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  • Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system
    Glycobiology (IF 3.112) Pub Date : 2018-01-11
    Adrian J Jervis, Alison G Wood, Joel A Cain, Jonathan A Butler, Helen Frost, Elizabeth Lord, Rebecca Langdon, Stuart J Cordwell, Brendan W Wren, Dennis Linton

    N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of individual gene products in the PglB1-dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system.

    更新日期:2018-01-11
  • Keratan Sulphate, a complex Glycosaminoglycan with Unique Functional Capability
    Glycobiology (IF 3.112) Pub Date : 2018-01-11
    Bruce Caterson, James Melrose

    From an evolutionary perspective keratan sulphate (KS) is the newest glycosaminoglycan (GAG) but the least understood. KS is a sophisticated molecule with a diverse structure, and unique functional roles continue to be uncovered for this GAG. The cornea is the richest tissue source of KS in the human body but the central and peripheral nervous systems also contain significant levels of KS and a diverse range of KS-proteoglycans with essential functional roles. KS also displays important cell regulatory properties in epithelial and mesenchymal tissues and in bone and in tumour development of diagnostic and prognostic utility. Corneal KS-I displays variable degrees of sulphation along the KS chain ranging from non-sulphated polylactosamine, mono-sulphated and di-sulphated disaccharide regions. Skeletal KS-II is almost completely sulphated consisting of disulphated disaccharides interrupted by occasional mono-sulphated N-acetyllactosamine residues. KS-III also contains highly sulphated KS disaccharides but differs from KS-I and KS-II through 2-O-mannose linkage to serine or threonine core protein residues on proteoglycans such as phosphacan and abakan in brain tissue. Historically, the major emphasis on the biology of KS has focused on its sulphated regions for good reason. The sulphation motifs on KS convey important molecular recognition information and direct cell behavior through a number of interactive proteins. Emerging evidence also suggest functional roles for the poly N-acetyllactosamine regions of KS requiring further investigation. Thus further research is warranted to better understand the complexities of KS.

    更新日期:2018-01-11
  • A Comprehensive Caenorhabditis elegans N-glycan Shotgun Array
    Glycobiology (IF 3.112) Pub Date : 2018-01-09
    Ewa Jankowska, Lisa M Parsons, Xuezheng Song, Dave F Smith, Richard D Cummings, John F Cipollo

    Here we present a Caenorhabditis elegans N-glycan shotgun array. This nematode serves as a model organism for many areas of biology including but not limited to tissue development, host-pathogen interactions, innate immunity, and genetics. C. elegans N-glycans contain structural motifs that are also found in other nematodes as well as trematodes and lepidopteran species. Glycan binding toxins that interact with C. elegans glycoconjugates also do so with some agriculturally relevant species, such as Haemonchus contortus, Ascaris suum, Oesophagostomum dentatum and Trichoplusia ni. This situation implies that protein carbohydrate interactions seen with C. elegans glycans may also occur in other species with related glycan structures. Therefore, this array may be useful to study these relationships in other nematodes as well as trematode and insect species. The array contains 134 distinct glycomers spanning a wide range of C. elegans N-glycans including the subclasses high mannose, pauci mannose, high fucose, mammalian-like complex, and phosphorylcholine substituted forms. The glycans presented on the array have been characterized by two-dimensional separation, ion trap mass spectrometry, and lectin affinity. High fucose glycans were well represented and contain many novel core structures found in C. elegans as well as other species. This array should serve as an investigative platform for carbohydrate binding proteins that interact with N-glycans of C. elegans and over a range of organisms that contain glycan motifs conserved with this nematode.

    更新日期:2018-01-09
  • Galectin-3 and Cancer Stemness
    Glycobiology (IF 3.112) Pub Date : 2018-01-05
    Pratima Nangia-Makker, Victor Hogan, Avraham Raz

    Over the last few decades galectin-3, a carbohydrate binding protein, with affinity for N-acetyllactosamine residues, has been unique due to the regulatory roles it performs in processes associated with tumor progression and metastasis such as cell proliferation, homotypic/ heterotypic aggregation, dynamic cellular transformation, migration and invasion, survival and apoptosis. Structure-function association of galectin-3 reveals that it consists of a short amino terminal motif, which regulates its nuclear-cytoplasmic shuttling; a collagen α-like domain, susceptible to cleavage by matrix metalloproteases and prostate specific antigen (PSA); accountable for its oligomerization and lattice formation, and a carbohydrate recognition/binding domain containing the anti-death motif of the Bcl2 protein family. This structural complexity permits galectin-3 to associate with numerous molecules utilizing protein-protein and/or protein-carbohydrate interactions in the extracellular as well as intracellular milieu and regulate diverse signaling pathways, a number of which appear directed towards Epithelial-Mesenchymal Transition (EMT) and cancer stemness. Self-renewal, differentiation, long-term culturing and drug-resistance potential characterize Cancer Stem Cells (CSCs), a small cell subpopulation within the tumor that is thought to be accountable for heterogeneity, recurrence and metastasis of tumors. Despite the fact that association of galectin-3 to the tumor stemness phenomenon is still in its infancy, there is sufficient direct evidence of its regulatory roles in CSC-associated phenotypes and signaling pathways. In this review, we have highlighted the available data on galectin-3 regulated functions pertinent to cancer stemness and explored the opportunities of its exploitation as a cancer stem cell marker and a therapeutic target.

    更新日期:2018-01-05
  • A validated gRNA library for CRISPR/Cas9 targeting of the human glycosyltransferase genome
    Glycobiology (IF 3.112) Pub Date : 2018-01-05
    Yoshiki Narimatsu, Hiren J Joshi, Yang Zhang, Catarina Gomes, Yen-Hsi Chen, Flaminia Lorenzetti, Sanae Furukawa, Katrine Schjoldager, Lars Hansen, Henrik Clausen, Eric P Bennett, Hans H Wandall

    Over 200 glycosyltransferases are involved in the orchestration of the biosynthesis of the human glycome , which is comprised of all glycan structures found on different glycoconjugates in cells. The glycome is vast, and despite advancements in analytic strategies it continues to be difficult to decipher biological roles of glycans with respect to specific glycan structures, type of glycoconjugate, particular glycoproteins, and distinct glycosites on proteins. In contrast to this, the number of glycosyltransferase genes involved in the biosynthesis of the human glycome is manageable, and the biosynthetic roles of most of these enzymes are defined or can be predicted with reasonable confidence. Thus, with the availability of the facile CRISPR/Cas9 gene editing tool it now seems easier to approach investigation of the functions of the glycome through genetic dissection of biosynthetic pathways, rather than by direct glycan analysis. However, obstacles still remain with design and validation of efficient gene targeting constructs, as well as with the interpretation of results from gene targeting and the translation of gene function to glycan structures. This is especially true for glycosylation steps covered by isoenzyme gene families. Here, we present a library of validated high-efficiency gRNA designs suitable for individual and combinatorial targeting of the human glycosyltransferase genome together with a global view of the predicted functions of human glycosyltransferases to facilitate and guide gene targeting strategies in studies of the human glycome.

    更新日期:2018-01-05
  • Lipoteichoic acid mediates binding of a Lactobacillus S-layer protein
    Glycobiology (IF 3.112) Pub Date : 2018-01-04
    Eva Bönisch, Yoo Jin Oh, Julia Anzengruber, Fiona F Hager, Arturo Lopez-Guzman, Sonja Zayni, Peter Hinterdorfer, Paul Kosma, Paul Messner, Katarzyna A Duda, Christina Schäffer

    The Gram-positive lactic acid bacterium Lactobacillus buchneri CD034 is covered by a two-dimensional crystalline, glycoproteinaceous cell surface (S-) layer lattice. While lactobacilli are extensively exploited as cell surface display systems for applied purposes, questions about how they stick their cell wall together are remaining open. This also includes the identification of the S-layer cell wall ligand. In this study, lipoteichoic acid was isolated from the L. buchneri CD034 cell wall as a significant fraction of the bacterium's cell wall glycopolymers, structurally characterized and analyzed for its potential to mediate binding of the S-layer to the cell wall. Combined component analyses and 1D- and 2D-nuclear magnetic resonance spectroscopy (NMR) revealed the lipoteichoic acid to be composed of on average 31 glycerol-phosphate repeating units partially substituted with α-d-glucose, and with an α-d-Galp(1→2)-α-d-Glcp(1→3)−1,2-diacyl-sn-Gro glycolipid anchor. The specificity of binding between the Lactobacillus buchneri CD034 S-layer protein and purified lipoteichoic acid as well as their interaction force of about 45 pN were obtained by single-molecule force spectroscopy; this value is in the range of typical ligand-receptor interactions. This study sheds light on a functional implication of Lactobacillus cell wall architecture by showing direct binding between lipoteichoic acid and the S-layer of L. buchneri CD034.

    更新日期:2018-01-04
  • A Competitive AlphaScreen Assay for Detection of Hyaluronan
    Glycobiology (IF 3.112) Pub Date : 2017-12-29
    Xiayun Huang, Tannin A Schmidt, Claire Shortt, Shivani Arora, Akira Asari, Thorsten Kirsch, Mary K Cowman

    A method for specific quantification of hyaluronan (HA) concentration using AlphaScreen® (Amplified Luminescent Proximity Homogeneous Assay) technology is described. Two types of hydrogel-coated and chromophore-loaded latex nanobeads are employed. The proximity of the beads in solution is detected by excitation of the donor bead leading to the production of singlet oxygen, and chemiluminescence from the acceptor bead upon exposure to singlet oxygen. In the HA assay, the donor bead is modified with streptavidin, and binds biotin-labeled HA. The acceptor bead is modified with Ni(II), and is used to bind a specific recombinant HA-binding protein (such as HABP; aggrecan G1-IGD-G2) with a His-tag. Competitive inhibition of the HA-HABP interaction by free unlabeled HA in solution is used for quantification. The assay is specific for HA, and not dependent on HA molecular mass above the decasaccharide. HA can be quantified over a concentration range of approximately 30–1600 ng/mL using 2.5 μL of sample, for a detectable mass range of approximately 0.08–4 ng HA. This sensitivity of the AlphaScreen assay is greater than existing ELISA-like methods, due to the small volume requirements. HA can be detected in biological fluids using the AlphaScreen assay, after removal of bound proteins from HA and dilution or removal of other interfering proteins and lipids.

    更新日期:2017-12-31
  • Galectin-10: a new structural type of prototype galectin dimer and effects on saccharide ligand binding
    Glycobiology (IF 3.112) Pub Date : 2017-12-23
    Jiyong Su, Jin Gao, Yunlong Si, Linlin Cui, Chenyang Song, Yue Wang, Runjie Wu, Guihua Tai, Yifa Zhou

    Galectin-10 (Gal-10) which forms Charcot-Leyden crystals in vivo, is crucial to regulating lymph cell function. Here, we solved the crystal structures of Gal-10 and eight variants at resolutions of 1.55–2.00 Å. Structural analysis and size exclusion chromatography demonstrated that Gal-10 dimerizes with a novel global shape that is different from that of other prototype galectins (e.g., Gal-1, -2 and -7). In the Gal-10 dimer, Glu33 from one subunit modifies the carbohydrate binding site of another, essentially inhibiting disaccharide binding. Nevertheless, glycerol (and possibly other small hydroxylated molecules) can interact with residues at the ligand binding site, with His53 being the most crucial for binding. Alanine substitution of the conserved Trp residue (Trp72) that is crucial to saccharide binding in other galectins, actually leads to enhanced erythrocyte agglutination, suggesting that Trp72 negatively regulates Gal-10 ligand binding. Overall, our crystallographic and biochemical results provide insight into Gal-10 ligand binding specificity.

