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  • 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 (MHCII), 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:2017-11-17
  • 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:2017-11-17
  • 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:2017-11-17
  • 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:2017-11-17
  • 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:2017-11-17
  • 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:2017-11-17
  • 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
  • Novel polysaccharide binding to the N-terminal tail of galectin-3 is likely modulated by proline isomerization
    Glycobiology (IF 3.112) Pub Date : 2017-08-10
    Michelle C Miller, Yi 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 HSQC 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:2017-08-10
  • 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:2017-08-09
  • Characterization of tools to detect and enrich human and mouse O-GlcNAcase
    Glycobiology (IF 3.112) Pub Date : 2017-06-24
    Jennifer A Groves, Natasha E Zachara

    O-linked β-N-acetylglucosamine (O-GlcNAc) is an essential regulatory post-translational modification of thousands of nuclear, cytoplasmic and mitochondrial proteins. O-GlcNAc is dynamically added and removed from proteins by the O-GlcNAc transferase and the O-GlcNAcase (OGA), respectively. Dysregulation of O-GlcNAc-cycling is implicated in the etiology of numerous diseases including tumorigenesis, metabolic dysfunction and neurodegeneration. To facilitate studies focused on the role of O-GlcNAc and OGA in disease, we sought to identify commercially available antibodies that enable the enrichment of full-length OGA (fOGA) from lysates of mouse and human origin. Here, we report that antibodies from Abcam and Bethyl Laboratories can be used to immunoprecipitate OGA to near-saturation from human and mouse cell lysates. However, western blotting analysis indicates that both antibodies, as well as three noncommercially available antibodies (345, 346, 352), detect fOGA and numerous cross-reacting proteins. These nonspecific signals migrate similarly to fOGA and are detected robustly, suggesting that the use of appropriate controls is essential to avoid the misidentification of OGA.

    更新日期:2017-08-07
  • Planning, evaluating and vetting receptor signaling studies to assess hyaluronan size-dependence and specificity
    Glycobiology (IF 3.112) Pub Date : 2017-07-01
    Paul H Weigel

    Exciting discoveries in many diverse fields of hyaluronan (HA) biology over the last 40 years have centered around the ability of HA to bind cell surface HA receptors (e.g., CD44, Layilin, LYVE-1, HARE/Stab2 and RHAMM) and sometimes also to activate intracellular signal transduction pathways, frequently involving ERK1/2. Although perplexing, a major characteristic of HA-mediated signal pathway activation for some receptors has been a dependence on the size of the bound HA. Receptors that directly interact with HA, which may not include TLR2/4, bind very well to any HA molecule >8–20 sugars, depending on the receptor. Despite their ability to bind virtually any size HA, only HA chains of a particular mass range can activate receptor-mediated cell signaling. Many studies have demonstrated parts of this emerging story by utilizing different: HA receptors, cell types, animal models, HA sources, HA sizes, assays to assess HA mass and varying controls to verify HA specificity or HA size-dependence. Recent reports have highlighted issues with potential endotoxin contamination of HA fragments, especially those generated by hyaluronidase digestion. Also, researchers unfamiliar with HA polydispersity must adjust to working with, and interpreting data for, preparations without a unique molecular mass (molecular weight). The confusion, uncertainty and skepticism generated by these and other factors has hindered the development of a general consensus about HA-specific and HA-size dependent receptor activation. An overview of issues, suggested strategies and validating controls is presented to aid those planning an HA-mediated receptor signaling study or those trying to evaluate the literature.

    更新日期:2017-08-07
  • Identification of lectin counter-receptors on cell membranes by proximity labeling
    Glycobiology (IF 3.112) Pub Date : 2017-07-28
    Gang Wu, Manjula Nagala, Paul R Crocker

    Lectin–glycan interactions play important roles in many biological systems, but the nature of glycoprotein counter-receptors expressed on cell membranes is often poorly understood. To help overcome this problem, we developed a method based on proximity labeling technology. Using a peroxidase-coupled lectin, addition of H2O2 and tyramide-biotin substrates leads to generation of short-range biotin radicals that biotinylate proteins in the immediate vicinity of the bound lectin, which can subsequently be identified. As a proof-of-principle, sialoadhesin-horseradish peroxidase-human IgG1 Fc recombinant protein constructs were precomplexed with anti-Fc antibodies, bound to human erythrocytes and reacted with H2O2 and tyramide-SS-biotin. The erythrocyte membrane protein with strongest biotinylation was identified as glycophorin A, in agreement with early studies using lectin overlay and reglycosylation approaches. As a further test of the method, the plant lectin MAL II was conjugated with horseradish peroxidase and used in proximity labeling of human erythrocytes. Glycophorin A was again selectively labeled, which is consistent with previous reports that MAL II has high affinity for glycophorin. This method could be applied to other lectins to identify their membrane counter-receptors.

