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  • 1. Bergstrom, M
    et al.
    Ryden, I
    Påhlsson, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Cell Biology.
    Ohlson, S
    Separation of protein glycoforms with affinity capillary electrophoresis2003In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 13, no 11, p. 76-Conference paper (Other academic)
  • 2. Broeker, N. K.
    et al.
    Gohlke, U.
    Müller, J. J.
    Uetrecht, Charlotte
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
    Heinemann, U.
    Seckler, R.
    Barbirz, S.
    Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity2013In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 23, no 1, p. 59-68Article in journal (Refereed)
    Abstract [en]

    Bacteriophage HK620 recognizes and cleaves the O-antigen polysaccharide of Escherichia coli serogroup O18A1 with its tailspike protein (TSP). HK620TSP binds hexasaccharide fragments with low affinity, but single amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants. Isothermal titration calorimetry showed that only small amounts of heat were released upon complex formation via a large number of direct and solvent-mediated hydrogen bonds between carbohydrate and protein. At room temperature, association was both enthalpy- and entropy-driven emphasizing major solvent rearrangements upon complex formation. Crystal structure analysis showed identical protein and sugar conformers in the TSP complexes regardless of their hexasaccharide affinity. Only in one case, a TSP mutant bound a different hexasaccharide conformer. The extended sugar binding site could be dissected in two regions: first, a hydrophobic pocket at the reducing end with minor affinity contributions. Access to this site could be blocked by a single aspartate to asparagine exchange without major loss in hexasaccharide affinity. Second, a region where the specific exchange of glutamate for glutamine created a site for an additional water molecule. Side-chain rearrangements upon sugar binding led to desolvation and additional hydrogen bonding which define this region of the binding site as the high-affinity scaffold.

  • 3.
    Conze, Tim
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Carvalho, Ana Sofia
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    Almeida, Raquel
    Reis, Celso A.
    David, Leonor
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
    MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas2010In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 2, p. 199-206Article in journal (Refereed)
    Abstract [en]

    Changes in mucin protein expression and in glycosylation are common features in pre-neoplastic lesions and cancer and are therefore used as cancer-associated markers. De novo expression of intestinal mucin MUC2 and cancer-associated sialyl-Tn antigen are frequently observed in intestinal metaplasia (IM) and gastric cancer. However, despite that these antigens often co-localize, MUC2 has not been demonstrated to be a carrier of sialyl-Tn. By using the in situ proximity ligation assay (in situ PLA), we herein could show that MUC2 is a major carrier of the sialyl-Tn antigen in all IM cases and in most gastric carcinoma cases. The requirement by in situ PLA for the presence of both antigens in close proximity increases the selectivity compared to measurement of co-localization, as determined by immunohistochemistry. Identification of the mucin which is the carrier of a carbohydrate structure offers unique advantages for future development of more accurate diagnostic and prognostic markers.

  • 4.
    de Oliveira, Felipe Marques Souza
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Mereiter, Stefan
    Univ Porto, i3S, Oporto, Portugal.;Univ Porto, IPATIMUP Inst Mol Pathol & Immunol, Oporto, Portugal..
    Persson, Nina
    Univ Copenhagen, Dept Chem, Copenhagen, Denmark..
    Blixt, Ola
    Univ Copenhagen, Dept Chem, Copenhagen, Denmark..
    Reis, Celso A.
    Univ Porto, i3S, Oporto, Portugal.;Univ Porto, IPATIMUP Inst Mol Pathol & Immunol, Oporto, Portugal..
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Detection of post-translational modification of cancer biomarkers via proximity ligation assay2016In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 26, no 12, p. 1455-1455Article in journal (Refereed)
  • 5. Fagerberg, David
    et al.
    Angström, Jonas
    Halim, Adnan
    Hultberg, Anna
    Rakhimova, Lena
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hammarström, Lennart
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Teneberg, Susann
    Novel Leb-like Helicobacter pylori-binding glycosphingolipid created by the expression of human alpha-1,3/4-fucosyltransferase in FVB/N mouse stomach.2009In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 19, no 2, p. 182-191Article in journal (Refereed)
    Abstract [en]

    The "Le(b) mouse" was established as a model for investigations of the molecular events following Le(b)-mediated adhesion of Helicobacter pylori to the gastric epithelium. By the expression of a human alpha-1,3/4-fucosyltransferase in the gastric pit cell lineage of FVB/N transgenic mice, a production of Le(b) glycoproteins in gastric pit and surface mucous cells was obtained in this "Le(b) mouse," as demonstrated by binding of monoclonal anti-Le(b) antibodies. To explore the effects of the human alpha-1,3/4-fucosyltransferase on glycosphingolipid structures, neutral glycosphingolipids were isolated from stomachs of transgenic alpha-1,3/4-fucosyltransferase-expressing mice. A glycosphingolipid recognized by BabA-expressing H. pylori was isolated and characterized by mass spectrometry and proton NMR as Fuc alpha 2Gal beta 3(Fuc alpha 4)GalNAc beta 4 Gal beta 4 Glc beta 1Cer, i.e., a novel Le(b)-like glycosphingolipid on a ganglio core. In addition, two other novel glycosphingolipids were isolated from the mouse stomach epithelium that were found to be nonbinding with regard to H. pylori. The first was a pentaglycosylceramide, GalNAc beta 3 Gal alpha 3(Fuc alpha 2)Gal beta 4 Glc beta 1Cer, in which the isoglobotetrasaccharide has been combined with Fuc alpha 2 to yield an isoglobotetraosylceramide with an internal blood group B determinant. The second one was an elongated fucosyl-gangliotetraosylceramide, GalNAc beta 3(Fuc alpha 2)Gal beta 3GalNAc beta 4Gal beta 4 Glc beta 1Cer.

  • 6. Fairweather, J. K.
    et al.
    Him, J. L. K.
    Heux, L.
    Driguez, H.
    Bulone, Vincent
    KTH, Superseded Departments, Biotechnology.
    Structural characterization by C-13-NMR spectroscopy of products synthesized in vitro by polysaccharide synthases using C-13-enriched glycosyl donors: application to a UDP-glucose:(1 -> 3)-beta-D-glucan synthase from blackberry (Rubus fruticosus)2004In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 14, no 9, p. 775-781Article in journal (Refereed)
    Abstract [en]

    A simple and sensitive method for the characterization of products synthesized in vitro by polysaccharide synthases is described. it relies on the use of C-13-enriched nucleotide sugars as substrates and on the analysis of the newly synthesized polysaccharides by C-13-nuclear magnetic resonance (NMR) spectroscopy. The method was validated with a (1-->3)-beta-D-glucan synthase from blackberry, but it may be applied to the study of any glycosyltransferase. The chemical synthesis of UDP-D-[U-C-13]glucose was achieved in a classical procedure with an overall yield of 50%. A uniformly labeled (1-->3)-beta-D-glucan was synthesized from this substrate, using detergent extracts of blackberry cell membranes as a source of synthase. One hundred micrograms of product was sufficient for liquid and solid-state C-13-NMR spectroscopy analyses. The method is at least 100 times more sensitive than in the case of non-enriched polysaccharides. It allows the unequivocal identification and direct structural characterization of the products synthesized in vitro, as opposed to conventional methods that rely on the use of radioactive substrates and enzymatic hydrolysis of the polysaccharides with specific glycoside hydrolases. The method proves that the glycan analyzed was synthesized de novo because the final product is enriched in C-13. Information on the 3D organization of the polymer may also be obtained by solid-state NMR spectroscopy.