    更新日期:2017-12-23
  • Targeting sialic acid-Siglec interactions to reverse immune suppression in cancer
    Glycobiology (IF 3.112) Pub Date : 2017-12-22
    Olivia Joan Adams, Michal A Stanczak, Stephan von Gunten, Heinz Läubli

    Changes in sialic acids in cancer have been observed for many years. In particular, the increase of sialoglycan density or hypersialylation in tumors has been described. Recent studies have identified mechanisms for immune evasion based on sialoglycan interactions with immunoregulatory Siglec receptors that are exploited by tumor cells and microorganisms alike. Siglecs are mostly inhibitory receptors similar to known immune checkpoints including PD-1 or CTLA-4 that are successfully targeted with blocking antibodies for cancer immunotherapy. Here, we summarize the known changes of sialic acids in cancer and the role Siglec receptors play in cancer immunity. We also focus on potential ways to target these Siglec receptors or sialoglycans in order to improve anti-cancer immunity.

    更新日期:2017-12-22
  • Induction of human tolerogenic dendritic cells by 3’-sialyllactose via TLR4 is explained by LPS contamination
    Glycobiology (IF 3.112) Pub Date : 2017-12-21
    Olaf Perdijk, R J Joost van Neerven, Ben Meijer, Huub F J Savelkoul, Sylvia Brugman

    The human milk oligosaccharide 3’sialyllactose (3’SL) has previously been shown to activate murine dendritic cells (DC) in a TLR4-mediated manner ex vivo. In this study we aimed to investigate whether 3’SL has similar immunomodulatory properties on human DC. 3’SL was shown to induce NF-κB activation via human TLR4. However, LPS was detected in the commercially obtained 3’SL from different suppliers. After the removal of LPS from 3’SL, we studied its ability to modify DC differentiation in vitro. In contrast to LPS and 3’SL, LPS-free 3’SL did not induce functional and phenotypical changes on immature DC (iDC). iDC that were differentiated in the presence of LPS or 3’SL showed a semi-mature phenotype (i.e., fewer CD83+CD86+ DC), produced IL-10 and abrogated IL-12p70 and TNF levels upon stimulation with several TLR ligands. Differentiation into these tolerogenic DC was completely abrogated by LPS removal from 3’SL. In contrast to previous reports in mice, we found that LPS-free 3’SL does not activate NF-κB via human TLR4. In conclusion, removing LPS from (oligo)saccharide preparations is necessary to study their potential immunomodulatory function.

    更新日期:2017-12-21
  • Selectins in cancer immunity
    Glycobiology (IF 3.112) Pub Date : 2017-12-20
    Lubor Borsig

    Selectins are vascular adhesion molecules that mediate physiological responses such as inflammation, immunity and hemostasis. During cancer progression selectins promote various steps enabling the interactions between tumor cells and the blood constituents, including platelets, endothelial cells, and leukocytes. Selectins are carbohydrate-binding molecules that bind to sialylated, fucosylated glycan structures. The increased selectin ligand expression on tumor cells correlates with enhanced metastasis and poor prognosis for cancer patients. While, many studies focused on the role of selectin as a mediator of tumor cell adhesion and extravasation during metastasis, there is evidence for selectins to activate signaling cascade that regulates immune responses within a tumor microenvironment. L-selectin binding induces activation of leukocytes, which can be further modulated by selectin-mediated interactions with platelets and endothelial cells. Selectin ligand on leukocytes, PSGL-1, triggers intracellular signaling in leukocytes that are induced through platelet's P-selectin or endothelial E-selectin binding. In this review I summarize the evidence for selectin-induced immune modulation in cancer progression that represents a possible target for controlling tumor immunity.

    更新日期:2017-12-20
  • GlycoDomainViewer: A bioinformatics tool for contextual exploration of glycoproteomes
    Glycobiology (IF 3.112) Pub Date : 2017-12-18
    Hiren J Joshi, Anja Jørgensen, Katrine T Schjoldager, Adnan Halim, Leo A Dworkin, Catharina Steentoft, Hans H Wandall, Henrik Clausen, Sergey Y Vakhrushev

    The GlycoDomainViewer is a bioinformatic tool to aid in the mining of glycoproteomic data sets from different sources and facilitate incorporation of glycosylation into studies of protein structure and function. We present a version 2.0 of GlycoDomainViewer incorporating a number of advanced features, which enhances visibility and accessibility of the wealth of glycoproteomic data being generated. The GlycoDomainViewer enables visual exploration of glycoproteomic data, incorporating information from recent N- and O-glycoproteome studies on human and animal cell lines and some organs and body fluids. The initial data comprises sites of glycosylation for N-linked, O-GalNAc, O-Fucose, O-Xyl, O-Mannose (in both human and yeast) and cytosolic O-GlcNAc type. The data made available via this tool will be regularly updated to improve the coverage of known glycosylation sites and data sets, reflecting the advances currently being made in characterisation of glycoproteomes. The tool is available at https://glycodomain.glycomics.ku.dk.

    更新日期:2017-12-18
  • Ten years of CAZypedia: a living encyclopedia of carbohydrate-active enzymes
    Glycobiology (IF 3.112) Pub Date : 2017-10-11

    CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate-active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification and degradation of complex carbohydrates. CAZypedia is closely connected with the actively curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.

    更新日期:2017-12-15
  • Concerted mass spectrometry-based glycomic approach for precision mapping of sulfo sialylated N-glycans on human peripheral blood mononuclear cells and lymphocytes
    Glycobiology (IF 3.112) Pub Date : 2017-10-26
    Jian-You Chen, Hsin-Hung Huang, Shin-Yi Yu, Shang-Ju Wu, Reiji Kannagi, Kay-Hooi Khoo

    Despite well-recognized biological importance, mass spectrometry (MS)-based glycomic identification of sulfo-, sialylated terminal glyco-epitopes on the N-glycans of various immune cell types remains technically challenging and rarely reported. Previous studies with monoclonal antibody have implicated a regulated expression of 6-sulfo-α2-6-sialyl LacNAc on B cells in peripheral lymph nodes and the circulating peripheral blood lymphocytes but its occurrence on leukemia cells or lymphomas have not been critically addressed. In this study, we have extended our previously developed MS-based sulfoglycomic platform by incorporating additional complementary analytical approaches in order to achieve a high sensitivity mapping and relative quantification of the detected sulfated glycotopes down to the level of defining their sialyl linkages. We showed that discovery mode sulfoglycomics and precise location of sulfate were best achieved by multimode MS analyses of fractionated, permethylated sulfated N-glycans. On the other hand, the relative degree of sulfation on individual N-glycans could be more efficiently inferred from the respective extracted ion chromatograms of native, non-sulfated and sulfated target N-glycans in single LC–MS/MS runs. The GlcNAc-6-O-sulfated α2-6-sialyl LacNAc, which constitutes the higher affinity ligand for the human inhibitory co-receptor of B cells, CD22, was found to be commonly carried on a range of complex type N-glycans from human CD19+ and CD4+ lymphocytes. We further showed that its occurrence on the most abundant α2-6-disialylated biantennary structure from the peripheral blood mononuclear cells of patients diagnosed as B-cell chronic lymphocytic leukemia varied within ±2-fold abundance from the mean value determined for isolated CD19+ lymphocytes and cultured B-CLL cells.

    更新日期:2017-12-15
  • Lectin ZG16p inhibits proliferation of human colorectal cancer cells via its carbohydrate-binding sites
    Glycobiology (IF 3.112) Pub Date : 2017-10-23
    Akiko Mito, Yukiko Nakano, Takako Saitoh, Sabine S S Gouraud, Yoshiki Yamaguchi, Toshiro Sato, Nobuo Sasaki, Kyoko Kojima-Aikawa

    Zymogen granule protein 16 (ZG16p) is a soluble lectin that binds to both mannose and heparin/heparan sulfate. It is highly expressed in the human digestive tract and is secreted into the mucus. In this study, we investigated the effect of ZG16p on the proliferation of human colorectal cancer cells. Overexpression of ZG16p in Caco-2 cells decreased cell growth. Recombinant ZG16p markedly inhibited proliferation of Caco-2, LS174T, HCT116 and HCT15 cells. Caco-2 cell growth was not inhibited by two mutated ZG16p proteins, D151A and M5 (K36A, R37A, R53A, R55A and R79A) lacking mannose- and heparin-binding activities, respectively. Immunofluorescent cell staining revealed that ZG16p-D151A maintained its binding to the Caco-2 cell surface, whereas ZG16p-M5 failed to bind to the cells. These results suggest that ZG16p interacts with the cell surface via basic amino acids substituted in ZG16p-M5 and inhibits Caco-2 cell proliferation via Asp151. In addition, growth of patient-derived colorectal tumor organoids in a 3D intestinal stem cell system was suppressed by ZG16p but not by ZG16p-M5. Taken together, our findings indicate that ZG16p inhibits the growth of colorectal cancer cells via its carbohydrate-binding sites in vitro and ex vivo. In this study, a novel pathway in cancer cell growth regulation through cell surface carbohydrate chains is suggested.

    更新日期:2017-12-15
  • Loss of heparan sulfate in the niche leads to tumor-like germ cell growth in the Drosophila testis
    Glycobiology (IF 3.112) Pub Date : 2017-10-23
    Daniel C Levings, Hiroshi Nakato

    The stem cell niche normally prevents aberrant stem cell behaviors that lead to cancer formation. Recent studies suggest that some cancers are derived from endogenous populations of adult stem cells that have somehow escaped from normal control by the niche. However, the molecular mechanisms by which the niche retains stem cells locally and tightly controls their divisions are poorly understood. Here, we demonstrate that the presence of heparan sulfate (HS), a class glygosaminoglycan chains, in the Drosophila germline stem cell niche prevents tumor formation in the testis. Loss of HS in the niche, called the hub, led to gross changes in the morphology of testes as well as the formation of both somatic and germline tumors. This loss of hub HS resulted in ectopic signaling events in the Jak/Stat pathway outside the niche. This ectopic Jak/Stat signaling disrupted normal somatic cell differentiation, leading to the formation of tumors. Our finding indicates a novel non-autonomous role for niche HS in ensuring the integrity of the niche and preventing tumor formation.

