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  • Role of proteoglycans and glycosaminoglycans in Duchenne muscular dystrophy
    Glycobiology (IF 3.112) Pub Date : 2018-06-19
    Laurino Carmen, Vadala Maria, Julio Cesar Morales-Medina, Annamaria Vallelunga, Beniamino Palmieri, Tommaso Iannitti

    Duchenne Muscular dystrophy (DMD) is an inherited fatal X-linked myogenic disorder with a prevalence of 1 in 3500 male live births. It affects voluntary muscles, and heart and breathing muscles. DMD is characterised by continuous degeneration and regeneration cycles resulting in extensive fibrosis and a progressive reduction in muscle mass. Since the identification of a reduction in dystrophin protein as the cause of this disorder, numerous innovative and experimental therapies, focusing on increasing the levels of dystrophin, have been proposed, but the clinical improvement has been unsatisfactory. Dystrophin forms the dystrophin-associated glycoprotein complex and its proteins have been studied as a promising novel therapeutic target to treat DMD. Among these proteins, cell surface glycosaminoglycans (GAGs) are found almost ubiquitously on the surface and in the extracellular matrix of mammalian cells. These macromolecules interact with numerous ligands, including extracellular matrix constituents, adhesion molecules and growth factors that play a crucial role in muscle development and maintenance. In this article, we have reviewed in vitro, in vivo and clinical studies focused on the functional role of GAGs in the pathophysiology of DMD with the final aim of summarising the state of the art of GAG dysregulation within the extracellular matrix in DMD and discussing future therapeutic perspectives.

    更新日期:2018-06-19
  • Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways
    Glycobiology (IF 3.112) Pub Date : 2018-06-19
    Anabel Gonzalez-Gil, Ryan N Porell, Steve M Fernandes, Yadong Wei, Huifeng Yu, Daniela J Carroll, Ryan McBride, James C Paulson, Michael Tiemeyer, Kazuhiro Aoki, Bruce S Bochner, Ronald L Schnaar

    Human siglecs are a family of 14 sialic acid binding proteins, most expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells – primarily eosinophils and mast cells – where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size exclusion chromatography and composite agarose-acrylamide gel electrophoresis, blotted, and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated keratan sulfate proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

    更新日期:2018-06-19
  • Tn and STn are members of a family of carbohydrate tumor antigens that possess carbohydrate–carbohydrate interactions
    Glycobiology (IF 3.112) Pub Date : 2018-04-03
    Marit Sletmoen, Thomas A Gerken, Bjørn T Stokke, Joy Burchell, C Fred Brewer

    The mucin-type O-glycome in cancer aberrantly expresses the truncated glycans Tn (GalNAcα1-Ser/Thr) and STn (Neu5Acα2,6GalNAcα1-Ser/Thr). However, the role of Tn and STn in cancer and other diseases is not well understood. Our recent discovery of the self-binding properties (carbohydrate–carbohydrate interactions, CCIs) of Tn (Tn–Tn) and STn (STn–STn) provides a model for their possible roles in cellular transformation. We also review evidence that Tn and STn are members of a larger family of glycan tumor antigens that possess CCIs, which may participate in oncogenesis.

    更新日期:2018-06-15
  • Global aspects of viral glycosylation
    Glycobiology (IF 3.112) Pub Date : 2018-03-21
    Ieva Bagdonaite, Hans H Wandall

    Enveloped viruses encompass some of the most common human pathogens causing infections of different severity, ranging from no or very few symptoms to lethal disease as seen with the viral hemorrhagic fevers. All enveloped viruses possess an envelope membrane derived from the host cell, modified with often heavily glycosylated virally encoded glycoproteins important for infectivity, viral particle formation and immune evasion. While N-linked glycosylation of viral envelope proteins is well characterized with respect to location, structure and site occupancy, information on mucin-type O-glycosylation of these proteins is less comprehensive. Studies on viral glycosylation are often limited to analysis of recombinant proteins that in most cases are produced in cell lines with a glycosylation capacity different from the capacity of the host cells. The glycosylation pattern of the produced recombinant glycoproteins might therefore be different from the pattern on native viral proteins. In this review, we provide a historical perspective on analysis of viral glycosylation, and summarize known roles of glycans in the biology of enveloped human viruses. In addition, we describe how to overcome the analytical limitations by using a global approach based on mass spectrometry to identify viral O-glycosylation in virus-infected cell lysates using the complex enveloped virus herpes simplex virus type 1 as a model. We underscore that glycans often pay important contributions to overall protein structure, function and immune recognition, and that glycans represent a crucial determinant for vaccine design. High throughput analysis of glycosylation on relevant glycoprotein formulations, as well as data compilation and sharing is therefore important to identify consensus glycosylation patterns for translational applications.

    更新日期:2018-06-15
  • TCA cycle-powered synthesis of fucosylated oligosaccharides
    Glycobiology (IF 3.112) Pub Date : 2018-05-24
    Ningzi Guan, Hyun-Dong Shin, Lingfeng Long, Parastoo Azadi, Rachel Chen

    Microbial catalysis has recently emerged as one of the most promising approaches in oligosaccharide synthesis. However, despite significant progress, microbial synthesis still requires much improvement in efficiency and in reduction of process complexity. Additionally, given the stunning diversity and many varied applications of glycans, broadening the range of glycans accessible via microbial synthesis is of paramount importance. Major challenges in microbial synthesis include catabolite repression and high cellular energy requirement. Here we demonstrated a new approach to overcome these challenges by directly tapping into the cellular “power house,” the TCA cycle, to provide the cellular energy for synthesis. This approach not only circumvents catabolite repression but also eliminates acidic glycolysis by-products. As such, the whole-cell biocatalysis can be carried out without sophisticated fed-batch feeding and pH control in the synthesis stage. The system could achieve several grams per liter (3–4 g/L) within a 24-h period in shaker flask cultivation for two targets, fucosyllactose and fucosyllactulose, demonstrating efficiency of the biocatalyst developed and its applicability to both natural and non-natural targets. To the best of our knowledge, this is the first use of TCA cycle intermediates as the energy source for oligosaccharide synthesis and the first description of successful synthesis of fucosyllactulose with titers in several grams per liter.

    更新日期:2018-06-15
  • The parasitic nematode Oesophagostomum dentatum synthesizes unusual glycosaminoglycan-like O-glycans
    Glycobiology (IF 3.112) Pub Date : 2018-05-11
    Jorick Vanbeselaere, Shi Yan, Anja Joachim, Katharina Paschinger, Iain BH Wilson

    O-glycosylation is probably one of the most varied sets of post-translational modifications across all organisms, but amongst the most refractory to analyze. In animals, O-xylosylation of serine residues represents the first stage in the synthesis of glycosaminoglycans, whose repeat regions are generally analyzed as fragments resulting from enzymatic or chemical degradation, whereas their core regions can be isolated by β-elimination or endo-β-xylosidase digestion. In the present study, we show that hydrazinolysis can be employed for release of glycosaminoglycan-type oligosaccharides from nematodes prior to fluorescent labeling with 2-aminopyridine. While various [HexNAcHexA]nGal2Xyl oligosaccharides were isolated from the model organism Caenorhabditis elegans, more unusual glycosaminoglycan-type glycans were found to be present in the porcine parasite Oesophagostomum dentatum. In this case, as judged by MS/MS before and after hydrofluoric acid or β-galactosidase digestion, core sequences with extra galactose and phosphorylcholine residues were detected as [(±PC)HexNAcHexA]n(±PC)Galβ3-(±Galβ4)Galβ4Xyl. Thus, hydrazinolysis and fluorescent labeling can be combined to analyze unique forms of O-xylosylation, including new examples of zwitterionic glycan modifications.

    更新日期:2018-06-15
  • Generation and characterization of a site-specific antibody for SIRT1 O-GlcNAcylated at serine 549
    Glycobiology (IF 3.112) Pub Date : 2018-04-24
    Hui Shan, Jiahui Sun, Minghui Shi, Xue Liu, Zhu Shi, Wengong Yu, Yuchao Gu

    O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) is a dynamic post-translational modification that modifies thousands of proteins. However, the roles and mechanisms of O-GlcNAcylation have been clarified in only a few proteins, and one of the main reasons for this is the lack of site-specific anti-O-GlcNAc antibodies. Recently, we found that SIRT1, which is an NAD+-dependent deacetylase, is O-GlcNAcylated at the serine 549 site (S549) and plays a cytoprotective role under stress. However, the mechanism underlying the roles of SIRT1 O-GlcNAcylation remains unclear. Here, we describe a site-specific antibody for SIRT1 O-GlcNAcylated at S549, named SIRT1-549-O. This antibody can be used for immunoprecipitation and western blotting assays, and it can be used to recognize the endogenous levels of both human and mouse SIRT1 O-GlcNAcylation. Therefore, this antibody not only provides an effective method to further understand the roles of SIRT1 O-GlcNAcylation but also makes it possible to discover the genetic and pharmacological factors that could regulate SIRT1 activity by modulating its O-GlcNAcylation.