  • 7.
    Fontana, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Lundborg, Magnus
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Weintraub, Andrej
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Rapid structural elucidation of polysaccharides employing predicted functions of glycosyltransferases and NMR data: Application to the O-antigen of Escherichia coli O592014In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 24, no 5, p. 450-457Article in journal (Refereed)
    Abstract [en]

    A computerized method that uses predicted functions of glycosyltransferases (GTs) in conjunction with unassigned NMR data has been developed for the structural elucidation of bacterial polysaccharides (PSs). In this approach, information about the action of GTs (consisting of possible sugar residues used as donors and/or acceptors, as well as the anomeric configuration and/or substitution position in the respective glycosidic linkages) is extracted from the Escherichia coli O-antigen database and is submitted, together with the unassigned NMR data, to the CASPER program. This time saving methodology, which alleviates the need for chemical analysis, was successfully implemented in the structural elucidation of the O-antigen PS of E. coli O59. The repeating unit of the O-specific chain was determined using the O-deacylated PS and has a branched structure, namely, -> 6)[alpha-d-GalpA3Ac/4Ac-(1 -> 3)]-alpha-d-Manp-(1 -> 3)-alpha-d-Manp-(1 -> 3)-beta-d-Manp-(1 -> 3)-alpha-d-GlcpNAc-(1 ->. The identification of the O-acetylation positions was efficiently performed by comparison of the H-1,C-13 HSQC NMR spectra of the O-deacylated lipopolysaccharide and the lipid-free PS in conjunction with chemical shift predictions made by the CASPER program. The side-chain d-GalpA residue carries one equivalent of O-acetyl groups at the O-3 and O-4 positions distributed in the LPS in a 3:7 ratio, respectively. The presence of O-acetyl groups in the repeating unit of the E. coli O59 PS is consistent with the previously proposed acetyltransferase WclD in the O-antigen gene cluster.

  • 8.
    Fontana, Carolina
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Ramström, Kristoffer
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Weintraub, Andrej
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Structural studies of the O-antigen polysaccharide from Escherichia coli O115 and biosynthetic aspects thereof2013In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 23, no 3, p. 354-362Article in journal (Refereed)
    Abstract [en]

    The structure of the O-antigen polysaccharide (PS) of Escherichia coliO115 has been investigated using a combination of component analysis and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy experiments. The repeating unit of the O-antigen was elucidated using the O-deacetylated PS and has the following branched pentasaccharide structure: →3)[β-L-Rhap-(1 → 4)]-β-D-GlcpNAc-(1 → 4)-α-D-GalpA-(1 → 3)-α-D-Manp-(1 → 3)-β-D-GlcpNAc-(1→. Cross-peaks of low intensity, corresponding to a β-L-Rhap-(1 → 4)-β-D-GlcpNAc-(1→ structural element, were present in the NMR spectra and attributed to the terminal part of the PS; this information defines the biological repeating unit of the O-antigen by having a 3-substituted N-acetyl-D-glucosamine (GlcNAc) residue at its reducing end. Analysis of the NMR spectra of the native PS revealed O-acetyl groups distributed over different positions of theL-Rhap residue (∼0.70 per repeating unit) as well as at O-2 and O-3 of the D-GalpA residue (∼0.03 and ∼0.25 per repeating unit, respectively), which is in agreement with the presence of two acetyltransferases previously identified in the O-antigen gene cluster (Wang Q, Ruan X, Wei D, Hu Z, Wu L, Yu T, Feng L, Wang L. 2010. Mol Cell Probes. 24:286–290.). In addition, the four glycosyltransferases initially identified in the O-antigen gene cluster of E. coli O115 were analyzed using BLAST, and the function of two of them predicted on the basis of similarities with glycosyltransferases from Shigella dysenteriae type 5 and 12, as well as E. coli O58 and O152.

  • 9. Greffe, L.
    et al.
    Bessueille, L.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Synthesis, preliminary characterization, and application of novel surfactants from highly branched xyloglucan oligosaccharides2005In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 15, no 4, p. 437-445Article in journal (Refereed)
    Abstract [en]

    A novel class of nonionic, carbohydrate-based surfactants has been synthesized from the plant polysaccharide xyloglucan. Enzymatic hydrolysis of xyloglucan yielded a series of well-defined, highly branched oligosaccharides that, following reductive amination, were readily conjugated with fatty acids bearing C-8 to C-18 chains under mild conditions. The critical micelle concentration, determined by tensiometry and dye-inclusion measurements, showed a typical dependence on acyl chain length and was sensitive to the degree of galactosylation of the head group. Several compounds from this new group of surfactants, especially those with C-14 and C-16 chains, were useful for the extraction of membrane-bound enzyme markers from different plant cell compartments in catalytically active form.

  • 10. Gunnarsson, Lavinia Cicortas
    et al.
    Zhou, Qi
    KTH, School of Biotechnology (BIO), Glycoscience.
    Montanier, Cedric
    Karlsson, Eva Nordberg
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ohlin, Mats
    Engineered xyloglucan specificity in a carbohydrate-binding module2006In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 16, no 12, p. 1171-1180Article in journal (Refereed)
    Abstract [en]

    The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.

  • 11.
    Gunnarsson, Lavinia Cicortas
    et al.
    Department of Immunotechnology, Lund University, Lund, Sweden.
    Zhou, Qi
    School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.
    Montanier, Cedric
    Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, UK.
    Karlsson, Eva Nordberg
    Department of Biotechnology, Lund University, Lund, Sweden.
    Brumer, Harry
    School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.
    Ohlin, Mats
    Department of Immunotechnology, Lund University, Lund, Sweden.
    Engineered xyloglucan specificity in a carbohydrate-binding module2006In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 16, no 12, p. 1171-1180Article in journal (Refereed)
    Abstract [en]

    The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.

  • 12.
    Gustafsson, Anki
    et al.
    Recopharma AB, Huddinge.
    Sjöblom, Magnus
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Strindelius, Lena
    Recopharma AB, Huddinge.
    Johansson, Thomas
    Recopharma AB, Huddinge.
    Fleckenstein, Tilly
    Recopharma AB, Huddinge.
    Chatzissavidou, Nathalie
    Recopharma AB, Huddinge.
    Lindberg, Linda
    Absorber AB, Stockholm.
    Ångström, Jonas
    Göteborgs universitet.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Holgersson, Jan
    Karolinska Institutet.
    Pichia pastoris-produced mucin-type fusion proteins with multivalent O-glycan substitution as targeting molecules for mannose-specific receptors of the immune system2011In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 21, no 8, p. 1071-1086Article in journal (Refereed)
    Abstract [en]

    Mannose-binding proteins like the macrophage mannose receptor (MR), the dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose-binding lectin (MBL) play crucial roles in both innate and adaptive immune responses. Immunoglobulin fusion proteins of the P-selectin glycoprotein ligand-1 (PSGL-1/mIgG2b) carrying mostly O-glycans and, as a control, the a1-acid glycoprotein (AGP/mIgG2b) carrying mainly N-linked glycans were stably expressed in the yeast Pichia pastoris. P. pastoris-produced PSGL-1/mIgG2b was shown to carry O-glycans that mediated strong binding to mannose-specific lectins in a lectin array and were susceptible to cleavage by a-mannosidases including an a1,2- but not an a1,6-mannosidase. Electrospray ionization - ion trap mass spectrometry (ESI-MS) confirmed the presence of O-glycans containing up to 9 hexoses with the penta- and hexasaccharides being the predominant ones. a1,2- and a1,3-linked, but not a1,6-linked, mannose residues were detected by 1H-nuclear magnetic resonance (1H-NMR) spectroscopy confirming the results of the mannosidase cleavage. The apparent equilibrium dissociation constants for binding of PNGase F-treated mannosylated PSGL-1/mIgG2b to MR, DC-SIGN and MBL were shown by surface plasmon resonance to be 126, 56 and 16 nM, respectively. In conclusion, PSGL-1/mIgG2b expressed in P. pastoris carried O-glycans mainly comprised of a-linked mannoses and with up to nine residues. It bound mannose-specific receptors with high apparent affinity and may become a potent targeting molecule for these receptors in vivo.