    更新日期:2017-12-15
  • NMR interaction studies of Neu5Ac-α-(2,6)-Gal-β-(1-4)-GlcNAc with influenza-virus hemagglutinin expressed in transfected human cells
    Glycobiology (IF 3.112) Pub Date : 2017-10-26
    Francesca Vasile, Francesco Gubinelli, Maddalena Panigada, Elisa Soprana, Antonio Siccardi, Donatella Potenza

    The emergence of escape-mutants of influenza hemagglutinin (HA) following vaccination compels the yearly re-formulation of flu vaccines. Since binding the sialic acid receptor remains in all cases essential for infection, small-molecule inhibitors of HA binding to sialic acid could be interesting therapeutic complements or alternatives to immuno-prophylaxis in the control of flu epidemics. In this work, we made use of NMR spectroscopy to study the interaction between a derivative of sialic acid (the Neu5Ac-α-(2,6)-Gal-β-(1-4)-GlcNAc trisaccharide) and HAs (H1 and H5) from human and avian strains of influenza virus, directly expressed on the surface of stable transfected 293 T human cells. The HAs were shown to retain their native trimeric conformation and binding properties. Exploiting the magnetization transfer between the proteins and the ligand, we obtained evidence of the binding event and mapped the (non-identical) sugar epitopes recognized by the two HA species. The rapid and reliable method for screening sialic acid-related HA ligands we have developed could yield useful information for an efficient drug design.

    更新日期:2017-12-15
  • Polysaccharide-experienced effector T cells induce IL-10 in FoxP3+ regulatory T cells to prevent pulmonary inflammation
    Glycobiology (IF 3.112) Pub Date : 2017-10-26
    Jenny L Johnson, Mark B Jones, Brian A Cobb

    Inhibition of peripheral inflammatory disease by carbohydrate antigens derived from normal gut microbiota has been demonstrated for the GI tract, brain, peritoneum, and most recently the airway. We have demonstrated that polysaccharide A (PSA) from the commensal organism Bacteroides fragilis activates CD4+ T cells upon presentation by the class II major histocompatibility complex, and that these PSA-experienced T cells prevent the development of lung inflammation in murine models. While the PSA-responding T cells themselves are not canonical FoxP3+ regulatory T cells (Tregs), their ability to prevent inflammation is dependent upon the suppressive cytokine IL-10. Using an adoptive T cell transfer approach, we have discovered that PSA-experienced T cells require IL-10 expression by PSA-naïve recipient animals in order to prevent inflammation. A cooperative relationship was found between PSA-activated effector/memory T cells and tissue-resident FoxP3+ Tregs both in vivo and in vitro, and it is this cooperation that enables the suppressive activity of PSA outside of the gut environment where exposure takes place. These findings suggest that carbohydrate antigens from the normal microbiota communicate with peripheral tissues to maintain homeostasis through T cell-to-T cell cooperation.

    更新日期:2017-12-15
  • Interaction of Neisseria meningitidis Group X N-Acetylglucosamine-1-phosphotransferase with Its Donor Substrate
    Glycobiology (IF 3.112) Pub Date : 2017-12-08
    Shonoi A Ming, Ebony Cottman-Thomas, Natalee C Black, Yi Chen, Vamsee Veeramachineni, Dwight C Peterson, Xi Chen, Lauren M Tedaldi, Gerd K Wagner, Chao Cai, Robert J Linhardt, Willie F Vann

    Neisseria meningitidis Group X is an emerging cause of bacterial meningitis in the Sub-Saharan Africa. The capsular polysaccharide of Group X is a homopolymer of N-acetylglucosamine α(1–4) phosphate and is a vaccine target for prevention of disease associated with this meningococcal serogroup. We have demonstrated previously that the formation of the polymer is catalyzed by a phosphotransferase which transfers N- acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to the 4- hydroxyl of the N-acetylglucosamine on the non-reducing end of the growing chain. In this study, we use substrate analogs of UDP-GlcNAc to define the enzyme/donor substrate interactions critical for catalysis. Our kinetic analysis of the phosphotransferase reaction is consistent with a sequential mechanism of substrate addition and product release. We used novel uracil modified analogs designed by Wagner et al. enabled us to assess whether the CsxA-catalyzed reaction is consistent with a donor dependent conformational change. As expected with this model for glycosyltransferases, UDP-GlcNAc analogs with bulky uracil modifications are not substrates but are inhibitors. An analog with a smaller iodo uracil substitution is a substrate and a less potent inhibitor. Moreover, our survey of analogs with modifications on the N- acetylglucosamine residue of the sugar nucleotide donor highlights the importance of substituents at C-2 and C-4 of the sugar residue. The hydroxyl group at C4 and the structure of the acyl group at C2 are very important for specificity and substrate interactions during the polymerization reaction. While most analogs modified at C-2 were inhibitors, acetamido analogs were also substrates suggesting the importance of the carbonyl group.

    更新日期:2017-12-10
  • Vascular abnormalities in the placenta of Chst14−/− fetuses: implications in the pathophysiology of perinatal lethality of the murine model and vascular lesions in human CHST14/D4ST1 deficiency
    Glycobiology (IF 3.112) Pub Date : 2017-12-01
    Takahiro Yoshizawa, Shuji Mizumoto, Yuki Takahashi, Shin Shimada, Kazuyuki Sugahara, Jun Nakayama, Shin'ichi Takeda, Yoshihiro Nomura, Yuko Nitahara-Kasahara, Takashi Okada, Kiyoshi Matsumoto, Shuhei Yamada, Tomoki Kosho

    Collagen is one of the most important components of the extracellular matrix that is involved in the strength of tissues, cell adhesion, and cell proliferation. Mutations in several collagen and post-translational modification enzyme genes cause Ehlers-Danlos syndrome (EDS) characterized by joint and skin hyperextensibility as well as fragility of various organs. Carbohydrate sulfotransferase 14/dermatan 4-O-sulfotransferase-1 (CHST14/D4ST1) is a critical enzyme for biosynthesis of dermatan sulfate, a side chain of various proteoglycans including biglycan that regulates collagen fibrils through their interaction. Mutations in CHST14 were found to cause a new form of EDS, named musculocontractural type EDS (mcEDS-CHST14). Large subcutaneous hematomas are one of the most serious complications accompanied by decreased quality of life and potential lethality.

    更新日期:2017-12-01
  • Tunicamycin-Induced ER Stress in Breast Cancer Cells Neither Expresses GRP78 on the Surface Nor Secretes it into the Media
    Glycobiology (IF 3.112) Pub Date : 2017-12-01
    Jesús E Serrano-Negrón, Zhenbo Zhang, Andrea P Rivera-Ruiz, Aditi Banerjee, Eva C Romero-Nutz, Neysharie Sánchez-Torres, Krishna Baksi, Dipak K Banerjee

    GRP78 (an Mr 78 kDa calcium dependent glucose binding protein) located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn-X-Ser/Thr. Paraffin sections from N-glycosylation inhibitor tunicamycin treated ER-/PR-/HER2+ (double negative) breast tumor in athymic nude mice exhibited reduced N-glycan but increased GRP78 expression. We have evaluated the effect of tunicamycin on cellular localization of GRP78 in metastatic human breast cancer cells MDA-MB-231 (ER-/PR-/HER2-). Tunicamycin inhibited cell proliferation in a time and dose-dependent manner. Non-metastatic estrogen receptor positive (ER+) MCF-7 breast cancer cells were also equally effective. GRP78 expression (protein and mRNA) was higher in tunicamycin (1.0 μg/ml) treated MCF-7 and MDA-MB-231 cells. GRP78 is an ER stress marker, so we have followed its intracellular localization using immunofluorescence microscopy after subjecting the cancer cells to various stress conditions. Unfixed cells stained with either FITC-conjugated Concanavalin A (Con A), or Texas-red conjugated wheat germ agglutinin (WGA) exhibited surface expression of N-glycans but not GRP78. GRP78 became detectable only after a brief exposure of cells to ice-cold methanol. Western blotting did not detect GRP78 in conditioned media of cancer cells whereas it did for MMP-1. The conclusion, GRP78 is expressed neither on the outer-leaflet of the (ER-/PR-/HER2-) human breast cancer cells nor it is secreted into the culture media during tunicamycin-induced ER stress. Our study therefore suggests strongly that anti-tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast and other sold tumors.

    更新日期:2017-12-01
  • Distinct reaction mechanisms for hyaluronan biosynthesis in different kingdoms of life
    Glycobiology (IF 3.112) Pub Date : 2017-11-27
    Matthew R Blackburn, Caitlin Hubbard, Volker Kiessling, Yunchen Bi, Brian Kloss, Lukas Tamm, Jochen Zimmer

    Hyaluronan (HA) is an acidic high molecular weight cell surface polysaccharide ubiquitously expressed by vertebrates, some pathogenic bacteria, and even viruses. HA modulates many essential physiological processes and is implicated in numerous pathological conditions ranging from autoimmune diseases to cancer. In various pathogens, HA functions as a non-immunogenic surface polymer that reduces host immune responses. It is a linear polymer of strictly alternating glucuronic acid and N-acetylglucosamine units synthesized by HA synthase (HAS), a membrane-embedded family-2 glycosyltransferase. The enzyme synthesizes HA and secretes the polymer through a channel formed by its own membrane-integrated domain. To reveal how HAS achieves these tasks, we determined the biologically functional units of bacterial and viral HAS in a lipid bilayer environment by co-immunoprecipitation, single molecule fluorescence photobleaching, and site-specific cross-linking analyses. Our results demonstrate that bacterial HAS functions as an obligate homo-dimer with two functional HAS copies required for catalytic activity. In contrast, the viral enzyme, closely related to vertebrate HAS, functions as a monomer. Using site-specific cross-linking, we identify the dimer interface of bacterial HAS and show that the enzyme uses a reaction mechanism distinct from viral HAS that necessitates a dimeric assembly.

    更新日期:2017-11-28
  • Identification and characterization of the Streptococcus pneumoniae type 3 capsule-specific glycoside hydrolase of Paenibacillus species 32352
    Glycobiology (IF 3.112) Pub Date : 2017-11-27
    Dustin R Middleton, Xing Zhang, Paeton L Wantuch, Ahmet Ozdilek, Xinyue Liu, Rachel Lopilato, Nikhil Gangasani, Robert Bridger, Lance Wells, Robert J Linhardt, Fikri Y Avci

    Bacillus circulans Jordan 32352 was isolated from decaying organic matter in the New Jersey soil in the early 1930s. This soil-dwelling bacterium produced an enzyme capable of degrading the type 3 capsular polysaccharide (Pn3P) of Streptococcus pneumoniae (Spn). Early reports of this enzyme, Pn3Pase, demonstrated its inducibility by, and specificity for Pn3P. We set out to identify and clone this enzyme for its recombinant expression and characterization. We first sequenced the genome of this bacterial species, and reclassified the Pn3Pase producing bacterium as Paenibacillus species 32352. We identified the putative protein of Pn3Pase through mass spectrometry-based proteomics and cloned the gene for recombinant expression. We then characterized the oligosaccharide products generated upon the enzymatic depolymerization of Pn3P. Sequence analysis suggests that this glycoside hydrolase (GH) belongs to a new Carbohydrate-Active enzyme (CAZy) GH family. To our knowledge, this is the only enzyme to demonstrate Pn3P depolymerization activity.

    更新日期:2017-11-28
  • Imaging specific cellular glycan structures using glycosyltransferases via click chemistry
    Glycobiology (IF 3.112) Pub Date : 2017-11-25
    Zhengliang L Wu, Anthony Person, Matthew Anderson, Barbara Burroughs, Timothy Tatge, Kshitij Khatri, Yonglong Zou, Lianchun Wang, Todd Geders, Joseph Zaia, Robert Sackstein

    Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells. Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for O-glycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans.