    更新日期:2018-06-15
  • Quantifying the binding stoichiometry and affinity of histo-blood group antigen oligosaccharides for human noroviruses
    Glycobiology (IF 3.112) Pub Date : 2018-03-19
    Ling Han, Ruixiang Zheng, Michele R Richards, Ming Tan, Elena N Kitova, Xi Jiang, John S Klassen

    Human noroviruses (HuNoVs) are a major cause of acute gastroenteritis. Many HuNoVs recognize histo-blood group antigens (HBGAs) as cellular receptors or attachment factors for infection. It was recently proposed that HuNoV recognition of HBGAs involves a cooperative, multistep binding mechanism that exploits both known and previously unknown glycan binding sites. In this study, binding measurements, implemented using electrospray ionization mass spectrometry (ESI-MS) were performed on homodimers of the protruding domain (P dimers) of the capsid protein of three HuNoV strains [Saga (GII.4), Vietnam 026 (GII.10) and VA387 (GII.4)] with the ethyl glycoside of the B trisaccharide (α-d-Gal-(1→3)-[α-l-Fuc-(1→2)]-β-d-Gal-OC2H5) and free B type 1 tetrasaccharide (α-d-Gal-(1→3)-[α-l-Fuc-(1→2)]-β-d-Gal-(1→3)-d-GlcNAc) in an effort to confirm the existence of new HBGA binding sites. After correcting the mass spectra for nonspecific interactions that form in ESI droplets as they evaporate to dryness, all three P dimers were found to bind a maximum of two B trisaccharides at the highest concentrations investigated. The apparent affinities measured for stepwise binding of B trisaccharide suggest positive cooperativity. Similar results were obtained for B type 1 tetrasaccharide binding to Saga P dimer. Based on these results, it is proposed that HuNoV P dimers possess only two HBGA binding sites. It is also shown that nonspecific binding corrections applied to mass spectra acquired using energetic ion source conditions that promote in-source dissociation can lead to apparent HuNoV–HBGA oligosaccharide binding stoichiometries and affinities that are artificially high. Finally, evidence that high concentrations of oligosaccharide can induce conformational changes in HuNoV P dimers is presented.

    更新日期:2018-06-15
  • Fine-tuning the structure of glycosaminoglycans in living cells using xylosides
    Glycobiology (IF 3.112) Pub Date : 2018-05-24
    Andrea Persson, Ulf Ellervik, Katrin Mani

    Xylosides can induce the formation and secretion of xyloside-primed glycosaminoglycans when administered to living cells; however, their impact on the detailed glycosaminoglycan structure remains unknown. Here, we have systematically investigated how the xyloside concentration and the type of xyloside, as well as the cell type, influenced the structure of xyloside-primed glycosaminoglycans in terms of the heparan sulfate and chondroitin/dermatan sulfate proportion and disaccharide composition. We found that although greatest influence was exerted by the cell type, both the xyloside concentration and type of xyloside impacted the proportion of heparan sulfate and the complexity of chondroitin/dermatan sulfate. The disaccharide composition of the chondroitin/dermatan sulfate was influenced by the xyloside concentration and type of xyloside to a higher extent than that of the heparan sulfate; the proportion of 4S-sulfated disaccharides in the chondroitin/dermatan sulfate decreased and the proportions of 6S-sulfated and/or nonsulfated disaccharides increased both with increasing concentrations of xyloside and with increasing xyloside hydrophobicity, whereas the proportion of nonsulfated disaccharides was primarily altered in the heparan sulfate with increasing concentrations of xyloside. Our results indicate that it is feasible to not only produce large amounts of glycosaminoglycans in living cells but also to fine-tune their structures by using xylosides of different types and at different concentrations.

    更新日期:2018-06-15
  • Novel thioglycoside analogs of α-galactosylceramide stimulate cytotoxicity and preferential Th1 cytokine production by human invariant natural killer T cells
    Glycobiology (IF 3.112) Pub Date : 2018-05-06
    Ashanty M Melo, Lei Zhang, Éilis F Dockry, Andreea Petrasca, Yasmeen G Ghnewa, Eamon P Breen, Maria E Morrissey, Ciara O’Reilly, Robyn Bruen, Andrew O’Meara, Joanne Lysaght, Xiangming Zhu, Derek G Doherty

    Invariant natural killer T (iNKT) cells recognize glycolipid antigens bound to CD1d molecules on antigen-presenting cells. Therapeutic activation of iNKT cells with the xenogeneic glycolipid α-galactosylceramide (α-GalCer) can prevent and reverse tumor growth in murine models, but clinical trials using α-GalCer-stimulated human iNKT cells have shown limited efficacy. We synthesized a series of thioglycoside analogs of α-GalCer with different substituents to the galactose residue and found that two of these compounds, XZ7 and XZ11, bound to CD1d-transfected HeLa cells and activated lines of expanded human iNKT cells. Both compounds stimulated cytolytic degranulation by iNKT cells and while XZ7 preferentially stimulated the production of the antitumor cytokine interferon-γ (IFN-γ), XZ11 preferentially stimulated interleukin-4 (IL-4) production. This biased T helper type 1 effector profile of XZ7 was also evident when iNKT were stimulated with dendritic cells presenting this glycolipid. Separate analysis of the responses of CD4+, CD8α+ and CD4−CD8− iNKT cells indicated that XZ7 preferentially activated CD8α+ iNKT cells, and to a lesser degree, CD4−CD8− iNKT cells. The partial agonist effect of glycolipid XZ7, inducing cytotoxicity and IFN-γ production but not IL-4 production, indicates that specific protumour activities of iNKT cells can be abolished, while preserving their antitumor activities, by introducing structural modifications to α-GalCer. Since XZ7 was much less potent than α-GalCer as an iNKT cell agonist, it is unlikely to be superior to α-GalCer as a therapeutic agent for cancer, but may serve as a parent compound for developing more potent structural analogs.

    更新日期:2018-06-15
  • Distinct amino acid residues confer one of three UDP-sugar substrate specificities in Acinetobacter baumannii PglC phosphoglycosyltransferases
    Glycobiology (IF 3.112) Pub Date : 2018-04-13
    Christian M Harding, M Florencia Haurat, Evgeny Vinogradov, Mario F Feldman

    Acinetobacter baumannii is an opportunistic human pathogen with the highest reported rates of multidrug resistance among Gram-negative pathogens. The capsular polysaccharide of A. baumannii is considered one of its most significant virulence factors providing resistance against complemented-mediated killing. Capsule synthesis in A. baumannii is usually initiated by the phosphoglycosyltransferase PglC. PglC transfers a phosphosugar from a nucleotide diphosphate-sugar to a polyprenol phosphate generating a polyprenol diphosphate-linked monosaccharide. Traditionally, PglC was thought to have stringent specificity towards UDP-N-N′-diacetylbacillosamine (UDP-diNAcBac). In this work we demonstrate that A. baumannii PglC has the ability to utilize three different UDP-sugar substrates: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc) or UDP-diNAcBac. Using phylogenetic analyses, we first demonstrate that A. baumannii PglC orthologs separate into three distinct clades. Moreover, all members within a clade are predicted to have the same preference for one of the three possible sugar substrates. To experimentally determine the substrate specificity of each clade, we utilized in vivo complementation models and NMR analysis. We demonstrate that UDP-diNAcBac is accommodated by all PglC orthologs, but some orthologs evolved to utilize UDP-GlcNAc or UDP-GalNAc in a clade-dependent manner. Furthermore, we show that a single point mutation can modify the sugar specificity of a PglC ortholog specific for UDP-diNAcBac and that introduction of a non-native PglC ortholog into A. baumannii can generate a new capsule serotype. Collectively, these studies begin to explain why A. baumannii strains have such highly diverse glycan repertoires.

    更新日期:2018-06-15
  • Genetic and enzymatic characterization of 3-O-sulfotransferase SNPs associated with Plasmodium falciparum parasitaemia
    Glycobiology (IF 3.112) Pub Date : 2018-04-28
    Ngoc Thy Nguyen, Romain R Vivès, Magali Torres, Vincent Delauzun, Els Saesen, Véronique Roig-Zamboni, Hugues Lortat-Jacob, Pascal Rihet, Yves Bourne

    The HS3ST3A1/B1 genes encode two homologous 3-O-sulfotransferases involved in the late modification step during heparan sulfate (HS) biosynthesis. In addition to the single nucleotide polymorphisms (SNPs) rs28470223 (C > T) in the promoter region of both HS3ST3A1 and rs62636623 (Gly/Arg) in the stem region of HS3ST3B1, three missense mutations (rs62056073, rs61729712 and rs9906590) located within the catalytic sulfotransferase domain of 3-OST-B1 are linked and associated to Plasmodium falciparum parasitaemia. To ascertain the functional effects of these SNP associations, we investigated the regulatory effect of rs28470223 and characterized the enzymatic activity of the missense SNP rs61729712 (Ser279Asn) localized at proximity of the substrate binding cleft. The SNP rs28470223 results in decreased promoter activity of HS3ST3A1 in K562 cells, suggesting a reduced in vivo transcription activity of the target gene. A comparative kinetic analysis of wt HS3ST3B1 and the Ser269Asn variant (rs61729712) using a HS-derived oligosaccharide substrate reveals a slightly higher catalytic activity for the SNP variant. These genetic and enzymatic studies suggest that genetic variations in enzymes responsible of HS 3-O-sulfation can modulate their promoter and enzymatic activities and may influence P. falciparum parasitaemia.