    更新日期:2017-08-07
  • Recent advancements in understanding mammalian O-mannosylation
    Glycobiology (IF 3.112) Pub Date : 2017-07-20
    M Osman Sheikh, Stephanie M Halmo, Lance Wells

    The post-translational glycosylation of select proteins by O-linked mannose (O-mannose or O-man) is a conserved modification from yeast to humans and has been shown to be necessary for proper development and growth. The most well studied O-mannosylated mammalian protein is α-dystroglycan (α-DG). Hypoglycosylation of α-DG results in varying severities of congenital muscular dystrophies, cancer progression and metastasis, and inhibited entry and infection of certain arenaviruses. Defects in the gene products responsible for post-translational modification of α-DG, primarily glycosyltransferases, are the basis for these diseases. The multitude of clinical phenotypes resulting from defective O-mannosylation highlights the biomedical significance of this unique modification. Elucidation of the various O-mannose biosynthetic pathways is imperative to understanding a broad range of human diseases and for the development of novel therapeutics. In this review, we will focus on recent discoveries delineating the various enzymes, structures and functions associated with O-mannose-initiated glycoproteins. Additionally, we discuss current gaps in our knowledge of mammalian O-mannosylation, discuss the evolution of this pathway, and illustrate the utility and limitations of model systems to study functions of O-mannosylation.

    更新日期:2017-08-07
  • Bacterial phosphoglycosyl transferases: initiators of glycan biosynthesis at the membrane interface
    Glycobiology (IF 3.112) Pub Date : 2017-07-20
    Vinita Lukose, Marthe TC Walvoort, Barbara Imperiali

    Phosphoglycosyl transferases (PGTs) initiate the biosynthesis of both essential and virulence-associated bacterial glycoconjugates including lipopolysaccharide, peptidoglycan and glycoproteins. PGTs catalyze the transfer of a phosphosugar moiety from a nucleoside diphosphate sugar to a polyprenol phosphate, to form a membrane-bound polyprenol diphosphosugar product. PGTs are integral membrane proteins, which include between 1 and 11 predicted transmembrane domains. Despite this variation, common motifs have been identified in PGT families through bioinformatics and mutagenesis studies. Bacterial PGTs represent important antibacterial and virulence targets due to their significant role in initiating the biosynthesis of key bacterial glycoconjugates. Considerable effort has gone into mechanistic and inhibition studies for this class of enzymes, both of which depend on reliable, high-throughput assays for easy quantification of activity. This review summarizes recent advances made in the characterization of this challenging but important class of enzymes.

    更新日期:2017-08-07
  • Different properties of polysialic acids synthesized by the polysialyltransferases ST8SIA2 and ST8SIA4
    Glycobiology (IF 3.112) Pub Date : 2017-07-14
    Airi Mori, Masaya Hane, Yuki Niimi, Ken Kitajima, Chihiro Sato

    Polysialic acid (polySia) is mainly found as a modification of neural cell adhesion molecule (NCAM) in whole embryonic brains, as well as restricted areas of adult vertebrate brains, including the hippocampus. PolySia shows not only repulsive effects on NCAM-involved cell–cell interactions due to its bulky and hydrated properties, but also attractive effects on the interaction with neurologically active molecules, which exerts a reservoir function. Two different polysialyltransferases, ST8SIA2 and ST8SIA4, are involved in the synthesis of polySia chains; however, to date, the differences of the properties between polySia chains synthesized by these two enzymes remain unknown. In this study, to clarify this point, we first prepared polySia-NCAMs from HEK293 cells stably expressing ST8SIA4 and ST8SIA2, or ST8SIA2 (SNP-7), a mutant ST8SIA2 derived from a schizophrenia patient. The conventional sensitive chemical and immunological characterizations showed that the quantity and quality (structural features) of polySia are not so much different between ST8SIA4- and ST8SIA2-synthesized ones, apart from those of ST8SIA2 (SNP-7). Then, we assessed the homophilic and heterophilic interactions mediated by polySia-NCAM by adopting a surface plasmon resonance measurement as an in vitro analytical method. Our novel findings are as follows: (i) the ST8SIA2- and ST8SIA4-synthesized polySia-NCAMs exhibited different attractive and repulsive effects than each other; (ii) both polySia- and oligoSia-NCAMs synthesized by ST8SIA2 were able to bind polySia-NCAMs; (iii) the polySia-NCAM synthesized by a ST8SIA2 (SNP-7) showed markedly altered attractive and repulsive properties. Collectively, polySia-NCAM is suggested to simultaneously possess both attractive and repulsive properties that are highly regulated by the two polysialyltransferases.