  • 13.
    Harn, Donald
    et al.
    Univ Georgia, Dept Infect Dis, Athens, GA 30602 USA..
    Ramadhin, Jessica
    Univ Georgia, Dept Infect Dis, Athens, GA 30602 USA..
    Meagher, Richard
    Univ Georgia, Dept Genet, Athens, GA 30602 USA..
    Ambati, Suresh
    Univ Georgia, Dept Genet, Athens, GA 30602 USA..
    Filipov, Nikolay
    Univ Georgia, Dept Physiol & Pharm, Athens, GA 30602 USA. Univ Georgia, Coll Family & Consumer Sci, Athens, GA 30602 USA..
    Shollenberger, Lisa
    Univ Georgia, Dept Infect Dis, Athens, GA 30602 USA..
    Norberg, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    LNFPIII-Dex conjugates function in vivo to normalize metabolic function in High-Fat Diet Obese mice2017In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 27, no 12, p. 1190-1191Article in journal (Other academic)
  • 14. Hood, D W
    et al.
    Cox, A D
    Wakarchuk, W W
    Schur, M
    Schweda, Elke K H
    Södertörn University, Avdelning Naturvetenskap.
    Walsh, S L
    Deadman, M E
    Martin, A
    Moxon, E R
    Richards, J C
    Genetic basis for expression of the major globotetraose-containing lipopolysaccharide from H-influenzae strain Rd (RM118)2001In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 11, no 11, p. 957-967Article in journal (Refereed)
    Abstract [en]

    A genetic basis for the biosynthetic assembly of the globotetraose containing lipopolysaccharide (LPS) of Haemophilus influenzae strain RM118 (Rd) was determined by structural analysis of LPS derived from mutant strains. We have previously shown that the parent strain RM118 elaborates a population of LPS molecules made up of a series of related glycoforms differing in the degree of oligosaccharide chain extension from the distal heptose residue of a conserved phosphorylated inner-core element, L-alpha -D-Hepp-(1-->2)-L-alpha -D-Hepp-(1-->3)-[beta -D-Glcp-(1-->4)-]-L-alpha -D-Hepp-(1-->5)-alpha -Kdo. The fully extended LPS glycoform expresses the globotetraose structure, beta -D-GalpNAc-(1-->3)-alpha -D-Galp(1-->4)-beta -D-Galp-(1-->4)-beta -D-Glcp. A fingerprinting strategy was employed to establish the structure of LPS from strains mutated in putative glycosyltransferase genes compared to the parent strain. This involved glycose and linkage analysis on intact LPS samples and analysis of O-deacylated LPS samples by electrospray ionization mass spectrometry and 1D H-1-nuclear magnetic resonance spectroscopy. Four genes, lpsA, lic2A, lgtC, and lgtD, were required for sequential addition of the glycoses to the terminal inner-core heptose to give the globotetraose structure. lgtC and lgtD were shown to encode glycosyltransferases by enzymatic assays with synthetic acceptor molecules. This is the first genetic blueprint determined for H. influenzae LPS oligosaccharide biosynthesis, identifying genes Involved in the addition of each glycose residue.

  • 15. Ito, Yuki
    et al.
    Vela, Jose Luis
    Matsumura, Fumiko
    Hoshino, Hitomi
    Lee, Heeseob
    Kobayashi, Motohiro
    Bao, Xingfeng
    Boren, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Jin, Rongsheng
    Seeberger, Peter H.
    Nakayama, Jun
    Kronenberg, Mitchell
    Fukuda, Minoru
    Immunological functions of cholesteryl alpha-glucosides in helicobacter pylori-associated inflammation2011In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 21, no 11, p. 1485-1486Article in journal (Refereed)
  • 16. Ito, Yuki
    et al.
    Vela, Jose Luis
    Matsumura, Fumiko
    Hoshino, Hitomi
    Tyznik, Aaron
    Lee, Heeseob
    Girardi, Enrico
    Zajonc, Dirk M.
    Kobayashi, Motohiro
    Bao, Xingfeng
    Boren, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Jin, Rongsheng
    Seeberger, Peter H.
    Nakayama, Jun
    Kronenberg, Mitchell
    Fukuda, Minoru
    Helicobacter pylori cholesteryl alpha-glucosides are critical for bacterial growthand activation of invariant NKT cells2012In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 22, no 11, p. 1635-1636Article in journal (Other academic)
  • 17.
    Julenius, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    NetCGlyc 1.0: prediction of mammalian C-mannosylation sites2007In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 17, no 8, p. 868-876Article in journal (Refereed)
    Abstract [en]

    C-mannosylation is the attachment of an alpha-mannopyranose to a tryptophan via a C-C linkage. The sequence WXXW, in which the first Trp becomes mannosylated, has been suggested as a consensus motif for the modification, but only two-thirds of known sites follow this rule. We have gathered a data set of 69 experimentally verified C-mannosylation sites from the literature. We analyzed these for sequence context and found that apart from Trp in position + 3, Cys is accepted in the same position. We also find a clear preference in position + 1, where a small and/or polar residue (Ser, Ala, Gly, and Thr) is preferred and a Phe or a Len residue discriminated against. The Protein Data Bank was searched for structural information, and five structures of C-mannosylated proteins were obtained. We showed that modified tryptophan residues are at least partly solvent exposed. A method predicting the location of C-mannosylation sites in proteins was developed using a neural network approach. The best overall network used a 21-residue sequence input window and information on the presence/ absence of the WXXW motif. NetCGlyc 1.0 correctly predicts 93% of both positive and negative C-mannosylation sites. This is a significant improvement over the WXXW consensus motif itself, which only identifies 67% of positive sites. NetCGlyc 1.0 is available at http://www.cbs.dtu.dk/ services/NetCGlyc/. Using NetCGlyc 1.0, we scanned the human genome and found 2573 exported or transmembrane transcripts with at least one predicted C-mannosylation site.

  • 18.
    Keser, Toma
    et al.
    Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia.
    Akmacic, Irena Trbojevic
    Genos Glycoscience Research Laboratory, Zagreb, Croatia.
    Ventham, Nicholas T.
    Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.
    Theodoratou, Evropi
    Centre for Population Health Sciences, University of Edinburgh, Edinburgh, UK.
    Vuckovic, Frano
    Genos Glycoscience Research Laboratory, Zagreb, Croatia.
    Kennedy, Nicholas A.
    Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.
    Nimmo, Elaine R.
    Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.
    Kalla, Rahul
    Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.
    Drummond, Hazel
    Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.
    Stambuk, Jerko
    Genos Glycoscience Research Laboratory, Zagreb, Croatia.
    Campbell, Harry
    Centre for Population Health Sciences, University of Edinburgh, Edinburgh, UK.
    Hedin, Charlotte
    Diabetes and Nutritional Sciences Division, School of Medicine, King ’ s College London, London, UK; Centre for Digestive Diseases, Blizard Institute, Queen Mary University of London, London, UK.
    D'Amato, Mauro
    Department of Biosciences and Nutrition, Karolinska Institute, Stockholm, Sweden.
    Halfvarson, Jonas
    Örebro University, School of Medicine, Örebro University, Sweden. Division of Gastroenterology, Örebro University Hospital, Örebro, Sweden.
    Satsangi, Jack
    Centre for Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.
    Lauc, Gordan
    Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia; Genos Glycoscience Research Laboratory, Zagreb, Croatia.
    Changes in the immunoglobulin G glycome associated with inflammatory bowel disease2015In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 25, no 11, p. 1242-1242Article in journal (Other academic)
  • 19. Kulseth, Mari Ann
    et al.
    Mustorp, Stina Lund
    Uhlin-Hansen, Lars
    Öberg, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kolset, Svein Olav
    Serglycin expression during differentiation of U937-1 cells1998In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 8, no 8, p. 747-753Article in journal (Refereed)
    Abstract [en]

    Serglycin is the major proteoglycan in most hematopoietic cells, including monocytes and macrophages. The monoblastic cell line U937-1 was used to study the expression of serglycin during proliferation and differentiation. In unstimulated proliferating U937-1 cells serglycin mRNA is nonconstitutively expressed. The level of serglycin mRNA was found to correlate with the synthesis of chondroitin sulfate proteoglycan (CSPG). The U937-1 cells were induced to differentiate into different types of macrophage-like cells by exposing the cells to PMA, RA, or VitD3. These inducers of differentiation affected the expression of serglycin mRNA in three different ways. The initial upregulation seen in the normally proliferating cells was not observed in PMA treated cells. In contrast, RA increased the initial upregulation, giving a reproducible six times increase in serglycin mRNA level from 4 to 24 h of incubation, compared to a four times increase in the control cells. VitD3 had no effect on the expression of serglycin mRNA. The incorporation of (35S)sulfate into CSPG decreased approximately 50% in all three differentiated cell types. Further, the (35S)CSPGs expressed were of larger size in PMA treated cells than controls, but smaller after RA treatment. This was due to the expression of CSPGs, with CS-chains of 25 and 5 kDa in PMA and RA treated cells, respectively, compared to 11 kDa in the controls. VitD3 had no significant effect on the size of CSPG produced. PMA treated cells secreted 75% of the (35S)PGs expressed, but the major portion was retained in cells treated with VitD3 or RA. The differences seen in serglycin mRNA levels, the macromolecular properties of serglycin and in the PG secretion patterns, suggest that serglycin may have different functions in different types of macrophages.