    更新日期:2017-11-25
  • Society for Glycobiology Awards—2017
    Glycobiology (IF 3.112) Pub Date : 2017-10-27

    The Society for Glycobiology is pleased to announce Dr. Jamey Marth as the recipient of the 2017 Karl Meyer Lectureship Award and Dr. Gillian Air as the recipient of the 2017 Rosalind Kornfeld Award for Lifetime Achievement in Glycobiology.2 The Karl Meyer Award was established in 1990 to honor the distinguished career of Karl Meyer and his outstanding contributions to the field of Glycobiology. This international award is given to well-established scientists with currently active research programs who have made widely recognized major contributions to the field of Glycobiology. The Rosalind Kornfeld Award was established in 2008 to honor the distinguished scientific career and service to the Society by Dr. Rosalind Kornfeld. The award is given by the Society to scientists who have made significant contributions with an important impact on the field of Glycobiology over their professional lifetimes.

    更新日期:2017-11-07
  • Sialylation of extracellular superoxide dismutase (EC-SOD) enhances furin-mediated cleavage and secretion
    Glycobiology (IF 3.112) Pub Date : 2017-10-19
    Fumi Ota, Yasuhiko Kizuka, Miyako Nakano, Yoshiki Yamaguchi, Shinobu Kitazume, Tomomi Ookawara, Naoyuki Taniguchi

    Extracellular superoxide dismutase (EC-SOD, SOD3) protects tissues against oxidative damage by detoxifying superoxide anions, particularly in the lungs and cardiovascular system. EC-SOD undergoes several posttranslational modifications including N-glycosylation and proteolytic cleavage. While the roles of proteolytic cleavage have been well studied, the structure and function of EC-SOD N-glycans are poorly understood. Here we analyzed glycan structures on native EC-SOD purified from human sera, and identified sialylated biantennary structures. Using glycan maturation-defective CHO mutant cells, we further revealed that the presence of terminal sialic acids in the N-glycans of EC-SOD enhanced both the secretion and furin-mediated C-terminal cleavage of EC-SOD. These results provide new insights into how the posttranslational modifications of EC-SOD control its functions.

    更新日期:2017-11-07
  • Novel roles of glycosaminoglycans in the degradation of type I collagen by cathepsin K
    Glycobiology (IF 3.112) Pub Date : 2017-10-25
    Yota Tatara, Shinichiro Suto, Ken Itoh

    Glycosaminoglycans (GAGs) and collagen are the major organic components of bone matrix. However, their roles and functional relationships remain elusive. To investigate the role of GAGs in bone matrix degradation, the effects of GAGs on collagen were examined under acidic conditions that recapitulate the microenvironment of osteoclast resorption pits. We found that sulfated GAGs protect collagen fibrils against acid denaturation. Scanning electron microscopy demonstrated that collagen fibrils retain the fibril structure at pH 4.0 in the presence of chondroitin 6-sulfate. By surface plasmon resonance analysis, we found that sulfated GAGs, but not non-sulfated GAGs, bind to triple-helix type I collagen below pH 4.5. The binding of collagen in an acidic solution was dependent upon the GAG sugar chain length. Functionally, the acid-resistant collagen fibrils generated in the presence of sulfated GAGs were resistant to cathepsin K degradation in vitro below pH 4.0. As the pH increased from 4.0 to 5.0, the acid-resistant collagen fibrils were degraded by cathepsin K. Our results highlight the possibility that the interaction between GAGs and collagen under acidic conditions has a regulatory impact on cathepsin K-mediated bone degradation.

    更新日期:2017-11-07
  • High-throughput characterization of the functional impact of IgG Fc glycan aberrancy in juvenile idiopathic arthritis
    Glycobiology (IF 3.112) Pub Date : 2017-10-03
    Hao D Cheng, Henning Stöckmann, Barbara Adamczyk, Ciara A McManus, Altan Ercan, Ingrid A Holm, Pauline M Rudd, Margaret E Ackerman, Peter A Nigrovic

    Juvenile idiopathic arthritis (JIA) encompasses all forms of chronic idiopathic arthritis that arise before age 16. Previous studies have found JIA to be associated with lower Fc galactosylation of circulating IgG, but the overall spectrum of glycan changes and the net impact on IgG function are unknown. Using ultra performance liquid chromatography (UPLC), we compared IgG glycosylation in 54 subjects with recent-onset untreated JIA with 98 healthy pediatric controls, paired to biophysical profiling of affinity for 20 IgG receptors using a high-throughput multiplexed microsphere assay. Patients with JIA exhibited an increase in hypogalactosylated and hyposialylated IgG glycans, but no change in fucosylation or bisection, together with alteration in the spectrum of IgG ligand binding. Supervised machine learning demonstrated a robust capacity to discriminate JIA subjects from controls using either glycosylation or binding data. The binding signature was driven predominantly by enhanced affinity for Fc receptor like protein 5 (FcRL5), a noncanonical Fc receptor expressed on B cells. Affinity for FcRL5 correlated inversely with galactosylation and sialylation, a relationship confirmed through enzymatic manipulation. These results demonstrate the capacity of combined structural and biophysical IgG phenotyping to define the overall functional impact of IgG glycan changes and implicate FcRL5 as a potential cellular sensor of IgG glycosylation.

    更新日期:2017-11-07
  • The role of Zn2+, dimerization and N-glycosylation in the interaction of Auxin-Binding Protein 1 (ABP1) with different auxins
    Glycobiology (IF 3.112) Pub Date : 2017-10-06
    Cibele Tesser da Costa, Conrado Pedebos, Hugo Verli, Arthur Germano Fett-Neto

    Auxin is critical for plant growth and development. The main natural auxin is indole-3-acetic acid (IAA), whereas 1-naphthalene acetic acid (NAA) is a synthetic form. Auxin-Binding Protein 1 (ABP1) specifically binds auxins, presumably playing roles as receptor in nontranscriptional cell responses. ABP1 structure was previously established from maize at 1.9 Å resolution. To gain further insight on ABP1 structural biology, this study was carried out employing molecular dynamics simulations of the complete models of the oligomeric glycosylated proteins from maize and Arabidopsis thaliana with or without auxins. In maize, both Zn2+ coordination and glycosylation promoted conformational stability and most of such stabilization effect was located on the N-terminal region. The α-helix of C-terminal regions in ABP1 of both species unfolded during simulations, assuming a more extended structure in maize. In Arabidopsis, the helix appeared more stable, being preserved in most of the monomeric simulations and unfolding when the protein was in the dimeric form. In Arabidopsis ABP1 bound to IAA or NAA, glycosylation structures arranged around the protein, covering the putative site of entrance or egress of auxin. NAA bound protein folding was more similar to the crystal structure showing higher stability compared to that of IAA bound. The molecular structural differences of ABP1 found between the species and auxin types indicate that this auxin-binding protein shows functional specificities in dicots and monocots, as well as in auxin type binding.

    更新日期:2017-11-07
  • Distinct roles for each N-glycan branch interacting with mannose-binding type Jacalin-related lectins Orysata and Calsepa
    Glycobiology (IF 3.112) Pub Date : 2017-10-03
    Masamichi Nagae, Sushil K Mishra, Shinya Hanashima, Hiroaki Tateno, Yoshiki Yamaguchi

    Mannose-binding type Jacalin-related lectins (mJRLs) bind to branched N-glycans via conserved sugar-binding sites. Despite, significant 3D structural similarities, each mJRL is known to have a unique binding preference toward various N-glycans. However, the molecular basis of varying binding preference is substantially unknown. Here, we report a detailed comparison of N-glycan-binding preference for two mJRLs, Orysata and Calsepa using frontal affinity chromatography (FAC), X-ray and molecular modeling. The FAC analysis using a panel of N-glycans shows difference in N-glycan-binding preference between the lectins. Orysata shows broader specificity toward most high-mannose-type glycans as well as biantennary complex-type glycans bearing an extension on the Manα1–6 branch. Whereas, Calsepa shows narrow specificity to complex-type glycans with bisecting GlcNAc. The X-ray crystallographic structure reveals that two Orysata lectins bind to one biantennary N-glycan (2:1 binding) where one lectin binds to mannose of the α1–3 branch, while the other interacts with an N-acetylglucosamine of the α1–6 branch. In contrast, Calsepa shows 1:1 binding where α1–3 branch and core chitobiose region N-glycan interacts with lectin, while α1–6 branch is flipped-back to the chitobiose core. Molecular dynamics study of Orysata bound to N-glycans substantiate possibility of two-binding modes for each N-glycan. Binding free energies calculated separately for α1–3 and α1–6 branches of each N-glycan suggest both branches can bind to Orysata. Overall these results suggest that each branch of N-glycan has a distinct role in binding to mJRLs and the nonbinding branch can contribute significantly to the binding affinity and hence to the specificity.

    更新日期:2017-11-07
  • Lectin-binding characterizes the healthy human skeletal muscle glycophenotype and identifies disease-specific changes in dystrophic muscle
    Glycobiology (IF 3.112) Pub Date : 2017-10-03
    Brian J McMorran, M Carrie Miceli, Linda G Baum

    Our understanding of muscle glycosylation to date has derived from studies in mouse models and a limited number of human lectin histochemistry studies. As various therapeutic approaches aimed at treating patients with muscular dystrophies are being translated from rodent models to human, it is critical to better understand human muscle glycosylation and relevant disease-specific differences between healthy and dystrophic muscle. Here, we report the first quantitative characterization of human muscle glycosylation, and identify differentiation- and disease-specific differences in human muscle glycosylation. Utilizing a panel of 13 lectins with varying glycan specificities, we surveyed lectin binding to primary and immortalized myoblasts and myotubes from healthy and dystrophic sources. Following differentiation of primary and immortalized healthy human muscle cells, we observed increased binding of Narcissus pseudonarcissus agglutinin (NPA), PNA, MAA-II and WFA to myotubes compared to myoblasts. Following differentiation of immortalized healthy and dystrophic human muscle cells, we observed disease-specific differences in binding of NPA, Jac and Tricosanthes japonica agglutinin-I (TJA-I) to differentiated myotubes. We also observed differentiation- and disease-specific differences in binding of NPA, Jac, PNA, TJA-I and WFA to glycoprotein receptors in muscle cells. Additionally, Jac, PNA and WFA precipitated functionally glycosylated α-DG, that bound laminin, while NPA and TJA-I did not. Lectin histochemistry of healthy and dystrophic human muscle sections identified disease-specific differences in binding of O-glycan and sialic acid-specific lectins between healthy and dystrophic muscle. These results indicate that specific and discrete changes in glycosylation occur following differentiation, and identify specific lectins as potential biomarkers sensitive to changes in healthy human muscle glycosylation.