    更新日期:2017-08-07
  • Enterocyte glycosylation is responsive to changes in extracellular conditions: implications for membrane functions
    Glycobiology (IF 3.112) Pub Date : 2017-06-26
    Dayoung Park, Gege Xu, Mariana Barboza, Ishita M Shah, Maurice Wong, Helen Raybould, David A Mills, Carlito B Lebrilla

    Epithelial cells in the lining of the intestines play critical roles in maintaining homeostasis while challenged by dynamic and sudden changes in luminal contents. Given the high density of glycosylation that encompasses their extracellular surface, environmental changes may lead to extensive reorganization of membrane-associated glycans. However, neither the molecular details nor the consequences of conditional glycan changes are well understood. Here we assessed the sensitivity of Caco-2 and HT-29 membrane N-glycosylation to variations in (i) dietary elements, (ii) microbial fermentation products and (iii) cell culture parameters relevant to intestinal epithelial cell growth and survival. Based on global LC–MS glycomic and statistical analyses, the resulting glycan expression changes were systematic, dependent upon the conditions of each controlled environment. Exposure to short chain fatty acids produced significant increases in fucosylation while further acidification promoted hypersialylation. Notably, among all conditions, increases of high mannose type glycans were identified as a major response when extracellular fructose, galactose and glutamine were independently elevated. To examine the functional consequences of this discrete shift in the displayed glycome, we applied a chemical inhibitor of the glycan processing mannosidase, globally intensifying high mannose expression. The data reveal that upregulation of high mannose glycosylation has detrimental effects on basic intestinal epithelium functions by altering permeability, host–microbe associations and membrane protein activities.

    更新日期:2017-08-07
  • Hyaluronic acid conjugation facilitates clearance of intracellular bacterial infections by streptomycin with neglectable nephrotoxicity
    Glycobiology (IF 3.112) Pub Date : 2017-07-20
    Yuanhao Qiu, Yilin Hou, Feifei Sun, Peng Chen, Dongdong Wang, Haibo Mu, Xiaoli Zhang, Kan Ding, Jinyou Duan

    Antibiotics such as β-lactams and aminoglycosides are often subtherapeutic to intracellular infections due to their high hydrophilicity, resulting in low effectiveness against intracellular pathogens and the emergence of antibiotic resistance. Here we reported that an endogenous aminoglycan, hyaluronic acid could be an effective carbohydrate carrier of the aminoglycoside antibiotic, streptomycin against intracellular pathogens. This conjugation could enhance phagocytic activity, and facilitated the entry of streptomycin into host cells via a CD44-mediated pathway. It appeared that this conjugate could clear intracellular bacteria in phagocytic or nonphagocytic cells in a short-term therapy (4 h) at a lower effective dose. In addition, this conjugate was more efficient in reducing bacteria burden in an in vivo acute infection model than streptomycin did. Interestingly, subcutaneous injection of this conjugate at an excess amount had undetectable side effects such as nephrotoxicity. These results suggested that hyaluronic acid might be an efficient Trojan horse for the delivery of hydrophilic antibiotics to deal with intracellular infections.