  • 20. Kumar, Archana Vijaya
    et al.
    Gassar, Ezeddin Salem
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Stock, Christian
    Kiesel, Ludwig
    Yip, George
    Goette, Martin
    HS3ST2 overexpression increases invasiveness of MDA-MB-231 breast cancercells via up-regulation of protease expression and MAPK signaling2012In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 22, no 11, p. 1556-1556Article in journal (Other academic)
  • 21. Li, J J
    et al.
    Bauer, S H J
    Månsson, Martin
    Södertörn University, Avdelning Naturvetenskap.
    Moxon, E R
    Richards, J C
    Schweda, Elke K H
    Södertörn University, Avdelning Naturvetenskap.
    Glycine is a common substituent of the inner core in Haemophilus influenzae lipopolysaccharide2001In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 11, no 12, p. 1009-1015Article in journal (Refereed)
    Abstract [en]

    A survey of both typeable and nontypeable strainsof Haemophilus influenzae indicated that they contain glycine (Gly) in their lipopolysaccharide (LPS). Significant amounts (30-250 pmol Gly/mug LPS) were determined by high-performance anion-exchange chromatography using pulsed amperometric detection after treatment of the LPS with mild alkali. Oligosaccharides obtained from LPS after mild acid hydrolysis and gel filtration chromatography were investigated by electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis (CE) ESI-MS. In all cases, molecular ions corresponding to the major glycoforms were identified and were accompanied by ions differing by 57 Da, thus indicating the presence of glycine. The position of glycine in these glycoforms was determined by CE-ESI-MS/MS analyses. It was found that, depending on strain, glycine can substitute each of the heptoses of the inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-L-alpha-D-Hepp- (1-->5)-alpha-Kdo of H. influenzae LPS as well as Kdo. In some strains, mixtures of monosubstituted Gly-containing glycoforms having different substitution patterns were identified.

  • 22.
    Lind, Thomas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Falk, Elisabet
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hjertson, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kusche-Gullberg, Marion
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lidholt, Kerstin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    cDNA cloning and expression of UDP-glucose dehydrogenase from bovine kidney1999In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 9, no 6, p. 595-600Article in journal (Refereed)
    Abstract [en]

    We have isolated a cDNA encoding UDP-glucose dehydrogenase from a bovine kidney cDNA-library, the first mammalian cDNA clone published. [After submission of the manuscript, a study appeared describing the molecular cloning and characterization of  the human and mouse UDP-glucose dehydrogenase genes(Spicer et al., 1998).] The enzyme catalyzes the conversion of UDP-glucose to UDP-glucuronicacid, an essential precursor in glycosaminoglycan biosynthesis. The cDNA has an open reading frame of 1482 nucleotides coding for a 55 kDa protein. Expression of the enzyme in COS-7 cells showed a 3-fold increase inUDP-glucose dehydrogenase activity; also, the C-terminal 23 amino acidswas shown not to be necessary for enzyme activity. Northernblots from human and mouse tissues reveal high expression in liver and low in skeletal muscle. Human tissues have a majortranscript size of 3.2 kilobases and a minor of 2.6 whereas mousetissues have a single 2.6 kilobase transcript. We have also developed a sensitive and direct assay using UDP-[14C]Glc as a substrate for detection of small amounts of UDPGDH activity. 

  • 23. Liu, Bin
    et al.
    Perepelov, Andrei V.
    Svensson, Mona V.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Shevelev, Sergei D.
    Guo, Dan
    Senchenkova, Sof'ya N.
    Shashkov, Alexander S.
    Weintraub, Andrej
    Feng, Lu
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Knirel, Yuriy A.
    Wang, Lei
    Genetic and structural relationships of Salmonella O55 and Escherichia coli O103 O-antigens and identification of a 3-hydroxybutanoyltransferase gene involved in the synthesis of a Fuc3N derivative2010In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 6, p. 679-688Article in journal (Refereed)
    Abstract [en]

    O-antigen (O-polysaccharide), a part of the outer membrane of Gram-negative bacteria, is one of the most variable cell constituents and is related to bacterial virulence. O-antigen diversity is almost entirely due to genetic variations in O-antigen gene clusters. In this study, the O-polysaccharide structures of Salmonella O55 and Escherichia coli O103 were elucidated by chemical analysis and nuclear magnetic resonance spectroscopy. It was found that the O-polysaccharides have similar pentasaccharide O-units, which differ only in one sugar (glucose versus N-acetylglucosamine) and in the N-acyl group (acetyl versus 3-hydroxybutanoyl) on 3-amino-3,6-dideoxy-d-galactose (d-Fuc3N). The Salmonella O55 antigen gene cluster was sequenced and compared with the E. coli O103 antigen gene cluster reported previously. The two gene clusters were found to share high-level similarity (DNA identity ranges from 53% to 76%), except for two putative acyl transferase genes (fdtC in Salmonella O55 and fdhC in E. coli O103) which show no similarity. Replacement of the fdtC gene in Salmonella O55 with the fdhC gene from E. coli O103 resulted in production of a modified O-antigen, which contains a 3-hydroxybutanoyl derivative of Fuc3N in place of 3-acetamido-3,6-dideoxygalactose. This finding strongly suggests that fdhC is a 3-hydroxybutanoyltransferase gene. The sequence similarity level suggested that the O-antigen gene clusters of Salmonella O55 and E. coli O103 originate from a common ancestor, and this evolutionary relationship is discussed.

  • 24. Liu, J N
    et al.
    Gustafsson, A
    Breimer, M E
    Kussak, Anders
    Södertörn University, School of Life Sciences.
    Holgersson, J
    Anti-pig antibody adsorption efficacy of alpha-Gal carrying recombinant P-selectin glycoprotein ligand-1/immunoglobulin chimeras increases with core 2 beta 1,6-N-acetylglucosaminyltransferase expression2005In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 15, no 6, p. 571-583Article in journal (Refereed)
    Abstract [en]

    We have previously described the construction of a P-selectin glycoprotein ligand-1-mouse immunoglobulin Fc fusion protein, which when transiently coexpressed with the porcine alpha 1,3 galactosyltransferase in COS cells becomes a very efficient adsorber of xenoreactive, anti-pig antibodies. To relate the adsorption capacity with the glycan expression of individual fusion proteins produced in different cell lines, stable CHO-K1, COS, and 293T cells producing this fusion protein have been engineered. On alpha 1,3 galactosyltransferase coexpression, high-affinity adsorbers were produced by both COS and 293T cells, whereas an adsorber of lower affinity was derived from CHO-K1 cells. Stable coexpression of a core 2 beta 1,6 N-acetylglucosaminyltransferase in CHO-K1 cells led to increased alpha-Gal epitope density and improved anti-pig antibody adsorption efficacy. ESI-MS/MS of O-glycans released from PSGL-1/mIgG(2b) produced in an alpha 1,3 galactosyl- and core 2 beta 1,6 N-acetylglucosaminyltransferase expressing CHO-K1 cell clone revealed a number of structures with carbohydrate sequences consistent with terminal Gal-Gal. In contrast, no O-glycan structures with terminal Gal-Gal were identified on the fusion protein when expressed alone or in combination with the alpha 1,3 galactosyltransferase in CHO-K1 cells. In conclusion, the density of alpha-Gal epitopes on PSGL-1/mIgG(2b) was dependent on the expression of O-linked glycans with core 2 structures and lactosamine extensions. The structural complexity of the terminal Gal-Gal expressing O-glycans with both neutral as well as sialic acid-containing structures is likely to contribute to the high adsorption efficacy.