    更新日期:2017-11-07
  • Sialylation of extracellular superoxide dismutase (EC-SOD) enhances furin-mediated cleavage and secretion
    Glycobiology (IF 3.112) Pub Date : 2017-10-04
    Fumi Ota, Yasuhiko Kizuka, Miyako Nakano, Yoshiki Yamaguchi, Shinobu Kitazume, Tomomi Ookawara, Naoyuki Taniguchi

    Extracellular superoxide dismutase (EC-SOD, SOD3) protects tissues against oxidative damage by detoxifying superoxide anions, particularly in the lungs and cardiovascular system. EC-SOD undergoes several posttranslational modifications including N-glycosylation and proteolytic cleavage. While the roles of proteolytic cleavage have been well studied, the structure and function of EC-SOD N-glycans are poorly understood. Here we analyzed glycan structures on native EC-SOD purified from human sera, and identified sialylated biantennary structures. Using glycan maturation-defective CHO mutant cells, we further revealed that the presence of terminal sialic acids in the N-glycans of EC-SOD enhanced both the secretion and furin-mediated C-terminal cleavage of EC-SOD. These results provide new insights into how the posttranslational modifications of EC-SOD control its functions.

    更新日期:2017-10-27
  • The role of Zn2+, dimerization and N-glycosylation in the interaction of Auxin Binding Protein 1 (ABP1) with different auxins
    Glycobiology (IF 3.112) Pub Date : 2017-09-07
    Cibele Tesser da Costa, Conrado Pedebos, Hugo Verli, Arthur Germano Fett-Neto

    Auxin is critical for plant growth and development. The main natural auxin is indole-3-acetic acid (IAA), whereas 1-naphthalene acetic acid (NAA) is a synthetic form. Auxin-Binding Protein 1 (ABP1) specifically binds auxins, presumably playing roles as receptor in nontranscriptional cell responses. ABP1 structure was previously established from maize at 1.9 Å resolution. To gain further insight on ABP1 structural biology, this study was carried out employing molecular dynamics simulations of the complete models of the oligomeric glycosylated proteins from maize and Arabidopsis thaliana with or without auxins. In maize, both Zn2+ coordination and glycosylation promoted conformational stability and most of such stabilization effect was located on the N-terminal region. The α-helix of C-terminal regions in ABP1 of both species unfolded during simulations, assuming a more extended structure in maize. In Arabidopsis, the helix appeared more stable, being preserved in most of the monomeric simulations and unfolding when the protein was in the dimeric form. In Arabidopsis ABP1 bound to IAA or NAA, glycosylation structures arranged around the protein, covering the putative site of entrance or egress of auxin. NAA bound protein folding was more similar to the crystal structure showing higher stability compared to that of IAA bound. The molecular structural differences of ABP1 found between the species and auxin types indicate that this auxin-binding protein shows functional specificities in dicots and monocots, as well as in auxin type binding.

    更新日期:2017-10-27
  • Society for Glycobiology Awards—2017
    Glycobiology (IF 3.112) Pub Date : 2017-10-04

    The Society for Glycobiology is pleased to announce Dr. Jamey Marth as the recipient of the 2017 Karl Meyer Lectureship Award and Dr. Gillian Air as the recipient of the 2017 Rosalind Kornfeld Award for Lifetime Achievement in Glycobiology.2 The Karl Meyer Award was established in 1990 to honor the distinguished career of Karl Meyer and his outstanding contributions to the field of Glycobiology. This international award is given to well-established scientists with currently active research programs who have made widely recognized major contributions to the field of Glycobiology. The Rosalind Kornfeld Award was established in 2008 to honor the distinguished scientific career and service to the Society by Dr. Rosalind Kornfeld. The award is given by the Society to scientists who have made significant contributions with an important impact on the field of Glycobiology over their professional lifetimes.

    更新日期:2017-10-27
  • Novel roles of glycosaminoglycans in the degradation of type I collagen by cathepsin K
    Glycobiology (IF 3.112) Pub Date : 2017-10-04
    Yota Tatara, Shinichiro Suto, Ken Itoh

    Glycosaminoglycans (GAGs) and collagen are the major organic components of bone matrix. However, their roles and functional relationships remain elusive. To investigate the role of GAGs in bone matrix degradation, the effects of GAGs on collagen were examined under acidic conditions that recapitulate the microenvironment of osteoclast resorption pits. We found that sulfated GAGs protect collagen fibrils against acid denaturation. Scanning electron microscopy demonstrated that collagen fibrils retain the fibril structure at pH 4.0 in the presence of chondroitin 6-sulfate. By surface plasmon resonance analysis, we found that sulfated GAGs, but not non-sulfated GAGs, bind to triple-helix type I collagen below pH 4.5. The binding of collagen in an acidic solution was dependent upon the GAG sugar chain length. Functionally, the acid-resistant collagen fibrils generated in the presence of sulfated GAGs were resistant to cathepsin K degradation in vitro below pH 4.0. As the pH increased from 4.0 to 5.0, the acid-resistant collagen fibrils were degraded by cathepsin K. Our results highlight the possibility that the interaction between GAGs and collagen under acidic conditions has a regulatory impact on cathepsin K-mediated bone degradation.

    更新日期:2017-10-27
  • High-throughput characterization of the functional impact of IgG Fc glycan aberrancy in juvenile idiopathic arthritis
    Glycobiology (IF 3.112) Pub Date : 2017-09-14
    Hao D Cheng, Henning Stöckmann, Barbara Adamczyk, Ciara A McManus, Altan Ercan, Ingrid A Holm, Pauline M Rudd, Margaret E Ackerman, Peter A Nigrovic

    Juvenile idiopathic arthritis (JIA) encompasses all forms of chronic idiopathic arthritis that arise before age 16. Previous studies have found JIA to be associated with lower Fc galactosylation of circulating IgG, but the overall spectrum of glycan changes and the net impact on IgG function are unknown. Using ultra performance liquid chromatography (UPLC), we compared IgG glycosylation in 54 subjects with recent-onset untreated JIA with 98 healthy pediatric controls, paired to biophysical profiling of affinity for 20 IgG receptors using a high-throughput multiplexed microsphere assay. Patients with JIA exhibited an increase in hypogalactosylated and hyposialylated IgG glycans, but no change in fucosylation or bisection, together with alteration in the spectrum of IgG ligand binding. Supervised machine learning demonstrated a robust capacity to discriminate JIA subjects from controls using either glycosylation or binding data. The binding signature was driven predominantly by enhanced affinity for Fc receptor like protein 5 (FcRL5), a noncanonical Fc receptor expressed on B cells. Affinity for FcRL5 correlated inversely with galactosylation and sialylation, a relationship confirmed through enzymatic manipulation. These results demonstrate the capacity of combined structural and biophysical IgG phenotyping to define the overall functional impact of IgG glycan changes and implicate FcRL5 as a potential cellular sensor of IgG glycosylation.

    更新日期:2017-10-27
  • Distinct roles for each N-glycan branch interacting with mannose-binding type Jacalin-related lectins Orysata and Calsepa
    Glycobiology (IF 3.112) Pub Date : 2017-09-07
    Masamichi Nagae, Sushil K Mishra, Shinya Hanashima, Hiroaki Tateno, Yoshiki Yamaguchi

    Mannose-binding type Jacalin-related lectins (mJRLs) bind to branched N-glycans via conserved sugar-binding sites. Despite, significant 3D structural similarities, each mJRL is known to have a unique binding preference toward various N-glycans. However, the molecular basis of varying binding preference is substantially unknown. Here, we report a detailed comparison of N-glycan-binding preference for two mJRLs, Orysata and Calsepa using frontal affinity chromatography (FAC), X-ray and molecular modeling. The FAC analysis using a panel of N-glycans shows difference in N-glycan-binding preference between the lectins. Orysata shows broader specificity toward most high-mannose-type glycans as well as biantennary complex-type glycans bearing an extension on the Manα1–6 branch. Whereas, Calsepa shows narrow specificity to complex-type glycans with bisecting GlcNAc. The X-ray crystallographic structure reveals that two Orysata lectins bind to one biantennary N-glycan (2:1 binding) where one lectin binds to mannose of the α1–3 branch, while the other interacts with an N-acetylglucosamine of the α1–6 branch. In contrast, Calsepa shows 1:1 binding where α1–3 branch and core chitobiose region N-glycan interacts with lectin, while α1–6 branch is flipped-back to the chitobiose core. Molecular dynamics study of Orysata bound to N-glycans substantiate possibility of two-binding modes for each N-glycan. Binding free energies calculated separately for α1–3 and α1–6 branches of each N-glycan suggest both branches can bind to Orysata. Overall these results suggest that each branch of N-glycan has a distinct role in binding to mJRLs and the nonbinding branch can contribute significantly to the binding affinity and hence to the specificity.

    更新日期:2017-10-27
  • Lectin binding characterizes the healthy human skeletal muscle glycophenotype and identifies disease specific changes in dystrophic muscle
    Glycobiology (IF 3.112) Pub Date : 2017-08-23
    Brian J. McMorran, M. Carrie Miceli, Linda G. Baum

    Our understanding of muscle glycosylation to date has derived from studies in mouse models and a limited number of human lectin histochemistry studies. As various therapeutic approaches aimed at treating patients with muscular dystrophies are being translated from rodent models to human, it is critical to better understand human muscle glycosylation and relevant disease-specific differences between healthy and dystrophic muscle. Here, we report the first quantitative characterization of human muscle glycosylation, and identify differentiation- and disease-specific differences in human muscle glycosylation. Utilizing a panel of 13 lectins with varying glycan specificities, we surveyed lectin binding to primary and immortalized myoblasts and myotubes from healthy and dystrophic sources. Following differentiation of primary and immortalized healthy human muscle cells, we observed increased binding of Narcissus pseudonarcissus agglutinin (NPA), PNA, MAA-II and WFA to myotubes compared to myoblasts. Following differentiation of immortalized healthy and dystrophic human muscle cells, we observed disease-specific differences in binding of NPA, Jac and Tricosanthes japonica agglutinin-I (TJA-I) to differentiated myotubes. We also observed differentiation- and disease-specific differences in binding of NPA, Jac, PNA, TJA-I and WFA to glycoprotein receptors in muscle cells. Additionally, Jac, PNA and WFA precipitated functionally glycosylated α-DG, that bound laminin, while NPA and TJA-I did not. Lectin histochemistry of healthy and dystrophic human muscle sections identified disease-specific differences in binding of O-glycan and sialic acid-specific lectins between healthy and dystrophic muscle. These results indicate that specific and discrete changes in glycosylation occur following differentiation, and identify specific lectins as potential biomarkers sensitive to changes in healthy human muscle glycosylation.

    更新日期:2017-10-27
  • New and updated glycoscience-related resources at NCBI
    Glycobiology (IF 3.112) Pub Date : 2017-10-06
    Ajit Varki

    The Symbol Nomenclature for Glycans (SNFG) (Varki et al. 2015) was originally developed as a collaboration amongst the Consortium of Glycobiology Editors of the recently released third edition of the Essentials of Glycobiologytextbook (published by Cold Spring Harbor Laboratory Press), and the Programs of Excellence in Glycoscience of the National Heart, Lung, and Blood Institute (NHLBI), following discussions among an extended community SNFG Discussion Group that represents some of the major contributors in the field.