    更新日期:2018-06-15
  • Glycoengineering design options for IgG1 in CHO cells using precise gene editing
    Glycobiology (IF 3.112) Pub Date : 2018-03-27
    Morten A Schulz, Weihua Tian, Yang Mao, Julie Van Coillie, Lingbo Sun, Joachim S Larsen, Yen-Hsi Chen, Claus Kristensen, Sergey Y Vakhrushev, Henrik Clausen, Zhang Yang

    Precise gene editing technologies are providing new opportunities to stably engineer host cells for recombinant production of therapeutic glycoproteins with different glycan structures. The glycosylation of recombinant therapeutics has long been a focus for both quality and consistency of products and for optimizing and improving pharmacokinetic properties as well as bioactivity. Structures of glycans on therapeutic glycoproteins are important for circulation, biodistribution and bioactivity. In particular, the latter has been demonstrated for therapeutic IgG1 antibodies where the core α1,6Fucose on the conserved N-glycan at Asn297 have remarkable dampening effects on antibody effector functions. We previously explored precise gene engineering and design options for N-glycosylation in CHO cells, and here we focus on engineering options possible for N-glycans on human IgG1. We demonstrate stable precise gene engineering of rather homogenous biantennary N-glycans with and without galactose (G0F, G2F) as well as the α2,6-linked monosialylated (G2FS1) glycoform. We were unable to introduce substantial disialylated glycoforms. Instead we engineered a novel monoantennary homogeneous N-glycan design with complete α2,6-linked sialic acid capping. All N-glycoforms may be engineered with and without core α1,6Fucose. The stably engineered design options enable production of human IgG antibodies with an array of distinct glycoforms for testing and selection of optimal design for different therapeutic applications.

    更新日期:2018-06-15
  • Structural basis of oligosaccharide processing by glycosaminoglycan sulfotransferases
    Glycobiology (IF 3.112) Pub Date : 2018-06-06
    Tarsis F Gesteira, Vivien J Coulson-Thomas

    Heparan sulfate (HS) is a sulfated polysaccharide that plays a key role in morphogenesis, physiology and pathogenesis. The biosynthesis of HS takes place in the Golgi apparatus by a group of enzymes that polymerize, epimerize and sulfate the sugar chain. This biosynthetic process introduces varying degrees of sulfate substitution, which are tightly regulated and directly dictate binding specificity to different cytokines, morphogens and growth factors. Here we report the use of molecular dynamics simulations to investigate the dynamics of substrate recognition of two glycosaminoglycan (GAG) sulfotransferases, N-deacetylase-N-sulfotransferase and 2-O-sulfotransferase to the HS chain during the biosynthetic process. We performed multiple simulations of the binding of the sulfotransferase domains to both the HS oligosaccharide substrate and sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPs). Analysis of extended simulations provide detailed and useful insights into the atomic interactions that are at work during oligosaccharide processing. The Fast Information Matching method was used to detect the enzyme global dynamics and to predict the pairwise contact of residues responsible for GAG-enzyme binding and unbinding. The correlation between HS displacement and the location of the modified GAG chain were calculated, indicating a possible route for HS and heparin during sulfotransferase processing. Our data also show sulfotransferases contain a conserved interspaced positively charged amino acid residues that form a patch which controls the protein-GAG binding equilibrium. Together, our findings provide further understanding on the fine-tuned complex mechanism of GAG biosynthesis. Our findings can also be extrapolated to other systems for calculating rates of protein-GAG binding.

    更新日期:2018-06-06
  • Addicted to sugar: roles of glycans in the order Mononegavirales
    Glycobiology (IF 3.112) Pub Date : 2018-06-06
    Victoria Ortega, Jacquelyn A Stone, Erik M Contreras, Ronald M Iorio, Hector C Aguilar

    Glycosylation is a biologically important protein modification process by which a carbohydrate chain is enzymatically added to a protein at a specific amino acid residue. This process plays roles in many cellular functions, including intracellular trafficking, cell-cell signaling, protein folding, and receptor binding. While glycosylation is a common host cell process, it is utilized by many pathogens as well. Protein glycosylation is widely employed by viruses for both host invasion and evasion of host immune responses. Thus better understanding of viral glycosylation functions has potential applications for improved antiviral therapeutic and vaccine development. Here, we summarize our current knowledge on the broad biological functions of glycans for the Mononegavirales, an order of enveloped negative-sense single-stranded RNA viruses of high medical importance that includes Ebola, Rabies, Measles, and Nipah viruses. We discuss glycobiological findings by genera in alphabetical order within each of eight Mononegavirales families, namely the bornaviruses, filoviruses, mymonaviruses, nyamiviruses, paramyxoviruses, pneumoviruses, rhabdoviruses, and sunviruses.

    更新日期:2018-06-06
  • Conserved residues Arg188 and Asp302 are critical for active site organization and catalysis in human ABO(H) blood group A and B glycosyltransferases
    Glycobiology (IF 3.112) Pub Date : 2018-06-05
    Susannah M L Gagnon, Max S G Legg, Robert Polakowski, James A Letts, Mattias Persson, Shuangjun Lin, Ruixiang Blake Zheng, Brian Rempel, Brock Schuman, Omid Haji-Ghassemi, Svetlana N Borisova, Monica M Palcic, Stephen V Evans

    Homologous glycosyltransferases GTA and GTB perform the final step in human ABO(H) blood group A and B antigen synthesis by transferring the sugar moiety from donor UDP-GalNAc/UDP-Gal to the terminal H antigen disaccharide acceptor. Like other GT-A fold family 6 glycosyltransferases, GTA and GTB undergo major conformational changes in two mobile regions, the C-terminal tail and internal loop, to achieve the closed, catalytic state. These changes are known to establish a salt bridge network among conserved active site residues Arg188, Asp211, and Asp302, which move to accommodate a series of discrete donor conformations while promoting loop ordering and formation of the closed enzyme state. However, the individual significance of these residues in linking these processes remains unclear. Here we report the kinetics and high-resolution structures of GTA/GTB mutants of residues 188 and 302. The structural data support a conserved salt bridge network critical to mobile polypeptide loop organization and stabilization of the catalytically competent donor conformation. Consistent with the X-ray crystal structures, the kinetic data suggest that disruption of this salt bridge network has a destabilizing effect on the transition state, emphasizing the importance of Arg188 and Asp302 in the glycosyltransfer reaction mechanism. The salt bridge network observed in GTA/GTB structures during substrate binding appears to be conserved not only among other CAZy family 6 glycosyltransferases, but also within both retaining and inverting GT-A fold glycosyltransferases. Our findings augment recently published crystal structures, which have identified a correlation between donor substrate conformational changes and mobile loop ordering.

    更新日期:2018-06-05
  • Selectins in cancer immunity
    Glycobiology (IF 3.112) Pub Date : 2017-12-20
    Lubor Borsig

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

    更新日期:2018-06-03
  • Cytosolic galectin-3 and -8 regulate antibacterial autophagy through differential recognition of host glycans on damaged phagosomes
    Glycobiology (IF 3.112) Pub Date : 2018-02-23
    I-Chun Weng, Hung-Lin Chen, Tzu-Han Lo, Wei-Han Lin, Huan-Yuan Chen, Daniel K Hsu, Fu-Tong Liu

    While glycans are generally displayed on the cell surface or confined within the lumen of organelles, they can become exposed to the cytosolic milieu upon disruption of organelle membrane by various stresses or pathogens. Galectins are a family of β-galactoside-binding animal lectins synthesized and predominantly localized in the cytosol. Recent research indicates that some galectins may act as “danger signal sensors” by detecting unusual exposure of glycans to the cytosol. Galectin-8 was shown to promote antibacterial autophagy by recognizing host glycans on ruptured vacuolar membranes and interacting with the autophagy adaptor protein NDP52. Galectin-3 also accumulates at damaged phagosomes containing bacteria; however, its functional consequence remains obscure. By studying mouse macrophages infected with Listeria monocytogenes (LM), we showed that endogenous galectin-3 protects intracellular LM by suppressing the autophagic response through a host N-glycan-dependent mechanism. Knock out of the galectin-3 gene resulted in enhanced LC3 recruitment to LM and decreased bacterial replication, a phenotype recapitulated when Galectin-8-deficient macrophages were depleted of N-glycans. Moreover, we explored the concept that alterations in cell surface glycosylation by extracellular factors can be deciphered by cytosolic galectins during the process of phagocytosis/endocytosis, followed by rupture of phagosomal/endosomal membrane. Notably, treatment of cells with sialidase, which removes sialic acid from glycans, resulted in increased galectin-3 accumulation and decreased galectin-8 recruitment at damaged phagosomes, and led to a stronger anti-autophagic response. Our findings demonstrate that cytosolic galectins may sense changes in glycosylation at the cell surface and modulate cellular response through differential recognition of glycans on ruptured phagosomal membranes.