  • 25.
    Lundborg, Magnus
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Modhukur, Vijayachitra
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Glycosyltransferase functions of E. coli O-antigens2010In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 3, p. 366-368Article in journal (Refereed)
    Abstract [en]

    ECODAB (the E. coli O-antigen database) has been expanded to include information about glycosyltransferases (GTs) involved in the assembly of the O-antigen polysaccharide. Similarity searches have been performed to be able to determine GT functions that have not been reported prior to this work. In addition to suggesting the function of 179 GTs, the approach leads to the prediction of part of the O-antigen structures of a number of serogroups. The procedure suggests a novel way of combining genetic information with experimental techniques in structural analysis of oligo- and polysaccharides.

  • 26. Löfling, Jonas
    et al.
    Diswall, Mette
    Eriksson, Sara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Breimer, Michael E
    Holgersson, Jan
    Studies of Lewis antigens and H. pylori adhesion in CHO cell lines engineered to express Lewis b determinants2008In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 18, no 7, p. 494-501Article in journal (Refereed)
    Abstract [en]

    Many microbes bind and adhere via adhesins to host cell carbohydrates as an initial step for infection. Therefore, cell lines expressing Lewis b (Le(b)) determinants were generated as a potential model system for Helicobacter pylori colonization and infection, and their expression of blood group Lewis determinants was characterized. CHO-K1 cells were stably transfected with selected glycosyltransferase cDNAs, and two Le(b) positive clones, 1C5 and 2C2, were identified. Expression of Lewis (Le(a), Le(b), Le(x), and Le(y)) determinants was analyzed by flow cytometry of intact cells, SDS-PAGE/Western blot of solubilized glycoproteins, and thin layer chromatography immunostaining of isolated glycolipids (GL). Binding of H. pylori to cells was examined by microscopy and quantified. Flow cytometry showed that 1C5 and 2C2 were Le(a) and Le(b) positive. 1C5 expressed Le(b) on O-linked, but not N-linked, glycans and only weakly on GLs. In contrast, 2C2 expressed Le(b) on N-, O-glycans, and GLs. Furthermore, both clones expressed Le(a) on N- and O-glycans but not on GLs. 2C2, but not 1C5, stained positively for Le(y) on N-linked glycans and GLs. Both clones, as well as the parental CHO-K1 cells, expressed Le(x) on GLs. A Le(b)-binding H. pylori strain bound to the 1C5 and 2C2 cells. In summary, two glycosyltransferase transfected CHO-K1 cell clones differed regarding Lewis antigen expression on N- and O-linked glycans as well as on GLs. Both clones examined supported adhesion of a Le(b)-binding H. pylori strain and may thus be a useful in vitro model system for H. pylori colonization/infection studies.

  • 27. Magalhaes, Ana
    et al.
    Marcos-Pinto, Ricardo
    Gomes, Joana
    Nairn, Alison V.
    Rossez, Yannick
    Robbe-Masselot, Catherine
    Maes, Emmanuel
    Bugaytsova, Zhanna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Figueiredo, Ceu
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Moremen, Kelley W.
    Reis, Celso A.
    The glycan receptors of Helicobacter pylori: decoding the pathways underlying gastric glycophenotype modulation2016In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 26, no 12, p. 1401-1402Article in journal (Refereed)
    Abstract [en]

    The gastrointestinal tract is covered by a complex extracellular mucus layer that protects the gastric epithelium fromexternal aggressions such as chemical agents, microorganismsand shear stress. Although this mucus barrier confers protec-tion against certain pathogens, it may also provide a niche formicrobial binding.Helicobacter pyloriexploits the host glycoconjugates present in the gastric mucus layer and lining thesurface epithelium of the gastric mucosa to colonize the stomach. Infection can persist for decades promoting chronicinflammation, and in a subset of individuals lesions cansilently progress to cancer. The secreted MUC5AC mucin isthe main component of the gastric mucus layer, andH. pyloriBabA-mediated binding to MUC5AC confers increased riskfor overt disease. We have shown that FUT2 determines theO-glycosylation pattern of Muc5ac, with Fut2 knock-outleading to a marked decrease inα1,2-fucosylated structuresand increased expression of the terminal type 1 glycan structure Lewisa. Importantly, for thefirst time, we structurallyvalidated the expression of Lewisain murine gastric mucosa(1). We further demonstrated that loss of mucin FUT2-mediated fucosylation impairs gastric mucosal binding ofH.pyloriBabA adhesin, which is a recognized feature of patho-genicity. UponH. pyloriinfection,concomitantly with tissueinflammation, there is a remodeling of the gastric glycopheno-type. We showed that increased expression of sialyl-Lewisa/xantigens is due to transcriptional up-regulation of theB3GNT5,B3GALT5,andFUT3genes. In addition, weobserved thatH. pyloriinfected individuals present a markedgastric local pro-inflammatory signature with significantlyhigher TNF-αlevels and demonstrated that TNF-induced activation of the NF-kappaB pathway results in B3GNT5 up-regulation (2). Furthermore, we showed that this gastric glycosylation shift, characterized by increased sialylation pat-terns, favors SabA-mediatedH. pyloriattachment to humaninflamed gastric mucosa. Our data provides clinically relevantinsights into the regulatory mechanisms underlyingH. pylorimodulation of host glycosylation machinery, and phenotypicalterations crucial for life-long infection and gastric disease.

  • 28. Magalhães, Ana
    et al.
    Gomes, Joana
    Ismail, Mohd Nazri
    Haslam, Stuart M
    Mendes, Nuno
    Osório, Hugo
    David, Leonor
    Le Pendu, Jacques
    Haas, Rainer
    Dell, Anne
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Reis, Celso A
    Fut2-null mice display an altered glycosylation profile and impaired BabA-mediated Helicobacter pylori adhesion to gastric mucosa2009In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 19, no 12, p. 1525-1536Article in journal (Refereed)
    Abstract [en]

    Glycoconjugates expressed on gastric mucosa play a crucial role in host-pathogen interactions. The FUT2 enzyme catalyzes the addition of terminal alpha(1,2)fucose residues, producing the H type 1 structure expressed on the surface of epithelial cells and in mucosal secretions of secretor individuals. Inactivating mutations in the human FUT2 gene are associated with reduced susceptibility to Helicobacter pylori infection. H. pylori infects over half the world's population and causes diverse gastric lesions, from gastritis to gastric cancer. H. pylori adhesion constitutes a crucial step in the establishment of a successful infection. The BabA adhesin binds the Le(b) and H type 1 structures expressed on gastric mucins, while SabA binds to sialylated carbohydrates mediating the adherence to inflamed gastric mucosa. In this study, we have used an animal model of nonsecretors, Fut2-null mice, to characterize the glycosylation profile and evaluate the effect of the observed glycan expression modifications in the process of H. pylori adhesion. We have demonstrated expression of terminal difucosylated glycan structures in C57Bl/6 mice gastric mucosa and that Fut2-null mice showed marked alteration in gastric mucosa glycosylation, characterized by diminished expression of alpha(1,2)fucosylated structures as indicated by lectin and antibody staining and further confirmed by mass spectrometry analysis. This altered glycosylation profile was further confirmed by the absence of Fucalpha(1,2)-dependent binding of calicivirus virus-like particles. Finally, using a panel of H. pylori strains, with different adhesin expression profiles, we have demonstated an impairment of BabA-dependent adhesion of H. pylori to Fut2-null mice gastric mucosa, whereas SabA-mediated binding was not affected.

  • 29.
    Malm, Linus
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hellman, Urban
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Larsson, Göran
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Size determination of hyaluronan using a gas-phase electrophoretic mobility molecular analysis2012In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 22, no 1, p. 7-11Article in journal (Refereed)
    Abstract [en]

    Hyaluronan (HA) is a linear non-sulfated polysaccharide mainly found in the extracellular matrix. The size of HA can vary from a fewq saccharides up to at least 25,000 units, reaching molecular weights of 10x103 kDa. HA has mainly biological functions, and both its size and tissue concentration play an important role in many physiological and phatological processes. It is relatively easy ti determine the HA concentration using enzyme-linked binding protein assays, but the molecular weight of HA has so far been shown to be a more challenging task to measure. Here, we present a method for size determination of HA using gas-phase electrophoretic mobility molecular analysis (GEMMA), which utilizes the electrophoretic mobility of molecules in air to estimate the molecular weight of the analyte. We show that this method gives reliable molecular weight estimations of HA in the range 30-2400 kDa, which covers almost its whole biological range. The average measuring time for one GEMMA spectrum is between 5 and 10 min using only 6 pg of HA. In addition the peak area in a GEMMA spectrum can be used to estimate the HA concentration in the sample. The high sensitivity and small sample volumes make GEMMA an excellent tool for both size determination and estimation of concentration of samples with low HA concentration, as is the case for HA extracted from small tissue samples.