    更新日期:2017-10-27
  • Immobilization alters heparin cleaving properties of heparinase I
    Glycobiology (IF 3.112) Pub Date : 2017-09-26
    Indu Bhushan, Alhumaidi Alabbas, Balagurunathan Kuberan, Ram B Gupta, Umesh R Desai

    We report here a novel observation that immobilization of heparinase I on CNBr-activated Sepharose results in heparin degradation properties that are different from heparinase I in the free solution form. Studies over a range of pHs (5–8) and temperatures (5–50°C) as well as under batch and flow conditions show that immobilized heparinase 1 displays altered pH and temperature optima, and a higher propensity for generation of longer chains (hexa- and octa-) with variable sulfation as compared to that in the free form, which is known to yield disaccharides. The immobilized enzyme retained good eliminase activity over at least five cycles of reuse. In combination, results suggest that heparinase I immobilization may offer a more productive route to longer, variably sulfated sequences.

    更新日期:2017-10-27
  • NFκB-mediated activation of the cellular FUT3, 5 and 6 gene cluster by herpes simplex virus type 1
    Glycobiology (IF 3.112) Pub Date : 2017-09-26
    Rickard Nordén, Ebba Samuelsson, Kristina Nyström

    Herpes simplex virus type 1 has the ability to induce expression of a human gene cluster located on chromosome 19 upon infection. This gene cluster contains three fucosyltransferases (encoded by FUT3, FUT5 and FUT6) with the ability to add a fucose to an N-acetylglucosamine residue. Little is known regarding the transcriptional activation of these three genes in human cells. Intriguingly, herpes simplex virus type 1 activates all three genes simultaneously during infection, a situation not observed in uninfected tissue, pointing towards a virus specific mechanism for transcriptional activation. The aim of this study was to define the underlying mechanism for the herpes simplex virus type 1 activation of FUT3, FUT5 and FUT6 transcription. The transcriptional activation of the FUT-gene cluster on chromosome 19 in fibroblasts was specific, not involving adjacent genes. Moreover, inhibition of NFκB signaling through panepoxydone treatment significantly decreased the induction of FUT3, FUT5 and FUT6 transcriptional activation, as did siRNA targeting of p65, in herpes simplex virus type 1 infected fibroblasts. NFκB and p65 signaling appears to play an important role in the regulation of FUT3, FUT5 and FUT6 transcriptional activation by herpes simplex virus type 1 although additional, unidentified, viral factors might account for part of the mechanism as direct interferon mediated stimulation of NFκB was not sufficient to induce the fucosyltransferase encoding gene cluster in uninfected cells.

    更新日期:2017-10-27
  • Core fucose is critical for CD14-dependent Toll-like receptor 4 signaling
    Glycobiology (IF 3.112) Pub Date : 2017-09-27
    Junko Iijima, Satoshi Kobayashi, Shinobu Kitazume, Yasuhiko Kizuka, Reiko Fujinawa, Hiroaki Korekane, Takuma Shibata, Shin-Ichiroh Saitoh, Sachiko Akashi-Takamura, Kensuke Miyake, Eiji Miyoshi, Naoyuki Taniguchi

    Core fucosylation, a posttranslational modification of N-glycans, modifies several growth factor receptors and impacts on their ligand binding affinity. Core-fucose-deficient mice generated by ablating the α1,6 fucosyltransferase enzyme, Fut8, exhibit severe pulmonary emphysema, partly due to impaired macrophage function, similar to aged Toll-like receptor 4 (Tlr4)–deficient mice. We therefore suspect that a lack of core fucose affects the TLR4-dependent signaling pathway. Indeed, upon lipopolysaccharide stimulation, Fut8-deficient mouse embryonic fibroblasts (MEFs) produced similar levels of interleukin-6 but markedly reduced levels of interferon-β (IFN-β) compared with wild-type MEFs. Lectin blot analysis of the TLR4 signaling complex revealed that core fucosylation was specifically found on CD14. Even though similar levels of TLR4/myeloid differentiation factor 2 (MD2) activation and dimerization were observed in Fut8-deficient cells after lipopolysaccharide stimulation, internalization of TLR4 and CD14 was significantly impaired. Given that internalized TLR4/MD2 induces IFN-β production, impaired IFN-β production in Fut8-deficient cells is ascribed to impaired TLR4/MD2 internalization. These data show for the first time that glycosylation critically regulates TLR4 signaling.

    更新日期:2017-10-27
  • Pectobacterium atrosepticum exopolysaccharides: identification, molecular structure, formation under stress and in planta conditions
    Glycobiology (IF 3.112) Pub Date : 2017-09-01
    Vladimir Gorshkov, Bakhtiyar Islamov, Polina Mikshina, Olga Petrova, Gennady Burygin, Elena Sigida, Alexander Shashkov, Amina Daminova, Marina Ageeva, Bulat Idiyatullin, Vadim Salnikov, Yuriy Zuev, Tatyana Gorshkova, Yuri Gogolev

    In the present study, we identified exopolysaccharides of the harmful phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043 and characterized the molecular structure of these polymers. The synthesis of the target polysaccharides was shown to be induced under starvation conditions. Moreover, intensive accumulation of exopolysaccharides occurred during the colonization by bacteria of the xylem vessels of infected plants, where microorganisms formed specific 3D “multicellular” structures—bacterial emboli. Thus, the identified polymers are likely to be involved in the adaptation and virulence of bacteria of Pectobacterium genus.

    更新日期:2017-10-27
  • Human norovirus GII.4(MI001) P dimer binds fucosylated and sialylated carbohydrates
    Glycobiology (IF 3.112) Pub Date : 2017-09-27
    Henrik Wegener, Álvaro Mallagaray, Tobias Schöne, Thomas Peters, Julia Lockhauserbäumer, Hao Yan, Charlotte Uetrecht, Grant S Hansman, Stefan Taube

    Human noroviruses (HuNoV), members of the family Caliciviridae, are the major cause of acute viral gastroenteritis worldwide. Successful infection is linked to the ability of the protruding (P) domain of the viral capsid to bind histo-blood group antigens (HBGA). Binding to gangliosides plays a major role for many nonhuman calici- and noroviruses. Increasing evidence points to a broader role of sialylated carbohydrates such as gangliosides in norovirus infection. Here, we compare HBGA and ganglioside binding of a GII.4 HuNoV variant (MI001), previously shown to be infectious in a HuNoV mouse model. Saturation transfer difference nuclear magnetic resonance spectroscopy, native mass spectrometry (MS) and surface plasmon resonance spectroscopy were used to characterize binding epitopes, affinities, stoichiometry and dynamics, focusing on 3′-sialyllactose, the GM3 ganglioside saccharide and B antigen. Binding was observed for 3′-sialyllactose and various HBGAs following a multistep binding process. Intrinsic affinities (Kd) of fucose, 3′-sialyllactose and B antigen were determined for the individual binding steps. Stronger affinities were observed for B antigen over 3′-sialyllactose and fucose, which bound in the mM range. Binding stoichiometry was analyzed by native MS showing the presence of four B antigens or two 3′-sialyllactose in the complex. Epitope mapping of 3′-sialyllactose revealed direct interaction of α2,3-linked sialic acid with the P domain. The ability of HuNoV to engage multiple carbohydrates emphasizes the multivalent nature of norovirus glycan-specificity. Our findings reveal direct binding of a GII.4 HuNoV P dimer to α2,3-linked sialic acid and support a broader role of ganglioside binding in norovirus infection.

    更新日期:2017-10-27
  • Novel polysaccharide binding to the N-terminal tail of galectin-3 is likely modulated by proline isomerization
    Glycobiology (IF 3.112) Pub Date : 2017-09-26
    Michelle C Miller, Y Zheng, Jingmin Yan, Yifa Zhou, Guihua Tai, Kevin H Mayo

    Interactions between galectins and polysaccharides are crucial to many biological processes, and yet these are some of the least understood, usually being limited to studies with small saccharides and short oligosaccharides. The present study is focused on human galectin-3 (Gal-3) interactions with a 60 kDa rhamnogalacturonan RG-I-4 that we use as a model to garner information as to how galectins interact with large polysaccharides, as well as to develop this agent as a therapeutic against human disease. Gal-3 is unique among galectins, because as the only chimera-type, it has a long N-terminal tail (NT) that has long puzzled investigators due to its dynamic, disordered nature and presence of numerous prolines. Here, we use 15N–1H heteronuclear single quantum coherence NMR spectroscopy to demonstrate that multiple sites on RG-I-4 provide epitopes for binding to three sites on 15N-labeled Gal-3, two within its carbohydrate recognition domain (CRD) and one at a novel site within the NT encompassing the first 40 residues that are highly conserved among all species of Gal-3. Moreover, strong binding of RG-I-4 to the Gal-3 NT occurs on a very slow time scale, suggesting that it may be mediated by cis–trans proline isomerization, a well-recognized modulator of many biological activities. The NT binding epitope within RG-I-4 appears to reside primarily in the side chains of the polysaccharide, some of which are galactans. Our results provide new insight into the role of the NT in Gal-3 function.

    更新日期:2017-10-27
  • Synthesis and characterization of heparosan-granulocyte-colony stimulating factor conjugates: a natural sugar-based drug delivery system to treat neutropenia
    Glycobiology (IF 3.112) Pub Date : 2017-09-08
    Wei Jing, Jonathan W Roberts, Dixy E Green, Andrew Almond, Paul L DeAngelis

    Many injectable drugs require delivery strategies for enhancing their pharmacokinetics due to rapid loss via renal filtration if possess low molecular weight (<60–70 kDa) and/or clearance by the body's components (e.g., proteases, antibodies, high-efficiency receptors) in their native form. FDA-approved polyethylene glycol (PEG) is a vehicle for improving therapeutics, but artificial polymers have potential biocompatibility and immunogenicity liabilities. Here, we utilized a natural vertebrate carbohydrate, heparosan (HEP), the biosynthetic precursor of heparan sulfate and heparin, to enhance performance of a biologic drug. The HEP polysaccharide was stable with a long half-life (~8 days for 99-kDa chain) in the nonhuman primate bloodstream, but was efficiently degraded to very short oligosaccharides when internalized by cells, and then excreted into urine and feces. Several HEP-modified human granulocyte-colony stimulating factor (G-CSF) conjugates were synthesized with defined quasi-monodisperse HEP polysaccharide chains. Single dosing of 55- or 99-kDa HEP-G-CSF in rats increased blood neutrophil levels comparable to PEG-G-CSF conjugates. Repeated dosing of HEP-G-CSF or HEP alone for 2 weeks did not cause HEP-specific toxic effects in rats. HEP did not possess the anticoagulant behavior of its daughter, heparin, based on testing in rats or clinical diagnostic assays with human plasma. Neither anti-HEP IgG nor IgM antibodies were detected in a long-term (9 doses over 7 months) immunogenicity study of the HEP-drug conjugate with rats. These proof-of-concept experiments with HEP-G-CSF indicate that it is a valid drug candidate for neutropenia and suggest the potential of this HEP-based platform as a safe alternative delivery vehicle for other therapeutics.