    更新日期:2017-08-07
  • What is special about 200 kDa hyaluronan that activates hyaluronan receptor signaling?
    Glycobiology (IF 3.112) Pub Date : 2017-05-25
    Paul H Weigel, Bruce A Baggenstoss

    The polydispersity of hyaluronan (HA) presents challenges for analyzing its solution properties, such as the relationship between mass and particle size. The broad mass range of natural HA (≤50-fold) makes molecular characterization difficult and ambiguous compared to molecules with known molecular weights (e.g., proteins). Biophysical studies show that large >MDa HA behaves like a random coil, whereas very small (e.g., 10 kDa) HA behaves like a rod. However, the mass range for this conformational transition is not easily determined in natural polydisperse HA. Some HA receptors (e.g., CD44 and HARE) initiate signaling responses upon binding HA in the 100–300 kDa range, but not larger MDa HA. Size-dependent responses are studied using nonnatural HA: purified narrow-size range HA [Pandey MS, Baggenstoss BA, Washburn J, Harris EN, Weigel PH. 2013. The hyaluronan receptor for endocytosis (HARE) activates NF-κB-mediated gene expression in response to 40–400 kDa, but not smaller or sarger, hyaluronans. J Biol Chem. 288:14068–14079] and very narrow size range Select-HA made chemo-enzymatically [Jing W, DeAngelis PL. 2004. Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers. J Biol Chem. 279:42345–42349]. Here, we used size exclusion chromatography and multiangle light scattering to determine the weight-average molar mass and diameter of ~60 very narrow size preparations from 29 to 1650 kDa. The ratio of HA mass to HA diameter showed a transition in the 150–250 kDa size range (~65 nm). The HA rod-to-coil transition occurs within the size range that specifically activates cell signaling by some receptors. Thus, size-specific signaling could be due to unique external receptor•HA conformation changes that enable transmembrane-mediated activation of cytoplasmic domains. Alternatively and more likely, transition-size HA may enable multiple receptors to bind the same HA, creating new internal signal-competent cytoplasmic domain complexes.

    更新日期:2017-08-07
  • Galectin-9 binds to O-glycans on protein disulfide isomerase
    Glycobiology (IF 3.112) Pub Date : 2017-07-28
    Katrin Schaefer, Nicholas E Webb, Mabel Pang, Jenny E Hernandez-Davies, Katharine P Lee, Pascual Gonzalez, Martin V Douglass, Benhur Lee, Linda G Baum

    Changes in the T cell surface redox environment regulate critical cell functions, such as cell migration, viral entry and cytokine production. Cell surface protein disulfide isomerase (PDI) contributes to the regulation of T cell surface redox status. Cell surface PDI can be released into the extracellular milieu or can be internalized by T cells. We have found that galectin-9, a soluble lectin expressed by T cells, endothelial cells and dendritic cells, binds to and retains PDI on the cell surface. While endogenous galectin-9 is not required for basal cell surface PDI expression, exogenous galectin-9 mediated retention of cell surface PDI shifted the disulfide/thiol equilibrium on the T cell surface. O-glycans on PDI are required for galectin-9 binding, and PDI recognition appears to be specific for galectin-9, as galectin-1 and galectin-3 do not bind PDI. Galectin-9 is widely expressed by immune and endothelial cells in inflamed tissues, suggesting that T cells would be exposed to abundant galectin-9, in cis and in trans, in infectious or autoimmune conditions.

    更新日期:2017-08-07
  • Structural and genetic analyses of glycan O-acetylation in a bacterial protein glycosylation system: evidence for differential effects on glycan chain length
    Glycobiology (IF 3.112) Pub Date : 2017-05-23
    Jan Haug Anonsen, Bente Børud, Åshild Vik, Raimonda Viburiene, Michael Koomey

    O-acetylation is a common modification of bacterial glycoconjugates. By modifying oligosaccharide structure and chemistry, O-acetylation has important consequences for biotic and abiotic recognition events and thus bacterial fitness in general. Previous studies of the broad-spectrum O-linked protein glycosylation in pathogenic Neisseria species (including N. gonorrhoeae and N. meningitidis) have revealed O-acetylation of some of their diverse glycoforms and identified the committed acetylase, PglI. Herein, we extend these observations by using mass spectrometry to examine a complete set of all glycan variants identified to date. Regardless of composition, all glycoforms and all sugars in the oligosaccharide are subject to acetylation in a PglI-dependent fashion with the only exception of di-N-acetyl-bacillosamine. Moreover, multiple sugars in a single oligosaccharide could be simultaneously modified. Interestingly, O-acetylation status was found to be correlated with altered chain lengths of oligosaccharides expressed in otherwise isogenic backgrounds. Models for how this unprecedented phenomenon might arise are discussed with some having potentially important implications for the membrane topology of glycan O-acetylation. Together, the findings provide better insight into how O-acetylation can both directly and indirectly govern glycoform structure and diversity.