    更新日期:2018-06-03
  • Airway glycomic and allergic inflammatory consequences resulting from keratan sulfate galactose 6-O-sulfotransferase (CHST1) deficiency
    Glycobiology (IF 3.112) Pub Date : 2018-04-06
    Tadahiro Kumagai, Takumi Kiwamoto, Mary E Brummet, Fan Wu, Kazuhiro Aoki, Zhou Zhu, Bruce S Bochner, Michael Tiemeyer

    Siglec-F is a pro-apoptotic receptor on mouse eosinophils that recognizes 6′-sulfated sialyl Lewis X and 6′-sulfated sialyl N-acetyl-lactosamine as well as multivalent sialyl N-acetyl-lactosamine structures on glycan arrays. We hypothesized that attenuation of the carbohydrate sulfotransferase 1 (CHST1) gene encoding keratan sulfate galactose 6-O-sulfotransferase, an enzyme likely required for 6′-sulfation of some of these putative Siglec-F glycan ligands, would result in decreased Siglec-F lung ligand levels and enhanced allergic eosinophilic airway inflammation. Tissue analysis detected CHST1 expression predominantly not only in parenchymal cells but not in airway epithelium, the latter being a location where Siglec-F ligands are located. Western blotting of lung extracts with Siglec-F–Fc fusion proteins detected ≈500 kDa and ≈200 kDa candidate Siglec-F ligands that were not appreciably altered in CHST1−/− lungs compared with normal mouse lungs. Characterization of the O-linked glycans of lung tissue and bronchoalveolar lavage fluid detected altered sialylation but minimal change in sulfation. Eosinophilic airway inflammation was induced in wild-type (WT) and CHST1−/− mice via sensitization to ovalbumin (OVA) and repeated airway challenge. After OVA sensitization and challenge, Siglec-F ligands on airway cells, and numbers of eosinophils and neutrophils accumulating in the airways, both increased to a similar degree in WT and CHST1−/− mouse lungs, while macrophages and lymphocytes increased significantly more in CHST1−/− mouse airway compared with normal mouse lungs. Therefore, keratan sulfate galactose 6-O-sulfotransferase does not contribute to the synthesis of glycan ligands for Siglec-F in the airways, although its absence results in exaggerated accumulation of airway macrophages and lymphocytes.

    更新日期:2018-06-03
  • Crystal structures of an archaeal chitinase ChiD and its ligand complexes
    Glycobiology (IF 3.112) Pub Date : 2018-03-21
    Yuichi Nishitani, Ayumi Horiuchi, Mehwish Aslam, Tamotsu Kanai, Haruyuki Atomi, Kunio Miki

    Chitinase D (designated as Pc-ChiD) was found in a hyperthermophilic archaeon, Pyrococcus chitonophagus (previously described as Thermococcus chitonophagus), that was isolated from media containing only chitin as carbon source. Pc-ChiD displays chitinase activity and is thermostable at temperatures up to 95°C, suggesting its potential for industrial use. Pc-ChiD has a secretion signal peptide and two chitin-binding domains (ChBDs) in the N-terminal domain. However, the C-terminal domain shares no sequence similarity with previously identified saccharide-degrading enzymes and does not contain the DXDXE motif conserved in the glycoside hydrolase (GH) 18 family chitinases. To elucidate its overall structure and reaction mechanism, we determined the first crystal structures of Pc-ChiD, both in the ligand-free form and in complexes with substrates. Structure analyses revealed that the C-terminal domain of Pc-ChiD, Pc-ChiD(ΔBD), consists of a third putative substrate-binding domain, which cannot be predicted from the amino acid sequence, and a catalytic domain structurally similar to that found in not the GH18 family but the GH23 family. Based on the similarity with GH23 family chitinase, the catalytic residues of Pc-ChiD were predicted and confirmed by mutagenesis analyses. Moreover, the specific C-terminal 100 residues of Pc-ChiD are important to fix the putative substrate-binding domain next to the catalytic domain, contributing to the structure stability as well as the long chitin chain binding. Our findings reveal the structure of a unique archaeal chitinase that is distinct from previously known members of the GH23 family.

    更新日期:2018-06-03
  • Sulfated fucans and a sulfated galactan from sea urchins as potent inhibitors of selectin-dependent hematogenous metastasis
    Glycobiology (IF 3.112) Pub Date : 2018-03-07
    Felipe C O B Teixeira, Eliene Oliveira Kozlowski, Kayene Vitória de A Micheli, Ana Cristina E S Vilela-Silva, Lubor Borsig, Mauro S G Pavão

    Metastasis is responsible for the majority of cancer-associated deaths, though only a very small number of tumor cells are able to efficiently complete all the steps of that process. Tumor cell survival in the bloodstream is one of the limiting aspects of the metastatic cascade. The formation of tumor cell–platelet complexes that promote tumor cell survival is facilitated by the binding of P-selectin on activated platelets to sialyl Lewis-containing oligosaccharides on the surface of tumor cells. Inhibition of this interaction has been shown to attenuate metastasis. Heparin is a potent selectin inhibitor and is capable to block platelet–tumor cell complex formation, thereby attenuating metastasis. Similarly, other sulfated polysaccharides isolated from marine invertebrates attenuate metastasis by a P-selectin-mediated mechanism. In this work, we investigated the selectin-dependent antimetastatic activity of sea urchin sulfated polysaccharides with slight structural differences: a sulfated fucan from Strongylocentrotus franciscanus; a sulfated fucan from Strongylocentrotus droebachiensis; and a sulfated galactan from Echinometra lucunter. The results demonstrate that these fucans and the galactan have different antiselectin activities despite being very similar molecules. Therefore, they may be interesting tools for studies on the structure–function relationship or even for future treatments.

    更新日期:2018-06-03
  • Recent Approaches for Directly Profiling Cell Surface Sialoform
    Glycobiology (IF 3.112) Pub Date : 2018-05-24
    Xiaoqing Zhang, Huan Nie, Joshua Whited, Dan Wang, Yu Li, Xue-Long Sun

    Sialic acids (SAs) are nine-carbon monosaccharides existing at the terminal location of glycan structures on the cell surface and secreted glycoconjugates. The expression levels and linkages of SAs on cells and tissues, collectively known as sialoform, present the hallmark of the cells and tissues of different systems and conditions. Accordingly, detecting or profiling cell surface sialoforms is very critical for understanding the function of cell surface glycans and glycoconjugates and even the molecular mechanisms of their underlying biological processes. Further, it may provide therapeutic and diagnostic applications for different diseases. In the past decades, several SA-specific binding molecules have been developed for detecting and profiling specific sialoforms of cells and tissues; the experimental materials have expanded from frozen tissue to living cells; and the analytical technologies have advanced from histochemistry to fluorescent imaging, flow cytometry and microarrays. This review summarizes the recent bio-affinity approaches for directly detecting and profiling specific SAs or sialylglycans, and their modifications of different cells and tissues.

    更新日期:2018-05-24
  • Through the barricades: Overcoming the barriers to effective antibody-based cancer therapeutics
    Glycobiology (IF 3.112) Pub Date : 2018-05-24
    Martin Dalziel, Stephen A Beers, Mark S Cragg, Max Crispin

    Since the turn of the century, cancer therapy has undergone a transformation in terms of new treatment modalities and renewed optimism in achieving long-lived tumour control and even cure. This is, in large part, thanks to the widespread incorporation of monoclonal antibodies (mAbs) into standard treatment regimens. These new therapies have, across many settings, significantly contributed to improved clinical responses, patient quality of life and survival. Moreover, the flexibility of the antibody platform has led to the development of a wide range of innovative and combinatorial therapies that continue to augment the clinician's armoury. Despite these successes, there is a growing awareness that in many cases mAb therapy remains suboptimal, primarily due to inherent limitations imposed by the immune system's own homeostatic controls and the immunosuppressive tumour microenvironment. Here, we discuss the principal barriers that act to constrain the tumour-killing activity of antibody-based therapeutics, particularly those involving antibody glycans, using illustrative examples from both pre-clinical and market approved mAbs. We also discuss strategies that have been, or are in development to overcome these obstacles. Finally, we outline how the growing understanding of the biological terrain in which mAbs function is shaping innovation and regulation in cancer therapeutics.

    更新日期:2018-05-24
  • Streptococcal Siglec-like Adhesins Recognize Different Subsets of Human Plasma Glycoproteins: Implications for Infective Endocarditis
    Glycobiology (IF 3.112) Pub Date : 2018-05-23
    Barbara A Bensing, Qiongyu Li, Dayoung Park, Carlito B Lebrilla, Paul M Sullam

    Streptococcus gordonii and Streptococcus sanguinis are typically found among the normal oral microbiota, but can also cause infective endocarditis. These organisms express cell-surface serine-rich repeat adhesins containing “Siglec-like” binding regions (SLBRs) that mediate attachment to α2-3 linked sialic acids on human glycoproteins. Two known receptors for the Siglec-like adhesins are the salivary mucin MG2/MUC7 and platelet GPIbα, and the interaction of streptococci with these targets may contribute to oral colonization and endocarditis, respectively. The SLBRs display a surprising diversity of preferences for defined glycans, ranging from highly selective to broader specificity. In this report, we characterize the glycoproteins in human plasma recognized by four SLBRs that prefer different α2-3 sialoglycan structures. We found that the SLBRs recognize a surprisingly small subset of plasma proteins that are extensively O-glycosylated. The preferred plasma protein ligands for a sialyl-T antigen-selective SLBR are proteoglycan 4 (lubricin) and inter-alpha-trypsin inhibitor heavy chain H4. Conversely, the preferred ligand for a 3’sialyllactosamine-selective SLBR is glycocalicin (the extracellular portion of platelet GPIbα). All four SLBRs recognize C1 inhibitor, but detect distinctly different glycoforms of this key regulator of the complement and kallikrein protease cascades. The four plasma ligands have potential roles in thrombosis and inflammation, and each has been cited as a biomarker for one or more vascular or other diseases. The combined results suggest that the interaction of Siglec-like adhesins with different subsets of plasma glycoproteins could have a significant impact on the propensity of streptococci to establish endocardial infections.