    更新日期:2017-10-27
  • Heparosan-coated liposomes for drug delivery
    Glycobiology (IF 3.112) Pub Date : 2017-09-08
    Rachel S Lane, F Michael Haller, Anais A E Chavaroche, Andrew Almond, Paul L DeAngelis

    Liposomal encapsulation is a useful drug delivery strategy for small molecules, especially chemotherapeutic agents such as doxorubicin. Doxil® is a doxorubicin-containing liposome (“dox-liposome”) that passively targets drug to tumors while reducing side effects caused by free drug permeating and poisoning healthy tissues. Polyethylene glycol (PEG) is the hydrophilic coating of Doxil® that protects the formulation from triggering the mononuclear phagocyte system (MPS). Evading the MPS prolongs dox-liposome circulation time thus increasing drug deposition at the tumor site. However, multiple doses of Doxil® sometimes activate an anti-PEG immune response that enhances liposome clearance from circulation and causes hypersensitivity, further limiting its effectiveness against disease. These side effects constrain the utility of PEG-coated liposomes in certain populations, justifying the need for investigation into alternative coatings that could improve drug delivery for better patient quality of life and outcome. We hypothesized that heparosan (HEP; [-4-GlcA-β1-4-GlcNAc-α1-]n) may serve as a PEG alternative for coating liposomes. HEP is a natural precursor to heparin biosynthesis in mammals. Also, bacteria expressing an HEP extracellular capsule during infection escape detection and are recognized as “self,” not a foreign threat. By analogy, coating drug-carrying liposomes with HEP should camouflage the delivery vehicle from the MPS, extending circulation time and potentially avoiding immune-mediated clearance. In this study, we characterize the postmodification insertion of HEP-lipids into liposomes by dynamic light scattering and coarse-grain computer modeling, test HEP-lipid immunogenicity in rats, and compare the efficacy of drug delivered by HEP-coated liposomes to PEG-coated liposomes in a human breast cancer xenograft mouse model.

    更新日期:2017-10-27
  • IMMOBILIZATION ALTERS HEPARIN CLEAVING PROPERTIES OF HEPARINASE I
    Glycobiology (IF 3.112) Pub Date : 2017-08-22
    Indu Bhushan, Alhumaidi Alabbas, Balagurunathan Kuberan, Ram B. Gupta, Umesh R. Desai

    We report here a novel observation that immobilization of heparinase I on CNBr-activated Sepharose results in heparin degradation properties that are different from heparinase I in the free solution form. Studies over a range of pHs (5–8) and temperatures (5–50°C) as well as under batch and flow conditions show that immobilized heparinase 1 displays altered pH and temperature optima, and a higher propensity for generation of longer chains (hexa- and octa-) with variable sulfation as compared to that in the free form, which is known to yield disaccharides. The immobilized enzyme retained good eliminase activity over at least five cycles of reuse. In combination, results suggest that heparinase I immobilization may offer a more productive route to longer, variably sulfated sequences.

    更新日期:2017-09-23
  • GlyTouCan: an accessible glycan structure repository
    Glycobiology (IF 3.112) Pub Date : 2017-08-09
    Michael Tiemeyer, Kazuhiro Aoki, James Paulson, Richard D Cummings, William S York, Niclas G Karlsson, Frederique Lisacek, Nicolle H Packer, Matthew P Campbell, Nobuyuki P Aoki, Akihiro Fujita, Masaaki Matsubara, Daisuke Shinmachi, Shinichiro Tsuchiya, Issaku Yamada, Michael Pierce, René Ranzinger, Hisashi Narimatsu, Kiyoko F Aoki-Kinoshita

    Rapid and continued growth in the generation of glycomic data has revealed the need for enhanced development of basic infrastructure for presenting and interpreting these datasets in a manner that engages the broader biomedical research community. Early in their growth, the genomic and proteomic fields implemented mechanisms for assigning unique gene and protein identifiers that were essential for organizing data presentation and for enhancing bioinformatic approaches to extracting knowledge. Similar unique identifiers are currently absent from glycomic data. In order to facilitate continued growth and expanded accessibility of glycomic data, the authors strongly encourage the glycomics community to coordinate the submission of their glycan structures to the GlyTouCan Repository and to make use of GlyTouCan identifiers in their communications and publications. The authors also deeply encourage journals to recommend a submission workflow in which submitted publications utilize GlyTouCan identifiers as a standard reference for explicitly describing glycan structures cited in manuscripts.

    更新日期:2017-09-11
  • Generation and characterization of a monoclonal antibody to the cytoplasmic tail of MUC16
    Glycobiology (IF 3.112) Pub Date : 2017-07-01
    Ilene K Gipson, Ulla Mandel, Balaraj Menon, Sandra Michaud, Ann Tisdale, Diana Campos, Henrik Clausen

    MUC16 is a large transmembrane mucin expressed on the apical surfaces of the epithelium covering the ocular surface, respiratory system and female reproductive tract. The transmembrane mucin is overexpressed by ovarian carcinomas, it is one of the most frequently used diagnostic markers for the disease and it is considered a promising target for immunotherapeutic intervention. Immunodetection of the mucin has to date been through antibodies that recognize its exceptionally large ectodomain. Similar to other membrane anchored mucins, MUC16 has a short cytoplasmic tail (CT), but studies of the biological relevance of the C-terminal domain of MUC16 has been limited by lack of availability of monoclonal antibodies that recognize the native CT. Here, we report the development of a novel monoclonal antibody to the CT region of the molecule that recognizes native MUC16 and its enzymatically released CT region. The antibody is useful for immunoprecipitation of the released CT domain as demonstrated with the OVCAR3 ovarian cancer cell line and can be used for detailed cytolocalization in cells as well as in frozen sections of ocular surface and uterine epithelium.

    更新日期:2017-09-11
  • O-GlcNAc transferase regulates transcriptional activity of human Oct4
    Glycobiology (IF 3.112) Pub Date : 2017-06-24
    Sandii Constable, Jae-Min Lim, Krithika Vaidyanathan, Lance Wells

    O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4.

    更新日期:2017-09-11
  • Genomic and biochemical characterization of sialic acid acetylesterase (siae) in zebrafish
    Glycobiology (IF 3.112) Pub Date : 2017-08-18
    Viola Ravasio, Eufemia Damiati, Daniela Zizioli, Flavia Orizio, Edoardo Giacopuzzi, Marta Manzoni, Roberto Bresciani, Giuseppe Borsani, Eugenio Monti

    Sialic acid acetylesterase (SIAE) removes acetyl moieties from the carbon 9 and 4 hydroxyl groups of sialic acid and recently a debate has been opened on its association to autoimmunity. Trying to get new insights on this intriguing enzyme we have studied siae in zebrafish (Danio rerio). In this teleost siae encodes for a polypeptide with a high degree of sequence identity to human and mouse counterparts. Zebrafish Siae behavior upon transient expression in COS7 cells is comparable to human enzyme concerning pH optimum of enzyme activity, subcellular localization and glycosylation. In addition, and as already observed in case of human SIAE, the glycosylated form of the enzyme from zebrafish is released into the culture media. During embryogenesis, in situ hybridization experiments demonstrate that siae transcript is always detectable during development, with a more specific expression in the central nervous system, in pronephric ducts and liver in the more advanced stages of the embryo development. In adult fish an increasing amount of siae mRNA is detectable in heart, eye, muscle, liver, brain, kidney and ovary. These results provide novel information about Siae and point out zebrafish as animal model to better understand the biological role(s) of this rather puzzling enzyme in vertebrates, regarding immune system function and the development of central nervous system.

    更新日期:2017-09-11
  • Colocalization of receptors for Shiga toxins with lipid rafts in primary human renal glomerular endothelial cells and influence of D-PDMP on synthesis and distribution of glycosphingolipid receptors
    Glycobiology (IF 3.112) Pub Date : 2017-06-08
    Nadine Legros, Gottfried Pohlentz, Jana Runde, Stefanie Dusny, Hans-Ulrich Humpf, Helge Karch, Johannes Müthing

    Damage of human renal glomerular endothelial cells (HRGECs) of the kidney represents the linchpin in the pathogenesis of the hemolytic uremic syndrome caused by Shiga toxins of enterohemorrhagic Escherichia coli (EHEC). We performed a comprehensive structural analysis of the Stx-receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα4Galβ4Glcβ1Cer) and globotetraosylceramide (Gb4Cer, GalNAcβ3Galα4Galβ4Glcβ1Cer) and their distribution in lipid raft analog detergent-resistant membranes (DRMs) and nonDRMs prepared from primary HRGECs. Predominant receptor lipoforms were Gb3Cer and Gb4Cer with Cer (d18:1, C16:0), Cer (d18:1, C22:0) and Cer (d18:1, C24:1/C24:0). Stx-receptor GSLs co-distribute with sphingomyelin (SM) and cholesterol as well as flotillin-2 in DRMs, representing the liquid-ordered membrane phase and indicating lipid raft association. Lyso-phosphatidylcholine (lyso-PC) was identified as a nonDRM marker phospholipid of the liquid-disordered membrane phase. Exposure of primary HRGECs to the ceramide analogon d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) reduced total Gb3Cer and Gb4Cer content, roughly calculated from two biological replicates, down to half and quarter of its primordial content, respectively, but strengthened their prevalence and cholesterol preponderance in DRMs. At the same time, the distribution of PC, SM and lyso-PC to subcellular membrane fractions remained unaffected by D-PDMP treatment. Defining the GSL composition and precise microdomain structures of primary HRGECs may help to develop novel therapeutic options to combat life-threatening EHEC infections.

    更新日期:2017-09-11
  • High-resolution crystal structures and STD NMR mapping of human ABO(H) blood group glycosyltransferases in complex with trisaccharide reaction products suggest a molecular basis for product release
    Glycobiology (IF 3.112) Pub Date : 2017-07-03
    Susannah M L Gagnon, Max S G Legg, Nora Sindhuwinata, James A Letts, Asha R Johal, Brock Schuman, Svetlana N Borisova, Monica M Palcic, Thomas Peters, Stephen V Evans

    The human ABO(H) blood group A- and B-synthesizing glycosyltransferases GTA and GTB have been structurally characterized to high resolution in complex with their respective trisaccharide antigen products. These findings are particularly timely and relevant given the dearth of glycosyltransferase structures collected in complex with their saccharide reaction products. GTA and GTB utilize the same acceptor substrates, oligosaccharides terminating with α-l-Fucp-(1→2)-β-d-Galp-OR (where R is a glycolipid or glycoprotein), but use distinct UDP donor sugars, UDP-N-acetylgalactosamine and UDP-galactose, to generate the blood group A (α-l-Fucp-(1→2)[α-d-GalNAcp-(1→3)]-β-d-Galp-OR) and blood group B (α-l-Fucp-(1→2)[α-d-Galp-(1→3)]-β-d-Galp-OR) determinant structures, respectively. Structures of GTA and GTB in complex with their respective trisaccharide products reveal a conflict between the transferred sugar monosaccharide and the β-phosphate of the UDP donor. Mapping of the binding epitopes by saturation transfer difference NMR measurements yielded data consistent with the X-ray structural results. Taken together these data suggest a mechanism of product release where monosaccharide transfer to the H-antigen acceptor induces active site disorder and ejection of the UDP leaving group prior to trisaccharide egress.