    更新日期:2017-08-07
  • The β-reducing end in α(2–8)-polysialic acid constitutes a unique structural motif
    Glycobiology (IF 3.112) Pub Date : 2017-04-13
    Hugo F Azurmendi, Marcos D Battistel, Jasmin Zarb, Flora Lichaa, Alejandro Negrete Virgen, Joseph Shiloach, Darón I Freedberg

    Over the years, structural characterizations of α(2–8)-polysialic acid (polySia) in solution have produced inconclusive results. Efforts for obtaining detailed information in this important antigen have focused primarily on the α-linked residues and not on the distinctive characteristics of the terminal ones. The thermodynamically preferred anomeric configuration for the reducing end of sialic acids is β, which has the [I]CO2– group equatorial and the OH ([I]OH2) axial, while for all other residues the CO2– group is axial. We show that this purportedly minor difference has distinct consequences for the structure of α(2–8)-polySia near the reducing end, as the β configuration places the [I]OH2 in a favorable position for the formation of a hydrogen bond with the carboxylate group of the following residue ([II]CO2–). Molecular dynamics (MD) simulations predicted the hydrogen bond, which we subsequently directly detected by NMR. The combination of MD and residual dipolar couplings shows that the net result for the structure of Sia2-βOH is a stable conformation with well-defined hydration and charge patterns, and consistent with experimental NOE-based hydroxyl and aliphatic inter-proton distances. Moreover, we provide evidence that this distinct conformation is preserved on Sia oligosaccharides, thus constituting a motif that determines the structure and dynamics of α(2–8)-polySia for at least the first two residues of the polymer. We suggest the hypothesis that this structural motif sheds light on a longtime puzzling observation for the requirement of 10 residues of α(2–8)-polySia in order to bind effectively to specific antibodies, about four units more than for analogous cases.

    更新日期:2017-08-07
  • Pectobacterium atrosepticum exopolysaccharides: identification, molecular structure, formation under stress and in planta conditions
    Glycobiology (IF 3.112) Pub Date : 2017-08-05
    Vladimir Gorshkov, Bakhtiyar Islamov, Polina Mikshina, Olga Petrova, Gennady Burygin, Elena Sigida, Alexander Shashkov, Amina Daminova, Marina Ageeva, Bulat Idiyatullin, Vadim Salnikov, Yuri 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:2017-08-05
  • Genomic and biochemical characterization of sialic acid acetyl esterase (siae) in zebrafish
    Glycobiology (IF 3.112) Pub Date : 2017-08-05
    Viola Ravasio, Eufemia Damiati, Daniela Zizioli, Flavia Orizio, Edoardo Giacopuzzi, Marta Manzoni, Roberto Bresciani, Giuseppe Borsani, Eugenio Monti

    Sialic acid acetyl esterase (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 behaviour 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:2017-08-05
  • A conserved DGGK motif is essential for the function of the PglB oligosaccharyltransferase from Campylobacter jejuni
    Glycobiology (IF 3.112) Pub Date : 2017-08-05
    Yasmin Barre, Harald Nothaft, Cody Thomas, Xin Liu, Jianjun Li, Kenneth K.S. 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 E. 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 C. 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:2017-08-05
  • Bacterial phosphoglycosyl transferases: initiators of glycan biosynthesis at the membrane interface
    Glycobiology (IF 3.112) Pub Date : 2017-07-15
    Vinita Lukose, Marthe TC Walvoort, Barbara Imperiali

    Phosphoglycosyl transferases (PGTs) initiate the biosynthesis of both essential and virulence-associated bacterial glycoconjugates including lipopolysaccharide, peptidoglycan and glycoproteins. PGTs catalyze the transfer of a phosphosugar moiety from a nucleoside diphosphate sugar to a polyprenol phosphate, to form a membrane-bound polyprenol diphosphosugar product. PGTs are integral membrane proteins, which include between 1 and 11 predicted transmembrane domains. Despite this variation, common motifs have been identified in PGT families through bioinformatics and mutagenesis studies. Bacterial PGTs represent important antibacterial and virulence targets due to their significant role in initiating the biosynthesis of key bacterial glycoconjugates. Considerable effort has gone into mechanistic and inhibition studies for this class of enzymes, both of which depend on reliable, high-throughput assays for easy quantification of activity. This review summarizes recent advances made in the characterization of this challenging but important class of enzymes.

    更新日期:2017-07-15
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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