    更新日期:2018-05-23
  • Identification of serum glycoprotein ligands for the immunomodulatory receptor blood dendritic cell antigen 2
    Glycobiology (IF 3.112) Pub Date : 2018-05-23
    Jong-won Kim, James Budzak, Yu Liu, Sabine A F Jégouzo, Kurt Drickamer, Maureen E Taylor

    Blood dendritic cell antigen 2 (BDCA-2) is a C-type lectin found on the surface of plasmacytoid dendritic cells. It functions as a glycan-binding receptor that down-regulates production of type I interferons and thus plays a role in oligosaccharide-mediated immunomodulation. The carbohydrate-recognition domain in BDCA-2 binds selectively to galactose-terminated bi-antennary glycans. Because the plasmacytoid dendritic cells function in a plasma environment rich in glycoproteins, experiments have been undertaken to identify endogenous ligands for BDCA-2. A combination of blotting, affinity chromatography and proteomic analysis reveals that serum glycoprotein ligands for BDCA-2 include IgG, IgA, and IgM. Compared to binding of IgG, which was previously described, IgA and IgM bind with higher affinity. The association constants for the different subclasses of immunoglobulins are below and roughly proportional to the serum concentrations of these glycoprotein ligands. Binding to the other main serum glycoprotein ligand, α2-macroglobulin, is independent of whether this protease inhibitor is activated. Binding to all of these glycoprotein ligands is mediated predominantly by bi-antennary glycans in which each branch bears a terminal galactose residue. The different affinities of the glycoprotein ligands reflect the different numbers of these galactose-terminated glycans and their degree of exposure on the native glycoproteins. The results suggests that normal serum levels of immunoglobulins could down-modulate interferon stimulation of further antibody production.

    更新日期:2018-05-23
  • A unique fucosylated chondroitin sulfate type II with strikingly homogeneous and neatly distributed α-fucose branches
    Glycobiology (IF 3.112) Pub Date : 2018-05-17
    Paulo AG Soares, Kátia A Ribeiro, Ana P Valente, Nina V Capillé, Stephan-Nicollas MCG Oliveira, Ana MF Tovar, Mariana S Pereira, Eduardo Vilanova, Paulo AS Mourão

    Fucosylated chondroitin sulfates (FCSs) and sulfated fucans (SFs) are conspicuous components of the body-wall of sea cucumbers (Holothuroidea). FCSs are composed of a central core of chondroitin sulfate decorated with branches of mono- or mono- and disaccharides of α-fucose (FCSs type I and II, respectively). FCSs type II have heterogeneous and irregularly distributed α-fucose branches; however, the novel FCS type II from Holothuria lentiginosa described herein via solution nuclear magnetic resonance has strikingly homogeneous α-fucose branches neatly distributed along its chondroitin sulfate core. This FCS is built-up of three distinct sequential units composed by the typical chondroitin sulfate disaccharides of FCSs, rich in β-galactosamine-4,6diS, decorated with branches of α-Fucp-2,4diS, α-Fucp-3,4diS or α-Fucp[1→3]α-Fucp-4S[1→ linked to the position 3- of the β-glucuronic acid. Conformational analyses of these repetitive units revealed a fairly rigid structure despite of the high sulfate content of their α-fucose branches. We also determined the structure of the SF from H. lentiginosa as a repetitive tetrasaccharide sequence composed of →3]α-Fucp-2,4diS[1→3]α-Fucp[1→3]α-Fucp-2S[1→3]α-Fucp-2S[1→. Furthermore, we determined that the non-sulfated α-fucose units present in FCSs type II did not interfere with their anticoagulant potencies and affinities to calcium. FCS is an autapomorphic molecular character of the class Holothuroidea and the composition of their α-fucose branches differ in a species-specific manner. Branches containing α-Fucp-2,4diS are the most common within the extant holothurians, being found in 90% of the FCSs characterized thus far.

    更新日期:2018-05-17
  • Quiescin Sulfhydryl Oxidase 1 (QSOX1) Glycosite Mutation Perturbs Secretion but Not Golgi Localization
    Glycobiology (IF 3.112) Pub Date : 2018-05-11
    Ben Horowitz, Gabriel Javitt, Tal Ilani, Yair Gat, David Morgenstern, Frederic A Bard, Deborah Fass

    Quiescin sulfhydryl oxidase 1 (QSOX1) catalyzes the formation of disulfide bonds in protein substrates. Unlike other enzymes with related activities, which are commonly found in the endoplasmic reticulum, QSOX1 is localized to the Golgi apparatus or secreted. QSOX1 is upregulated in quiescent fibroblast cells and secreted into the extracellular environment, where it contributes to extracellular matrix assembly. QSOX1 is also upregulated in adenocarcinomas, though the extent to which it is secreted in this context is currently unknown. To achieve a better understanding of factors that affect QSOX1 localization and function, we aimed to determine how post-translational modifications affect QSOX1 trafficking and activity. We found a highly conserved N-linked glycosylation site to be required for QSOX1 secretion from fibroblasts and other cell types. Notably, QSOX1 lacking a glycan at this site arrives at the Golgi, suggesting that it passes endoplasmic reticulum quality control but is not further transported to the cell surface for secretion. The QSOX1 transmembrane segment is dispensable for Golgi localization and secretion, as fully luminal and transmembrane variants displayed the same trafficking behavior. This study provides a key example of the effect of glycosylation on Golgi exit and contributes to an understanding of late secretory sorting and quality control.

    更新日期:2018-05-11
  • 更新日期:2018-05-04
  • β-Glucan-induced cooperative oligomerization of Dectin-1 C-type lectin-like domain
    Glycobiology (IF 3.112) Pub Date : 2018-04-20
    Hari P Dulal, Yoshiyuki Adachi, Naohito Ohno, Yoshiki Yamaguchi

    Dectin-1 is a C-type lectin-like pattern recognition receptor that recognizes β(1-3)-glucans present on non-self pathogens. It is of great importance in innate immunity to understand the mechanism whereby Dectin-1 senses β(1-3)-glucans and induces intracellular signaling. In this study, we characterize the ligand binding and ligand-induced oligomerization of murine Dectin-1 using its C-type lectin-like domain (CTLD). Interaction of CTLD with laminarin, a β-glucan ligand, induced a tetramer of CTLD, as evidenced by size exclusion chromatography and multi-angle light scattering. Component analysis suggested a stoichiometry of four CTLD molecules bound to four laminarin molecules. Dimers and trimers of CTLD were not detected suggesting cooperative oligomerization. In order to map the amino acid residues of CTLD involved in β-glucan binding and domain oligomerization, we performed site-directed mutagenesis on surface-exposed and most conserved amino acid residues. Among the mutants examined, W221 A, H223A and Y228A abolished oligomer formation. Since these residues are spatially arranged to form a hydrophobic groove, it is likely that W221, H223, and Y228 are directly involved in β-glucan binding. Interestingly, mutation of the residues on the other side of the hydrophobic groove, including Y141, R145 and E243, also exhibited reduced oligomer formation, suggesting involvement in protein-protein interactions guided by laminarin. Ligand titration using intrinsic tryptophan fluorescence revealed that wild-type CTLD binds laminarin cooperatively with a Hill coefficient of ~3, while the oligomer-reducing mutations, inside and outside the putative binding site abolish or decrease cooperativity. We suggest that the ligand-induced cooperative oligomer formation of Dectin-1 is physiologically relevant in sensing exogenous β-glucan and triggering intracellular signaling.

    更新日期:2018-04-20
  • Unraveling synthesis of the cryptococcal cell wall and capsule
    Glycobiology (IF 3.112) Pub Date : 2018-04-10
    Zhuo A Wang, Lucy X Li, Tamara L Doering

    Fungal pathogens cause devastating infections in millions of individuals each year, representing a huge but underappreciated burden on human health. One of these, the opportunistic fungus Cryptococcus neoformans, kills hundreds of thousands of patients annually, disproportionately affecting people in resource-limited areas. This yeast is distinguished from other pathogenic fungi by a polysaccharide capsule that is displayed on the cell surface. The capsule consists of two complex polysaccharide polymers: a mannan substituted with xylose and glucuronic acid, and a galactan with galactomannan side chains that bear variable amounts of glucuronic acid and xylose. The cell wall, with which the capsule is associated, is a matrix of alpha and beta glucans, chitin, chitosan, and mannoproteins. In this review, we focus on synthesis of the wall and capsule, both of which are critical for the ability of this microbe to cause disease and are distinct from structures found in either model yeasts or the mammals afflicted by this infection. Significant research effort over the last few decades has been applied to defining the synthetic machinery of these two structures, including nucleotide sugar metabolism and transport, glycosyltransferase activities, polysaccharide export, and assembly and association of structural elements. Discoveries in this area have elucidated fundamental biology and may lead to novel targets for antifungal therapy. In this review, we summarize the progress made in this challenging and fascinating area, and outline future research questions.