  • 30.
    Muiá, Romina P.
    et al.
    Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, B1650WGA San Martín, Argentina.
    Yu, Hai
    Department of Chemistry, University of California, CA95616 Davis, USA.
    Prescher, Jennifer A.
    Department of Chemistry, University of California, CA94720 Berkeley, USA.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Chen, Xi
    Department of Chemistry, University of California, CA95616 Davis, USA.
    Bertozzi, Carolyn R
    Department of Chemistry, University of California, CA94720 Berkeley, USA.
    Campetella, Oscar
    Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, B1650WGA San Martín, Argentina.
    Identification of glycoproteins targeted by Trypanosoma cruzi trans-sialidase, a virulence factor that disturbs lymphocyte glycosylation2010In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 7, p. 833-842Article in journal (Refereed)
    Abstract [en]

    Trypanosoma cruzi, the agent of the American trypanosomiasis or Chagas disease, bypasses its lack of de novo synthesis of sialic acids by expressing a surface-anchored trans-sialidase. This enzyme transfers sialic acid residues from the host's sialylglycoconjugates to the parasite's galactosylglycoconjugates. In addition to carrying out a pivotal role in parasite persistence/replication within the infected mammal, the trans-sialidase is shed into the bloodstream and induces alterations in the host immune system by modifying the sialylation of the immune cells. A major obstacle to understand these events is the difficulty to identify the transferred sialic acid among all those naturally occurring on the cell surface. Here, we report the use of azido-modified unnatural sialic acid to identify those molecules that act as cell surface acceptors of the sialyl residue in the trans-sialidase-catalyzed reaction, which might then be involved in the immune alterations induced. In living parasites, we readily observed the transfer of azido-sialic acid to surface mucins. When evaluating mouse thymocytes and splenocytes as acceptors of the azido-sugar, a complex pattern of efficiently tagged glycoproteins was revealed. In both leukocyte populations, the main proteins labeled were identified as different CD45 isoforms. Disruption of the cell architecture increased the number and the molecular weight distribution of azido-sialic acid tagged proteins. Nevertheless, CD45 remained to be the main acceptor. Mass spectrometry assays allowed us to identify other acceptors, mainly integrins. The findings reported here provide a molecular basis to understand the abnormalities induced in the immune system by the trans-sialidase during T. cruzi infection.

  • 31.
    Olausson, Johan
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Jonsson, Bengt-Harald
    Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
    Tibell, Lena
    Linköping University, Department of Science and Technology, Media and Information Technology. Linköping University, The Institute of Technology.
    Påhlsson, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Production and characterization of a monomeric form and a single-site form of Aleuria aurantia lectin2011In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 21, no 1, p. 34-44Article in journal (Refereed)
    Abstract [en]

    Lectins have been widely used in structural and functional studies of complex carbohydrates. Lectins usually bind carbohydrates with relatively low affinity but compensate for this by multivalency. When using lectins in different biological and analytical assays the multivalent nature of lectins can sometimes produce unwanted reactions such as agglutination or precipitation of target glycoproteins. The mushroom lectin Aleuria aurantia binds to fucose-containing oligosaccharides. It is composed of two identical subunits where each subunit contains five binding sites for fucose. In the present study two forms of recombinant AAL were produced using site-directed mutagenesis. A monomeric form of AAL was produced by exchange of Tyr6 to Arg6, and a monovalent fragment of AAL was produced by insertion of a NdeI restriction enzyme cleavage site and a stop codon in the coding sequence. The AAL forms were expressed as His-tagged proteins in E.coli and purified by affinity chromatography. Binding properties of the two AAL forms were performed using hemagglutination assay, surface plasmon resonance and enzyme-linked lectin assay analyses. Both the monomeric AAL form (mAAL) and the monovalent AAL form (S2-AAL) retained their capacity to bind fucosylated oligosaccharides. However, both constructs exhibited properties that differed from the intact recombinant AAL (rAAL). Monomeric AAL showed similar binding affinities to fucosylated oligosaccharides compared to rAAL but had less hemagglutinating capacity. S2-AAL showed a lower binding affinity to fucosylated oligosaccharides and, in contrast to rAAL and mAAL, S2-AAL did not bind to sialylated fuco-oligosaccharides such as sialyl-Lex. The study shows that molecular engineering techniques may be a tool for producing lectins with more defined properties such as decreased valency and defined specificities and affinities. This may be very valuable for development of reliable diagnostic and biological assays for carbohydrate analysis.

  • 32. Pegeot, Mathieu
    et al.
    Sadir, Rabia
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Simorre, Jean-Pierre
    Gans, Pierre
    Lortat-Jacob, Hugues
    Profiling sulfation/epimerization pattern of full-length heparan sulfate by NMR following cell culture C-13-glucose metabolic labeling2015In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 25, no 2, p. 151-156Article in journal (Refereed)
    Abstract [en]

    Through its ability to interact with proteins, heparan sulfate (HS) fulfills a large variety of functions. Protein binding depends on the level of HS sulfation and epimerization which are cell specific and dynamically regulated. Characterization of this molecule, however, has been restricted to oligosaccharide fragments available in large amount for structural investigation or to sulfate distribution through compositional analysis. Here we developed a H-1-C-13 2D NMR-based approach, directly performed on HS isolated from C-13-labeled cells. By integrating the peak volumes measured at different chemical shifts, this non-destructive analysis allows us to determine both the sulfation and the iduronic/glucuronic profiles of the polysaccharide. Applied to wild-type and N-deacetylase/N-sulfotransferase-deficient fibroblasts as well as to epithelial cells differentiation, it also gives insights into the functional relationships existing between HS biosynthetic enzymes. This approach should be of significant interest to better understand HS changes that occur through physiologic regulations or during pathological development.

  • 33.
    Ramachandra, Rashmi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Namburi, Ramesh Babu
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dupont, Sam
    Department of Biological and Environmental Sciences, University of Gothenburg .
    Ortega-Martinez, Olga
    Department of Biological and Environmental Sciences, University of Gothenburg .
    Thorndyke, Michael
    Department of Biological and Environmental Sciences, University of Gothenburg .
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A Potential Role for Chondroitin Sulfate/Dermatan Sulfate in Arm Regeneration in Amphiura filiformis.2017In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 27, no 5, p. 438-449Article in journal (Refereed)
    Abstract [en]

    Glycosaminoglycans (GAGs), such as chondroitin sulfate (CS) and dermatan sulfate (DS) from various vertebrate and invertebrate sources are known to be involved in diverse cellular mechanisms during repair and regenerative processes. Recently, we have identified CS/DS as the major GAG in the brittlestar Amphiura filiformis, with high proportions of di- and tri-O-sulfated disaccharide units. As this echinoderm is known for its exceptional regeneration capacity, we aimed to explore the role of these GAG chains during A. filiformis arm regeneration. Analysis of CS/DS chains during the regeneration process revealed an increase in the proportion of the tri-O-sulfated disaccharides. Conversely, treatment of A. filiformis with sodium chlorate, a potent inhibitor of sulfation reactions in GAG biosynthesis, resulted in a significant reduction in arm growth rates with total inhibition at concentrations higher than 5 mM. Differentiation was less impacted by sodium chlorate exposure or even slightly increased at 1-2 mM. Based on the structural changes observed during arm regeneration we identified chondroitin synthase, chondroitin-4-O-sulfotransferase 2 and dermatan-4-O-sulfotransferase as candidate genes and sought to correlate their expression with the expression of the A. filiformis orthologue of bone morphogenetic factors, AfBMP2/4. Quantitative amplification by real-time PCR indicated increased expression of chondroitin synthase and chondroitin-4-O-sulfotransferase 2, with a corresponding increase in AfBMP2/4 during regeneration relative to nonregenerating controls. Our findings suggest that proper sulfation of GAGs is important for A. filiformis arm regeneration and that these molecules may participate in mechanisms controlling cell proliferation.

