta1-->4GlcNAcbeta1-->3(Galbeta1-->3)G alNAcalpha1-->OB 20 and Galbeta1-->4GlcNAcbeta1-->6(3-O-sulfoGalbeta1-->3)G alNAcalpha1-->OB 23. We herein report the synthesis of these two compounds and also that of other modified analogs that are highly specific acceptors for the two sulfotransferases. Appropriately protected 1-thio-glycosides 7, 8, and 10 were employed as glycosyl donors for the synthesis of our target compounds.

Substrate specificity of human α- l-fucosidase

Human α- l-fucosidase is a soluble lysosomal enzyme which hydrolyzes α- l-fucose residues linked to the 2 position of galactose or the 3, 4, or 6 position of N-acetylglucosamine. Demonstration of activity towards natural oligosaccharide or glycosphingolipid substrates was achieved by measuring liberated l-fucose by coupling to fucose dehydrogenase and NAD and measuring NADH production spectrophotometrically. Activity of purified human spleen, brain, and cultured skin fibroblast or crude cell extracts towards 4-methylumbelliferyl-α- l-fucoside had a pH optimum of 4.5 to 5.5 and was unaffected by the presence of neutral detergents such as Triton X-100. However, the addition of sodium taurocholate or other bile salts to the incubation mixture caused a marked inhibition at pH 5 and a shift in pH optimum to the pH 6–7 region. Sodium taurocholate effected a threefold reduction in the apparent K m for α- l-fucosidase at pH 6.0, but studies on fucosidosis tissue (α-fucosidase deficiency) or subcellular fractions derived from rat liver failed to indicate the existence of a membrane-bound α- l-fucosidase. The response of other lysosomal hydrolases to the presence of bile salts was investigated and was found to be variable, perhaps depending upon the hydrophilic or hydrophobic nature of the natural substrate and/or the state of association of the active enzyme.