    更新日期:2018-04-10
  • Demystifying O-GlcNAcylation: hints from peptide substrates
    Glycobiology (IF 3.112) Pub Date : 2018-03-22
    Jie Shi, Rob Ruijtenbeek, Roland J Pieters

    O-GlcNAcylation, analogous to phosphorylation, is an essential post-translational modification of proteins at Ser/Thr residues with a single β-N-acetylglucosamine moiety. This dynamic protein modification regulates many fundamental cellular processes and its deregulation has been linked to chronic diseases such as cancer, diabetes and neurodegenerative disorders. Reversible attachment and removal of O-GlcNAc is governed only by O-GlcNAc transferase and O-GlcNAcase, respectively. Peptide substrates, derived from natural O-GlcNAcylation targets, function in the catalytic cores of these two enzymes by maintaining interactions between enzyme and substrate, which makes them ideal models for the study of O-GlcNAcylation and deglycosylation. These peptides provide valuable tools for a deeper understanding of O-GlcNAc processing enzymes. By taking advantage of peptide chemistry, recent progress in the study of activity and regulatory mechanisms of these two enzymes has advanced our understanding of their fundamental specificities as well as their potential as therapeutic targets. Hence, this review summarizes the recent achievements on this modification studied at the peptide level, focusing on enzyme activity, enzyme specificity, direct function, site-specific antibodies and peptide substrate-inspired inhibitors.

    更新日期:2018-04-09
  • Subcellular Distribution of Endogenous Malectin under Rest and Stress Conditions Is Regulated by Ribophorin I
    Glycobiology (IF 3.112) Pub Date : 2018-04-02
    Qin-Peng Yang, Ming-Fen Fu, Hao Gao, Kazuo Yamamoto, Dan Hu, Sheng-Ying Qin

    Malectin is a newly discovered endoplasmic reticulum (ER)-resident lectin, which specifically recognizes Glc2Man9GlcNAc2 on newly synthesized glycoproteins. We have previously demonstrated that malectin forms a complex with ribophorin I for selective retention of misfolded glycoproteins inside the cell. Here, we showed that ribophorin I also functions to regulate the subcellular localization of malectin under various conditions. Even though malectin does not contain an ER-retention signal motif, we found that endogenous malectin mainly localizes in the ER, which is disrupted upon suppression of ribophorin I, leading to its movement from ER to Golgi. In contrast, under ER-stress conditions, malectin mainly localizes in the Golgi, which is restored to ER localization by over-expression of ribophorin I. These results indicate that the subcellular localization of malectin is accurately regulated by the expression level of ribophorin I, which will provide further insights into the understanding of the function of malectin.

    更新日期:2018-04-02
  • Glycosyltransferase genes that cause monogenic congenital disorders of glycosylation are distinct from glycosyltransferase genes associated with complex diseases
    Glycobiology (IF 3.112) Pub Date : 2018-03-22
    Hiren J Joshi, Lars Hansen, Yoshiki Narimatsu, Hudson H Freeze, Bernard Henrissat, Eric Bennett, Hans H Wandall, Henrik Clausen, Katrine T Schjoldager

    Glycosylation of proteins, lipids and proteoglycans in human cells involves at least 167 identified glycosyltransferases (GTfs), and these orchestrate the biosynthesis of diverse types of glycoconjugates and glycan structures. Mutations in this part of the genome—the GTf-genome—cause more than 58 rare, monogenic congenital disorders of glycosylation (CDGs). They are also statistically associated with a large number of complex phenotypes, diseases or predispositions to complex diseases based on Genome-Wide Association Studies (GWAS). CDGs are extremely rare and often with severe medical consequences. In contrast, GWAS are likely to identify more common genetic variations and generally involve less severe and distinct traits. We recently confirmed that structural defects in GTf genes are extremely rare, which seemed at odds with the large number of GWAS pointing to GTf-genes. To resolve this issue, we surveyed the GTf-genome for reported CDGs and GWAS candidates; we found little overlap between the two groups of genes. Moreover, GTf-genes implicated by CDG or GWAS appear to constitute different classes with respect to their: (i) predicted roles in glycosylation pathways; (ii) potential for partial redundancy by closely homologous genes; and (iii) transcriptional regulation as evaluated by RNAseq data. Our analysis suggest that more complex traits are caused by dysregulation rather than structural deficiency of GTfs, which suggests that some glycosylation reactions may be predicted to be under tight regulation for fine-tuning of important biological functions.

    更新日期:2018-03-22
  • AANL (Agrocybe aegerita lectin 2) is a new facile tool to probe for O-GlcNAcylation
    Glycobiology (IF 3.112) Pub Date : 2018-03-19
    Wei Liu, Guanghui Han, Yalin Yin, Shuai Jiang, Guojun Yu, Qing Yang, Wenhui Yu, Xiangdong Ye, Yanting Su, Yajun Yang, Gerald W Hart, Hui Sun

    O-linked N-acetylglucosamine (O-GlcNAcylation) is an important post-translational modification on serine or threonine of proteins, mainly observed in nucleus or cytoplasm. O-GlcNAcylation regulates many cell processes, including transcription, cell cycle, neural development, and nascent polypeptide chains stabilization. However, the facile identification of O-GlcNAc is a major bottleneck in O-GlcNAcylation research. Herein, we report that a lectin, AANL (Agrocybe aegerita GlcNAc specific lectin), also reported as AAL2, can be used as a powerful probe for O-GlcNAc identification. Glycan array analyses and SPR (surface plasmon resonance) assays show that AANL binds to GlcNAc with a dissociation constant (KD) of 94.6 μM, which is consistent with the result tested through ITC assay reported before (Jiang et al. 2012). Confocal imaging shows that AANL co-localizes extensively with NUP62, a heavily O-GlcNAcylated and abundant nuclear pore glycoprotein. Furthermore, O-GlcNAc modified peptides could be effectively enriched in the late flow through peak from simple samples by using affinity columns Sepharose 4B-AANL or POROS-AANL. Therefore, using AANL affinity column, we identified 29 high-confidence O-linked HexNAc modified peptides mapped on 17 proteins involving diverse cellular progresses, including transcription, hydrolysis progress, urea cycle, alcohol metabolism and cell cycle. And most importantly, major proteins and sites were not annotated in the dbOGAP database. These results suggest that the AANL lectin is a new useful tool for enrichment and identification of O-GlcNAcylated proteins and peptides.

    更新日期:2018-03-19
  • Functional crosstalk among oxidative stress and O-GlcNAc signaling pathways: Abstract
    Glycobiology (IF 3.112) Pub Date : 2018-03-14
    Po-Han Chen, Jen-Tsan Chi, Michael Boyce

    In metazoans, thousands of intracellular proteins are modified with O-linked β-N-acetylglucosamine (O-GlcNAc) in response to a wide range of stimuli and stresses. In particular, a complex and evolutionarily conserved interplay between O-GlcNAcylation and oxidative stress has emerged in recent years. Here, we review the current literature on the connections between O-GlcNAc and oxidative stress, with a particular emphasis on major signaling pathways, such as KEAP1/NRF2, FOXO, NFκB, p53 and cell metabolism. Taken together, this work sheds important light on the signaling functions of protein glycosylation and the mechanisms of stress responses alike, and illuminates how the two are integrated in animal cell physiology.

    更新日期:2018-03-14
  • Specific storage of glycoconjugates with terminal α-galactosyl moieties in the exocrine pancreas of Fabry disease patients with blood group B
    Glycobiology (IF 3.112) Pub Date : 2018-03-14
    Jitka Rybová, Ladislav Kuchař, Helena Hůlková, Befekadu Asfaw, Robert Dobrovolný, Jakub Sikora, Vladimír Havlíček, Ľudovít Škultéty, Jana Ledvinová

    Blood group B glycosphingolipids (B-GSLs) are substrates of the lysosomal alpha-galactosidase A (AGAL). Similar to its major substrate - globotriaosylceramide (Gb3Cer) - B-GSLs are not degraded and accumulate in the cells of patients affected by an inherited defect of AGAL activity (Fabry disease - FD).

    更新日期:2018-03-14
  • Cancer glycan epitopes: biosynthesis, structure, and function
    Glycobiology (IF 3.112) Pub Date : 2018-03-13
    Oliver M Pearce

    Aberrant glycan epitopes are a classic hallmark of malignant transformation, yet their full clinical potential in cancer diagnostics and therapeutics is yet to be realized. This is partly because our understanding of how these epitopes are regulated remains poorly understood. In this review cancer glycan epitopes for the major glycan classes are summarized with a focus on their biosynthesis, structure, and role in cancer progression. Their application as cancer biomarkers, in particular the more recent work on cancer glycoforms, and the advantages these offer over the glycan or protein alone are discussed. Finally, emerging concepts which expand on the current view of the cancer glycan epitope beyond the single structure, to patterns and the whole glycocalyx, are described. These new approaches that consider the cancer glycan epitope as a glycoform, or as a pattern of many epitope structures, are providing new targets both for cancer biomarkers and therapeutics currently in development at the bench and the clinic.