    更新日期:2017-09-11
  • A conserved DGGK motif is essential for the function of the PglB oligosaccharyltransferase from Campylobacter jejuni
    Glycobiology (IF 3.112) Pub Date : 2017-08-18
    Yasmin Barre, Harald Nothaft, Cody Thomas, Xin Liu, Jianjun Li, Kenneth KS Ng, Christine M Szymanski

    In Campylobacter jejuni, the PglB oligosaccharyltransferase catalyzes the transfer of a heptasaccharide from a lipid donor to asparagine within the D/E-X1-N–X2-S/T sequon (X1,2 ≠ P) or releases this heptasaccharide as free oligosaccharides (fOS). Using available crystal structures and sequence alignments, we identified a DGGK motif near the active site of PglB that is conserved among all Campylobacter species. We demonstrate that amino acid substitutions in the aspartate and lysine residues result in loss of protein glycosylation in the heterologous Escherichia coli system. Similarly, complementation of a C. jejuni pglB knock-out strain with mutated pglB alleles results in reduced levels of N-linked glycoproteins and fOS in the native host. Analysis of the PglB crystal structures from Campylobacter lari and the soluble C-terminal domain from C. jejuni suggests a particularly important structural role for the aspartate residue and the two following glycine residues, as well as a more subtle, less defined role for the lysine residue. Limited proteolysis experiments indicate that conformational changes of wildtype PglB that are induced by the binding of the lipid-linked oligosaccharide are altered by changes in the DGGK motif. Related to these findings, certain Campylobacter species possess two PglB orthologues and we demonstrate that only the orthologue containing the DGGK motif is active. Combining the knowledge gained from the PglB structures and mutagenesis studies, we propose a function for the DGGK motif in affecting the binding of the undecaprenyl-pyrophosphate glycan donor substrate that subsequently influences N-glycan and fOS production.

    更新日期:2017-09-11
  • Synthesis and Characterization of Heparosan-Granulocyte-Colony Stimulating Factor Conjugates: a natural sugar-based drug delivery system to treat neutropenia
    Glycobiology (IF 3.112) Pub Date : 2017-08-23
    Wei Jing, Jonathan W. Roberts, Dixy E. Green, Andrew Almond, Paul L. DeAngelis

    Many injectable drugs require delivery strategies for enhancing their pharmacokinetics due to rapid loss via renal filtration if possess low molecular weight (<60–70 kDa) and/or clearance by the body’s components (e.g., proteases, antibodies, high-efficiency receptors) in their native form. FDA-approved polyethylene glycol (PEG) is a vehicle for improving therapeutics, but artificial polymers have potential biocompatibility and immunogenicity liabilities. Here, we utilized a natural vertebrate carbohydrate, heparosan (HEP), the biosynthetic precursor of heparan sulfate and heparin, to enhance performance of a biologic drug. The HEP polysaccharide was stable with a long half-life (~8 days for 99-kDa chain) in the nonhuman primate bloodstream, but was efficiently degraded to very short oligosaccharides when internalized by cells, and then excreted into urine and feces. Several HEP-modified human granulocyte-colony stimulating factor (G-CSF) conjugates were synthesized with defined quasi-monodisperse HEP polysaccharide chains. Single dosing of 55- or 99-kDa HEP-G-CSF in rats increased blood neutrophil levels comparable to PEG-G-CSF conjugates. Repeated dosing of HEP-G-CSF or HEP alone for 2 weeks did not cause HEP-specific toxic effects in rats. HEP did not possess the anticoagulant behavior of its daughter, heparin, based on testing in rats or clinical diagnostic assays with human plasma. Neither anti-HEP IgG nor IgM antibodies were detected in a long-term (9 doses over 7 months) immunogenicity study of the HEP-drug conjugate with rats. These proof-of-concept experiments with HEP-G-CSF indicate that it is a valid drug candidate for neutropenia and suggest the potential of this HEP-based platform as a safe alternative delivery vehicle for other therapeutics.

    更新日期:2017-09-08
  • Heparosan-Coated Liposomes for Drug Delivery
    Glycobiology (IF 3.112) Pub Date : 2017-08-10
    Rachel S Lane, F. Michael Haller, Anais A. E Chavaroche, Andrew Almond, Paul L DeAngelis

    Liposomal encapsulation is a useful drug delivery strategy for small molecules, especially chemotherapeutic agents such as doxorubicin. Doxil® is a doxorubicin-containing liposome (“dox-liposome”) that passively targets drug to tumors while reducing side effects caused by free drug permeating and poisoning healthy tissues. Polyethylene glycol (PEG) is the hydrophilic coating of Doxil® that protects the formulation from triggering the mononuclear phagocyte system (MPS). Evading the MPS prolongs dox-liposome circulation time thus increasing drug deposition at the tumor site. However, multiple doses of Doxil® sometimes activate an anti-PEG immune response that enhances liposome clearance from circulation and causes hypersensitivity, further limiting its effectiveness against disease. These side effects constrain the utility of PEG-coated liposomes in certain populations, justifying the need for investigation into alternative coatings that could improve drug delivery for better patient quality of life and outcome. We hypothesized that heparosan (HEP; [-4-GlcA-β1-4-GlcNAc-α1-]n) may serve as a PEG alternative for coating liposomes. HEP is a natural precursor to heparin biosynthesis in mammals. Also, bacteria expressing an HEP extracellular capsule during infection escape detection and are recognized as “self,” not a foreign threat. By analogy, coating drug-carrying liposomes with HEP should camouflage the delivery vehicle from the MPS, extending circulation time and potentially avoiding immune-mediated clearance. In this study, we characterize the postmodification insertion of HEP-lipids into liposomes by dynamic light scattering and coarse-grain computer modeling, test HEP-lipid immunogenicity in rats, and compare the efficacy of drug delivered by HEP-coated liposomes to PEG-coated liposomes in a human breast cancer xenograft mouse model.

    更新日期:2017-09-08
  • NFκB-mediated activation of the cellular FUT3, 5 and 6 gene cluster by herpes simplex virus type 1
    Glycobiology (IF 3.112) Pub Date : 2017-09-04
    Rickard Nordén, Ebba Samuelsson, Kristina Nyström

    Herpes simplex virus type 1 has the ability to induce expression of a human gene cluster located on chromosome 19 upon infection. This gene cluster contains three fucosyltransferases (encoded by FUT3, FUT5 and FUT6) with the ability to add a fucose to an N-acetylglucosamine residue. Little is known regarding the transcriptional activation of these three genes in human cells. Intriguingly, herpes simplex virus type 1 activates all three genes simultaneously during infection, a situation not observed in uninfected tissue, pointing towards a virus specific mechanism for transcriptional activation. The aim of this study was to define the underlying mechanism for the herpes simplex virus type 1 activation of FUT3, FUT5 and FUT6 transcription. The transcriptional activation of the FUT gene cluster on chromosome 19 in fibroblasts was specific, not involving adjacent genes. Moreover, inhibition of NFκB signaling through panepoxydone treatment significantly decreased the induction of FUT3, FUT5 and FUT6 transcriptional activation, as did siRNA targeting of p65 in herpes simplex virus type 1 infected fibroblasts. NFκB and p65 signaling appears to play an important role in the regulation of FUT3, FUT5 and FUT6 transcriptional activation by herpes simplex virus type 1 although additional, unidentified, viral factors might account for part of the mechanism as direct interferon mediated stimulation of NFκB was not sufficient to induce the fucosyltransferase encoding gene cluster in uninfected cells.

    更新日期:2017-09-04
  • Human Norovirus GII.4(MI001) P Dimer Binds Fucosylated and Sialylated Carbohydrates
    Glycobiology (IF 3.112) Pub Date : 2017-09-04
    Henrik Wegener, Álvaro Mallagaray, Tobias Schöne, Thomas Peters, Julia Lockhauserbäumer, Hao Yan, Charlotte Uetrecht, Grant Hansman, Stefan Taube

    Human noroviruses (HuNoV), members of the family Caliciviridae are the major cause of acute viral gastroenteritis worldwide. Successful infection is linked to the ability of the protruding (P) domain of the viral capsid to bind histo-blood group antigens (HBGA). Binding to gangliosides plays a major role for many non-human calici- and noroviruses. Increasing evidence points to a broader role of sialylated carbohydrates such as gangliosides in norovirus infection. Here, we compare HBGA and ganglioside binding of a GII.4 HuNoV variant (MI001), previously shown to be infectious in a HuNoV mouse model. Saturation transfer difference nuclear magnetic resonance spectroscopy (STD NMR), native mass spectrometry (MS), and surface plasmon resonance (SPR) were used to characterize binding-epitopes, affinities, stoichiometry, and dynamics, focusing on 3’-sialyllactose, the GM3 ganglioside saccharide, and B antigen. Binding was observed for 3’-sialyllactose and various HBGAs following a multi-step binding process. Intrinsic affinities (Kd) of fucose, 3’-sialyllactose, and B antigen were determined for the individual binding steps. Stronger affinities were observed for B antigen over 3’-sialyllactose and fucose, which bound in the mM range. Binding stoichiometry was analyzed by native MS showing the presence of four B antigens or two 3’-sialyllactose in the complex. Epitope mapping of 3’-sialyllactose revealed direct interaction of α2,3-linked sialic acid with the P domain. The ability of HuNoV to engage multiple carbohydrates emphasizes the multivalent nature of norovirus glycan-specificity. Our findings reveal direct binding of a GII.4 HuNoV P dimer to α2,3-linked sialic acid and support a broader role of ganglioside-binding in norovirus infection.

    更新日期:2017-09-04
  • Core fucose is critical for CD14-dependent Toll-like receptor 4 signaling
    Glycobiology (IF 3.112) Pub Date : 2017-08-29
    Junko Iijima, Satoshi Kobayashi, Shinobu Kitazume, Yasuhiko Kizuka, Reiko Fujinawa, Hiroaki Korekane, Takuma Shibata, Shin-Ichiroh Saitoh, Sachiko Akashi-Takamura, Kensuke Miyake, Eiji Miyoshi, Naoyuki Taniguchi

    Core fucosylation, a post-translational modification of N-glycans, modifies several growth factor receptors and impacts on their ligand binding affinity. Core-fucose-deficient mice generated by ablating the α1,6 fucosyltransferase enzyme, Fut8, exhibit severe pulmonary emphysema, partly due to impaired macrophage function, similar to aged Toll-like receptor 4 (Tlr4) -deficient mice. We therefore suspect that a lack of core fucose affects the TLR4-dependent signaling pathway. Indeed, upon lipopolysaccharide stimulation, Fut8-deficient mouse embryonic fibroblasts (MEFs) produced similar levels of interleukin-6 but markedly reduced levels of interferon-β (IFN-β) compared with wild-type MEFs. Lectin blot analysis of the TLR4 signaling complex revealed that core fucosylation was specifically found on CD14. Even though similar levels of TLR4/myeloid differentiation factor 2 (MD2) activation and dimerization were observed in Fut8-deficient cells after lipopolysaccharide stimulation, internalization of TLR4 and CD14 was significantly impaired. Given that internalized TLR4/MD2 induces IFN-β production, impaired IFN-β production in Fut8-deficient cells is ascribed to impaired TLR4/MD2 internalization. These data show for the first time that glycosylation critically regulates TLR4 signaling.

    更新日期:2017-08-29
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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