  • 34.
    Ramachandra, Rashmi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Namburi, Ramesh Babu
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ortega-Martinez, Olga
    Department of Biological and Environmental Sciences, University of Gothenburg .
    Shi, Xiaofeng
    Department of Biochemistry, Boston University.
    Zaia, Joseph
    Department of Biochemistry, Boston University.
    Dupont, Sam T.
    Department of Biological and Environmental Sciences, University of Gothenburg .
    Thorndyke, Michael
    Department of Biological and Environmental Sciences, University of Gothenburg .
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brittlestars contain highly sulfated chondroitin sulfates/dermatan sulfates that promote fibroblast growth factor 2-induced cell signaling2014In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 24, no 2, p. 195-207Article in journal (Refereed)
    Abstract [en]

    Glycosaminoglycans (GAGs) isolated from brittlestars, Echinodermata class Ophiuroidea, were characterized, as part of attempts to understand the evolutionary development of these polysaccharides. A population of chondroitin sulfate/dermatan sulfate (CS/DS) chains with a high overall degree of sulfation and hexuronate epimerization was the major GAG found, whereas heparan sulfate (HS) was below detection level. Enzymatic digestion with different chondroitin lyases revealed exceptionally high proportions of di- and trisulfated CS/DS disaccharides. The latter unit appears much more abundant in one of four individual species of brittlestars, Amphiura filiformis, than reported earlier in other marine invertebrates. The brittlestar CS/DS was further shown to bind to growth factors such as fibroblast growth factor 2 and to promote FGF-stimulated cell signaling in GAG-deficient cell lines in a manner similar to that of heparin. These findings point to a potential biological role for the highly sulfated invertebrate GAGs, similar to those ascribed to HS in vertebrates.

  • 35.
    Razi, Nahid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Lay, L
    Russo, G
    Panza, L
    Lindahl, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Identification of O-sulphate substituents on D-glucuronic acid units in heparin-related glycosaminoglycans using novel synthetic disaccharide standards.1995In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 5, no 8, p. 807-811Article in journal (Refereed)
    Abstract [en]

    The two disaccharides, methyl 4-O-(2-O-sulpho-beta-D-glucopyranosyl-uronic acid)-2-deoxy-2-amino-alpha-D-glucopyranoside and methyl 4-O-(3-O-sulpho-beta-D-glucopyranosyluronic acid)-2-deoxy-2-amino-alpha-D-glucopyranoside, were prepared by de novo synthesis, and converted to the corresponding 2,5-anhydro-D-[1-3H]mannitol derivatives by deamination with nitrous acid followed by reduction with NaB3H4. The resultant labelled products were used as standards in the identification, by anion-exchange high-performance liquid chromatography (HPLC), of disaccharides generated by HNO2/NaB3H4 treatment of heparan sulphate isolated from human brain. The two standards, containing 2-O- and 3-O-sulphated glucuronic acid, respectively, were clearly separated by the HPLC procedure. Comparison with the deamination products derived from heparan sulphate showed that the mono-O-sulphated disaccharide species containing a sulphated glucuronic acid unit co-eluted with the 2-O-sulphated standard. The corresponding component isolated from other heparan sulphate preparations, or from heparin, also eluted at the same position. No disaccharide derived from heparin or heparan sulphate appeared at the elution position of the 3-O-sulphated standard. It is concluded that D-glucuronic acid units in heparin-related glycosaminoglycans may be sulphated at C2, whereas no evidence has been found for sulphation at C3. By contrast, analysis of mono-O-sulphated disaccharides derived from a chemically sulphated, bacterial capsular polysaccharide (generated by Escherichia coli K5) clearly demonstrated the occurrence of O-sulphate groups at C-3 of D-glucuronic acid units.

  • 36. Rojas-Macias, Miguel A.
    et al.
    Ståhle, Jonas
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Luetteke, Thomas
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Development of the ECODAB into a relational database for Escherichia coli O-antigens and other bacterial polysaccharides2015In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 25, no 3, p. 341-347Article in journal (Refereed)
    Abstract [en]

    Escherichia coli O-antigen database (ECODAB) is aweb-based application to support the collection of E. coli O-antigen structures, polymerase and flippase amino acid sequences, NMR chemical shift data of O-antigens as well as information on glycosyltransferases (GTs) involved in the assembly of O-antigen polysaccharides. The database content has been compiled from scientific literature. Furthermore, the system has evolved from being a repository to one that can be used for generating novel data on its own. GT specificity is suggested through sequence comparison with GTs whose function is known. The migration of ECODAB to a relational database has allowed the automation of all processes to update, retrieve and present information, thereby, endowing the system with greater flexibility and improved overall performance. ECODAB is freely available at http://www.casper.organ.su.se/ECODAB/. Currently, data on 169 E. coli unique O-antigen entries and 338 GTs is covered. Moreover, the scope of the database has been extended so that polysaccharide structure and related information from other bacteria subsequently can be added, for example, from Streptococcus pneumoniae.

  • 37.
    Rydell, Gustaf E
    et al.
    Sahlgrens University Hospital.
    Nilsson, Jonas
    Sahlgrens University Hospital.
    Rodriguez, Jesus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Molecular Virology .
    Ruvoen-Clouet, Nathalie
    INSERM.
    Svensson, Lennart
    Linköping University, Department of Clinical and Experimental Medicine, Molecular Virology . Linköping University, Faculty of Health Sciences.
    Le Pendu, Jacques
    INSERM.
    Larson , Goran
    Sahlgrens University Hospital.
    Human noroviruses recognize sialyl Lewis x neoglycoprotein2009In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 19, no 3, p. 309-320Article in journal (Refereed)
    Abstract [en]

    The carbohydrate binding characteristics of a norovirus GII.3 (Chron1) and a GII.4 (Dijon) strain were investigated using virus-like particles (VLPs) and saliva samples from 81 individuals genotyped for FUT2 (secretor) and FUT3 (Lewis) and phenotyped for ABO and Lewis blood groups. The two VLPs showed a typical secretor-gene-dependent binding and bound significantly stronger to saliva from A, B, and AB than from O individuals (P < 0.0001 and P < 0.001) but did not bind to any samples from secretor-negative individuals. The GII.3 strain showed larger interindividual variation and bound stronger to saliva from B than from A(2) secretors (P < 0.01). When assaying for binding to neoglycoproteins, the GII.3 and GII.4 strains were compared with the Norwalk GI.1 prototype strain. Although all three strains bound to Lewis b (and H type 1 chain) glycoconjugates, only the two GII strains showed an additional binding to sialyl Lewis x. This novel binding was specific since the VLPs did not bind to structural analogs, e.g., Lewis x or sialyl Lewis a, but only to sialyl Lewis x, sialyl diLewis x and sialylated type 2 chain conjugates. In inhibition experiments, the sialyl Lewis x conjugate was the most potent inhibitor. The minimal requirement for this potential receptor structure is Neu5Ac alpha 3Gal beta 4(Fuc alpha 3)GlcNAc beta 3Gal beta- where Fuc is not absolutely necessary for binding. Our study shows that some human norovirus GII strains have at least two binding specificities: one secretor-gene-dependent related to alpha 1,2-fucosylated carbohydrates and another related to alpha 2,3-sialylated carbohydrates of the type 2 chain, e.g., sialyl Lewis x.

  • 38.
    Sandwall, Elina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Zhang, Xiao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lannfelt, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heparan sulfate mediates amyloid-beta internalization and cytotoxicity2010In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 5, p. 533-541Article in journal (Refereed)
    Abstract [en]

    Heparan sulfate (HS) has been found associated with amyloid deposits, including the toxic amyloid-beta (Abeta) peptide aggregates in cerebral vasculature and neuronal tissues in patients with Alzheimer's disease. However, the pathophysiological significance of the HS-Abeta interaction has remained unclear. In the present study, we applied cell models to gain insight into the roles of HS in relation to Abeta toxicity. Wild-type Chinese hamster ovary (CHO-WT) cells showed loss of viability following exposure to Abeta40, whereas the HS-deficient cell line, pgsD-677, was essentially resistant. Immunocytochemical analysis showed Abeta internalization by CHO-WT, but not pgsD-677 cells. Abeta40 toxicity was also attenuated in human embryonic kidney cells overexpressing heparanase. Finally, addition of heparin to human umbilical vein endothelial cells prevented internalization of added Abeta40 and protected against Abeta toxicity. Taken together, these findings suggest that cell-surface HS mediates Abeta internalization and toxicity.