    更新日期:2018-03-13
  • Understanding the glycome: an interactive view of glycosylation from glycocompositions to glycoepitopes
    Glycobiology (IF 3.112) Pub Date : 2018-03-06
    Davide Alocci, Marie Ghraichy, Elena Barletta, Alessandra Gastaldello, Julien Mariethoz, Frederique Lisacek

    Nowadays, due to the advance of experimental techniques in glycomics, large collections of glycan profiles are regularly published. The rapid growth of available glycan data accentuates the lack of innovative tools for visualizing and exploring large amount of information. Scientists resort to using general-purpose spreadsheet applications to create ad hoc data visualization. Thus, results end up being encoded in publication images and text, while valuable curated data is stored in tables and published as supplemental material. To tackle this problem, we have built an interactive pipeline composed with three tools: Glynsight, EpitopeXtractor and Glydin’. Glycan profile data can be imported in Glynsight, which generates a custom interactive glycan profile. Several profiles can be compared and glycan composition is integrated with structural data stored in databases. Glycan structures of interest can then be sent to EpitopeXtractor to perform a glycoepitope extraction. EpitopeXtractor results can be superimposed on the Glydin’ glycoepitope network. The network visualization allows fast detection of clusters of glycoepitopes and discovery of potential new targets. Each of these tools is standalone or can be used in conjunction with the others, depending on the data and the specific interest of the user. All the tools composing this pipeline are part of the Glycomics@ExPASy initiative and are available at https://www.expasy.org/glycomics

    更新日期:2018-03-06
  • Applications of a highly α2,6-selective pseudosialidase
    Glycobiology (IF 3.112) Pub Date : 2018-02-28
    Peter Both, Michel Riese, Christopher J Gray, Kun Huang, Edward G Pallister, Iaroslav Kosov, Louis P Conway, Josef Voglmeir, Sabine L Flitsch

    Within human biology, combinations of regioisomeric motifs of α2,6- or α2,3-sialic acids linked to galactose are frequently observed attached to glycoconjugates. These include glycoproteins and glycolipids, with each linkage carrying distinct biological information and function. Microbial linkage-specific sialidases have become important tools for studying the role of these sialosides in complex biological settings, as well as being used as biocatalysts for glycoengineering. However, currently, there is no α2,6-specific sialidase available. This gap has been addressed herein by exploiting the ability of a Photobacterium sp. α2,6-sialyltransferase to catalyse trans-sialidation reversibly and in a highly linkage-specific manner, acting as a pseudosialidase in the presence of cytidine monophosphate. Selective, near quantitative removal of α2,6-linked sialic acids was achieved from a wide range of sialosides including small molecules conjugates, simple glycan, glycopeptide and finally complex glycoprotein including both linkages.

    更新日期:2018-02-28
  • Overexpression of heparanase in mice promoted megakaryopoiesis
    Glycobiology (IF 3.112) Pub Date : 2018-02-19
    Ying-Xia Tan, Hao Cui, Lu-Ming Wan, Feng Gong, Xiao Zhang, Israel Vlodavsky, Jin-Ping Li

    Heparanase, an endo-glucuronidase that specifically cleaves heparan sulfate, is upregulated in several pathological conditions. In this study, we aimed to find a correlation of heparanase expression and platelets production. In the transgenic mice overexpressing human heparanase (Hpa-tg), hematological analysis of blood samples revealed a significantly higher number of platelets in comparison with wild-type (Ctr) mice, while no significant difference was found in leukocytes and red blood cell number between the two groups. Total number of thiazole-orange positive platelets was increased in Hpa-tg vs. Ctr blood, reflecting a higher rate of platelets production. Concomitantly, MK from Hpa-tg mice produced more and shorter HS fragments that were shed into the medium. Further, thrombopoietin (TPO) level was elevated in the liver and plasma of Hpa-tg mice. Together, the data indicate that heparanase expression promoted megakaryopoiesis, which may be through upregulated expression of TPO and direct effect of released HS fragments expressed in the MK.

    更新日期:2018-02-21
  • Variant in human POFUT1 reduces enzymatic activity and likely causes a recessive microcephaly, global developmental delay with cardiac and vascular features
    Glycobiology (IF 3.112) Pub Date : 2018-02-14
    Hideyuki Takeuchi, Derek Wong, Michael Schneider, Hudson H Freeze, Megumi Takeuchi, Steven J Berardinelli, Atsuko Ito, Hane Lee, Stanley F Nelson, Robert S Haltiwanger

    Protein O-fucosyltransferase-1 (POFUT1) adds O-fucose monosaccharides to epidermal growth factor-like (EGF) repeats found on approximately 100 mammalian proteins, including Notch receptors. Haploinsufficiency of POFUT1 has been linked to adult-onset Dowling Degos Disease (DDD) with hyperpigmentation defects. Homozygous deletion of mouse Pofut1 results in embryonic lethality with severe Notch-like phenotypes including defects in somitogenesis, cardiogenesis, vasculogenesis, and neurogenesis, but the extent to which POFUT1 is required for normal human development is not yet understood. Here we report a patient with a congenital syndrome consisting of severe global developmental delay, microcephaly, heart defects, failure to thrive, and liver disease with a previously unreported homozygous NM_015352.1: c.485 C>T variant (p.Ser162Leu) in POFUT1 detected by exome sequencing. Both parents are heterozygotes and neither manifests any signs of DDD. No other detected variant explained the phenotype. This variant eliminated a conserved N-glycosylation sequon at Asn160 in POFUT1 and profoundly decreased POFUT1 activity in patient fibroblasts compared to control fibroblasts. Purified p.Ser162Leu mutant protein also showed much lower POFUT1 activity with a lower affinity for EGF acceptor substrate than wild type POFUT1. Eliminating the N-glycan sequon by replacing Asn160 with Gln had little effect on POFUT1 activity, suggesting that loss of the glycan is not responsible for the defect. Furthermore, the p.Ser162Leu mutant showed weaker ability to rescue Notch activity in cell-based assays. These results suggest that this N-glycan of POFUT1 is not required for its proper enzymatic function, and that the p.Ser162Leu mutation of POFUT1 likely causes global developmental delay, microcephaly with vascular and cardiac defects.

    更新日期:2018-02-14
  • Apicomplexan C-Mannosyltransferases Modify Thrombospondin Type I-Containing Adhesins of the TRAP Family
    Glycobiology (IF 3.112) Pub Date : 2017-02-08
    Carolin M Hoppe, Andreia Albuquerque, Giulia Bandini, Deborah R Leon, Aleksandra Shcherbakova, Falk F R Buettner, Luis Izquierdo, Catherine E Costello, Hans Bakker, Françoise H Routier

    In many metazoan species, an unusual type of protein glycosylation, called C-mannosylation, occurs on adhesive thrombospondin type 1 repeats (TSRs) and type I cytokine receptors. This modification has been shown to be catalyzed by the Caenorhabditis elegans DPY-19 protein and orthologues of the encoding gene were found in the genome of apicomplexan parasites. Lately, the micronemal adhesin thrombospondin-related anonymous protein (TRAP) was shown to be C-hexosylated in Plasmodium falciparum sporozoites. Here, we demonstrate that also the micronemal protein MIC2 secreted by Toxoplasma gondii tachyzoites is C-hexosylated. When expressed in a mammalian cell line deficient in C-mannosylation, P. falciparum and T. gondii Dpy19 homologues were able to modify TSR domains of the micronemal adhesins TRAP/MIC2 family involved in parasite motility and invasion. In vitro, the apicomplexan enzymes can transfer mannose to a WXXWXXC peptide but, in contrast to C. elegans or mammalian C-mannosyltransferases, are inactive on a short WXXW peptide. Since TSR domains are commonly found in apicomplexan surface proteins, C-mannosylation may be a common modification in this phylum.

    更新日期:2018-02-08
  • Identification of internally sialylated carbohydrate tumor marker candidates, including Sda/CAD antigens, by focused glycomic analyses utilizing the substrate specificity of neuraminidase
    Glycobiology (IF 3.112) Pub Date : 2018-01-30
    Miki Tanaka-Okamoto, Ken Hanzawa, Mikio Mukai, Hidenori Takahashi, Masayuki Ohue, Yasuhide Miyamoto

    In our previous study, 14 sulfated carbohydrate tumor marker candidates were identified by focused glycomic analyses. Here, glycomic analyses focused on internally sialylated glycans to identify novel marker candidates. Internally sialylated glycans were enriched by digestion of pyridylaminated glycans prepared from sera withα-neuraminidase from Salmonella typhimurium, which did not cleave sialic acids linked to internal residues, followed by anion-exchange chromatography. Next, internally sialylated O-glycan profiles were constructed using two types of high performance liquid chromatography, which were compared between 20 healthy controls and 11 patients with gastric cancer and 9 patients with pancreatic cancer. In all, 17 marker candidates were identified. The structures of glycan candidates were precisely analyzed using enzymatic digestion, glycan synthesis, two-dimensional mapping and mass spectrometry. Among 17 candidates, one was STn, and the other 16 comprised 10 core1, 1 core2 and 5 core3 glycans. The various structures included a α2,6-sialylated reducing terminal GalNAc and α2,6-sialylated type1 N-acetyl-lactosamine. Eight candidates possessed the Sda/CAD antigen. The levels of these candidate glycans in sera from all 40 subjects were quantified using a selected reaction monitoring assay and found to be elevated in at least one or more patients. Although the serum levels of each candidate glycan varied between patients, those candidates having the same backbone or determinant, such as core3 backbone and core1 structures with extended type1 N-acetyl-lactosamine, displayed similar patterns of elevation. These results suggest that analysis of multiple markers may be an effective means of diagnosing various cancers.

    更新日期:2018-01-30
  • Isotopic Labeling with Cellular O-glycome Reporter/Amplification (ICORA) for Comparative O-glycomics of Cultured Cells
    Glycobiology (IF 3.112) Pub Date : 2018-01-30
    Matthew R Kudelka, Alison V Nairn, Mohammed Y Sardar, Xiaodong Sun, Elliot L Chaikof, Tongzhong Ju, Kelley W Moremen, Richard D Cummings

    Mucin-type O-glycans decorate >80% of secretory and cell surface proteins and contribute to health and disease. However, dynamic alterations in the O-glycome are poorly understood because current O-glycomic methodologies are not sufficiently sensitive nor quantitative. Here we describe a novel isotope labeling approach termed Isotope-Cellular O-glycome Reporter Amplification (ICORA) to amplify and analyze the O-glycome from cells. In this approach, cells are incubated with Ac3GalNAc-Bn (Ac3GalNAc-[1H7]Bn) or a heavy labeled Ac3GalNAc-BnD7 (Ac3GalNAc-[2D7]Bn) O-glycan precursor (7 Da mass difference), which enters cells and upon de-esterification is modified by Golgi enzymes to generate Bn-O-glycans secreted into the culture media. After recovery, heavy and light Bn-O-glycans from two separate conditions are mixed, analyzed by MS, and statistically interrogated for changes in O-glycan abundance using a semi-automated approach. ICORA enables ~100–1000 fold enhanced sensitivity and increased throughput compared to traditional O-glycomics. We validated ICORA with model cell lines and used it to define alterations in the O-glycome in colorectal cancer. ICORA is a useful tool to explore the dynamic regulation of the O-glycome in health and disease.

    更新日期:2018-01-30
  • NEU1 sialidase exists on the cell surface of the mouse thymocyte and CD5 is a natural substrate for it
    Glycobiology (IF 3.112) Pub Date : 1753-01-01
    Shigeko Kijimoto-Ochiai, Tokuko Matsumoto-Mizuno, Daisuke Kamimura, Masaaki Murakami, Miwako Kobayashi, Ichiro Matsuoka, Hiroshi Ochiai, Hideharu Ishida, Makoto Kiso, Keiko Kamimura, Toshiaki Koda

    Membrane-bound sialidases in the mouse thymus are unique and mysterious because their activity at pH 6.5 is equal to or higher than that in the acidic region. However, such activity has never been reported in membrane bound form. To clarify this entity, we studied the sialidase activities of crude membrane fractions from immature-T, mature-T and non-T cells from C57BL/6 mice and from SM/J mice, a strain with a defect in NEU1 activity. Non-T cells from C57BL/6 mice had high activity at pH 6.5, but those from SM/J mice did not. Neu1 and Neu3 mRNA was shown by real-time PCR to be expressed in T cells and also in non-T cells, whereas Neu2 was expressed mainly in non-T cells and Neu4 was scarcely expressed. However, the in situ hybridization study on the localization of four sialidases in the thymus showed that Neu4 was clearly expressed in the thymus. We then focused on a sialidase on the thymocyte surface because the possibility of the existence of a silaidase on thymocytes was suggested by PNA staining after incubation of the cells alone in PBS. This activity was inhibited by NEU1-selective sialidase inhibitor C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid. The natural substrate for the cell surface sialidase was identified as CD5 by PNA-blot analysis of anti-CD5 immunoprecipitate. We conclude that NEU1 exists on the surface of mouse thymocytes and CD5 is a natural substrate for it, although this is not the main entity of the mysterious membrane-bound thymus-sialidase.

    更新日期:2018-01-30
  • GLYCAN-DIRECTED CAR-T CELLS
    Glycobiology (IF 3.112) Pub Date : 2018-01-23
    Catharina Steentoft, Denis Migliorini, Tiffany R King, Ulla Mandel, Carl H June, Avery D Posey

    Cancer immunotherapy is rapidly advancing in the treatment of a variety of hematopoietic cancers, including pediatric acute lymphoblastic leukemia and diffuse large B cell lymphoma, with chimeric antigen receptor (CAR)-T cells. CARs are genetically encoded artificial T cell receptors that combine the antigen specificity of an antibody with the machinery of T cell activation. However, implementation of CAR technology in the treatment of solid tumors has been progressing much slower. Solid tumors are characterized by a number of challenges that need to be overcome, including cellular heterogeneity, immunosuppressive tumor microenvironment (TME), and, in particular, few known cancer-specific targets. Post-translational modifications that differentially occur in malignant cells generate valid cell surface, cancer-specific targets for CAR-T cells. We previously demonstrated that CAR-T cells targeting an aberrant O-glycosylation of MUC1, a common cancer marker associated with changes in cell adhesion, tumor growth, and poor prognosis, could control malignant growth in mouse models. Here, we discuss the field of glycan-directed CAR-T cells and review the different classes of antibodies specific for glycan-targeting, including the generation of high affinity O-glycopeptide antibodies. Finally, we discuss historic and recently investigated glycan targets for CAR-T cells and provide our perspective on how targeting the tumor glycoproteome and/or glycome will improve CAR-T immunotherapy.

    更新日期:2018-01-23
  • Heterologous Expression, Characterization and Possible Functions of the Chitin Deacetylases, Cda1 and Cda2, from Mushroom Coprinopsis cinerea
    Glycobiology (IF 3.112) Pub Date : 2018-01-23
    Yanxin Wang, Xin Niu, Xiaoli Guo, Han Yu, Zhonghua Liu, Zhenqing Zhang, Sheng Yuan

    Two chitin deacetylases, Cda1 and Cda2, from C. cinerea were expressed and characterized. Cda1 preferably deacetylates the non-reducing end residue of (GlcNAc)2, the internal or non-reducing end residue of (GlcNAc)3, and the non-reducing residue of (GlcNAc)6 after deacetylating the internal residues. In contrast, Cda2 preferably deacetylates the reducing end residue of (GlcNAc)2, the internal or reducing end residue of (GlcNAc)3, and the reducing residue of (GlcNAc)6 after deacetylating the internal residues. Furthermore, Cda1 prefers chitohexaose with higher degrees of acetylation for deacetylation, while Cda2 shows a weaker preference for chitohexaose with varying degrees of acetylation. The predicted Cda1 structure shows more hydrophobic aromatic amino acids on the surface near subsite +1 in the active site than on the surface near subsite −1, whereas the predicted Cda2 structure has more hydrophobic aromatic amino acids on the surface near subsite −1 than on the surface near subsite +1, which may be the molecular basis of the distinctive catalytic features between Cda1 and Cda2. Notably, Cda1 has a high transcription level in the non-elongating basal stipe region, whereas Cda2 has a high transcription level in the elongating apical stipe region, and the transcription level of the former is approximately five times that of the latter. Correspondingly, the molar ratio of GlcN/GlcNAc increased from 0.15 in the cell wall of the apical stipe region to 0.22 in the cell wall of the basal stipe region. Different modes of action of Cda1 and Cda2 may be related to their functions in the different stipe regions.

    更新日期:2018-01-23
  • Modification of histone by glyoxal: Recognition of glycated histone containing advanced glycation adducts by serum antibodies of type 1 diabetes patients
    Glycobiology (IF 3.112) Pub Date : 2018-01-19
    Nadeem Ahmad Ansari, Dharmendra Kumar Chaudhary, Debabrata Dash

    Dicarbonyl compounds react more rapidly, than glucose, with arginine and lysine in proteins to form advanced glycation end products (AGEs) and further produce free radicals which cause DNA damage. AGEs are reliable diagnostic biomarkers for most of the age-related diseases.

    更新日期:2018-01-19
  • Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system
    Glycobiology (IF 3.112) Pub Date : 2018-01-11
    Adrian J Jervis, Alison G Wood, Joel A Cain, Jonathan A Butler, Helen Frost, Elizabeth Lord, Rebecca Langdon, Stuart J Cordwell, Brendan W Wren, Dennis Linton

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

    更新日期:2017-12-21
  • GlycoDomainViewer: A bioinformatics tool for contextual exploration of glycoproteomes
    Glycobiology (IF 3.112) Pub Date : 2017-12-18
    Hiren J Joshi, Anja Jørgensen, Katrine T Schjoldager, Adnan Halim, Leo A Dworkin, Catharina Steentoft, Hans H Wandall, Henrik Clausen, Sergey Y Vakhrushev

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

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

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

    更新日期:2017-12-15
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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