  • 39. Senchenkova, Sof'ya N.
    et al.
    Zhang, Yuanyuan
    Perepelov, Andrei V.
    Guo, Xi
    Shashkov, Alexander S.
    Weintraub, Andrej
    Liu, Bin
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Knirel, Yuriy A.
    Structure and gene cluster of the O-antigen of Escherichia coli O165 containing 5-N-acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic acid2016In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 26, no 4, p. 335-342Article in journal (Refereed)
    Abstract [en]

    Upon mild acid degradation of the lipopolysaccharide of Escherichia coli O165, the O-polysaccharide chain was cleaved at the glycosidic linkage of 5-N-acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic acid (Pse5Hb7Ac). Analysis of the resulting linear tetrasaccharide and alkali-treated lipopolysaccharide by 1H/13C 1D and 2D nuclear magnetic resonance spectroscopy enabled elucidation of the following structure of the O-polysaccharide: →8)-α-Psep5Hb7Ac-(2 → 6)-β-D-Galp-(1 → 4)-β-D-Glсp-(1 → 3)-α-DGlсpNAc-(1→. The β-D-Galp-(1 → 4)-β-D-Glсp-(1 → 3)-D-GlсpNAc structural element is also present in the O-polysaccharide of E. coli O82. The content of the O-antigen gene cluster of E. coli O165 was found to be consistent with the O-polysaccharide structure established. Functions of proteins encoded in the gene cluster, including enzymes involved in the Pse5Hb7Ac biosynthesis and glycosyltransferases, were putatively assigned by comparison with sequences in available databases.

  • 40.
    Smeds, Emanuel
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Feta, A.
    Kusche-Gullberg, Marion
    Target selection of heparan sulfate hexuronic acid 2-O-sulfotransferase2010In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 20, no 10, p. 1274-1282Article in journal (Refereed)
    Abstract [en]

    The signaling of various molecules involved in development and regulation of cell growth are regulated by heparan sulfate (HS). Specific binding of HS to ligand proteins depends on the HS sulfation pattern, where the spacing and number of O-sulfate groups are of special importance. HS 2-O-sulfotransferase catalyzes 2-O-sulfation of glucuronic and iduronic acid residues with a 5-fold higher preference for iduronic acid, as inferred from previously determined kinetic parameters. To study in more detail the regulation of HS hexuronic acid 2-O-sulfation, we tested the ability of the enzyme to catalyze glucuronic acid 2-O-sulfation in polysaccharide mixtures with different glucuronic acid/iduronic acid ratios, using 3'-phosphoadenosine 5'-phospho[S-35]sulfate as sulfate donor. The 2-O-sulfotransferase revealed a more pronounced preference for 2-O-sulfation of iduronic acid than predicted. Even incubations with a 99:1 ratio of glucuronic acid to iduronic acid resulted in almost exclusive iduronic acid 2-O-sulfation. Unexpectedly, when the 2-O-sulfotransferase was co-immunoprecipitated with the glucuronyl C5-epimerase (that converts glucuronic acid to iduronic acid), both glucuronic acid and iduronic acid residues were sulfated to the same extent when a polysaccharide containing only glucuronic acid was used as a substrate. Attempting to understand the mechanism by which extended regions of iduronic acid 2-O-sulfation are formed during HS biosynthesis, a H-3-labeled N-sulfated iduronic acid containing octasaccharide substrate was incubated with the 2-O-sulfotransferase and 3'-phosphoadenosine 5'-phosphosulfate. The 2-O-sulfotransferase showed a preference for mono-2-O-sulfated substrates as compared with octasaccharides with no 2-O-sulfate group.

  • 41.
    Ståhle, Jonas
    et al.
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Fontana, Carolina
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Weintraub, Andrej
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Elucidation of the O-antigen structure of Escherichia coli O632019In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 29, no 2, p. 179-187Article in journal (Refereed)
    Abstract [en]

    The structure of the O-antigen polysaccharide (PS) from the Shiga-toxin producing Escherichia coli O63 has been elucidated using a combination of bioinformatics, component analyses and NMR spectroscopy. The O-antigen is comprised of tetrasaccharide repeating units with the following structure: 2)--d-Quip3N(d-allo-ThrAc)-(12)--d-Ribf-(14)--d-Galp-(13)--d-GlcpNAc-(1 in which the N-acetylated d-allo-threonine is amide-linked to position 3 of the 3-amino-3-deoxy-d-Quip sugar residue. The presence of a predicted flippase and polymerase encoded in the O63 gene cluster is consistent with the Wzx/Wzy biosynthetic pathway and consequently the biological repeating unit has likely an N-acetyl-d-glucosamine residue at its reducing end. A bioinformatics approach based on predictive glycosyltransferase function present in ECODAB (E. coli O-antigen database) suggested the structural element -d-Galp-(13)-d-GlcpNAc in the O-antigen. Notably, multiple gene sequence alignment of fdtA and qdtA from E. coli to that in E. coli O63 resulted in discrimination between the two, confirmation of the latter in E. coli O63, and consequently, together with qdtB, biosynthesis of dTDP-d-Quip3N. The E. coli O63 O-antigen polysaccharide differs in two aspects from that of E. coli O114 where the latter carries instead an l-serine residue, and the glycosidic linkage positions to and from the Quip3N residue are both changed. The structural characterization of the O63 antigen repeat supports the predicted functional assignment of the O-antigen cluster genes.

  • 42.
    Tan, Ying-Xia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Beijing Inst Transfus Med, Dept Tissue Engn, 27 Taiping Rd, Beijing 100850, Peoples R China.
    Cui, Hao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab. Jiangxi Normal Univ, Coll Life Sci, 99 Ziyang Rd, Nanchang 330022, Jiangxi, Peoples R China.
    Wan, Lu-Ming
    Beijing Inst Transfus Med, Dept Tissue Engn, 27 Taiping Rd, Beijing 100850, Peoples R China.
    Gong, Feng
    Beijing Inst Transfus Med, Dept Tissue Engn, 27 Taiping Rd, Beijing 100850, Peoples R China.
    Zhang, Xiao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
    Vlodavsky, Israel
    Technion, Fac Med, Canc & Vasc Biol Res Ctr Rappaport, Box 9649, IL-31096 Haifa, Israel.
    Li, Jin-Ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Overexpression of heparanase in mice promoted megakaryopoiesis2018In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 28, no 5, p. 269-275Article in journal (Refereed)
    Abstract [en]

    Heparanase, an endo-glucuronidase that specifically cleaves heparan sulfate (HS), 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, megakaryocytes 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 megakaryocytes.

  • 43. von der Lieth, Claus-Wilhelm
    et al.
    Ardà Freire, Ana
    Blank, Dennis
    Campbell, Matthew P.
    Ceroni, Alessio
    Damerell, David R.
    Dell, Anne
    Dwek, Raymond A.
    Ernst, Beat
    Fogh, Rasmus
    Frank, Martin
    Geyer, Hildegard
    Geyer, Rudolf
    Harrison, Mathew J.
    Henrick, Kim
    Herget, Stefan
    Hull, William E.
    Ionides, John
    Joshi, Hiren J.
    Kamerling, Johannis P.
    Leeflang, Bas R.
    Lütteke, Thomas
    Lundborg, Magnus
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Maass, Kai
    Merry, Anthony
    Ranzinger, René
    Rosen, Jimmy
    Royle, Louise
    Rudd, Pauline M.
    Schloissnig, Siegfried
    Stenutz, Roland
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Vranken, Wim F.
    Widmalm, Göran
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Haslam, Stuart M.
    EUROCarbDB: an open-access platform for glycoinformatics2011In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 21, no 4, p. 493-502Article in journal (Refereed)
    Abstract [en]

    The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocarb.

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