| 1. |
- Blixt, Ola, et al.
(författare)
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Pathogen specific carbohydrate antigen microarrays : a chip for detection of Salmonella O-antigen specific antibodies
- 2008
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Ingår i: Glycoconjugate Journal. - : Springer. - 0282-0080 .- 1573-4986. ; 25:1, s. 27-36
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Tidskriftsartikel (refereegranskat)abstract
- A Salmonella O-antigen microarray was developed by covalent coupling of oligosaccharide antigens specific for serogroups Salmonella enterica sv. Paratyphi (group A), Typhimurium (group B) and Enteritidis (group D). Antibodies were correctly detected in sera from patients with culture verified salmonellosis. High serogroup-specificity was seen with the disaccharide antigens. With the larger antigens, containing the backbone sequence Man alpha 1-2Rha alpha 1-2Gal (MRG), common backbone-specific antibodies (O-antigen 12) were also detected. This is "proof of principle" that pathogen-specific carbohydrate antigen microarrays constitute a novel technology for rapid and specific serological diagnosis in either individual patients or larger sero-epidemiological and vaccine studies.
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| 2. |
- Breimer, Michael, 1951, et al.
(författare)
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Glycosphingolipids of human embryonic stem cells.
- 2017
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Ingår i: Glycoconjugate journal. - : Springer Science and Business Media LLC. - 1573-4986 .- 0282-0080. ; 34:6, s. 713-723
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Forskningsöversikt (refereegranskat)abstract
- The application of human stem cell technology offers theoretically a great potential to treat various human diseases. However, to achieve this goal a large number of scientific issues remain to be solved. Cell surface carbohydrate antigens are involved in a number of biomedical phenomena that are important in clinical applications of stem cells, such as cell differentiation and immune reactivity. Due to their cell surface localization, carbohydrate epitopes are ideally suited for characterization of human pluripotent stem cells. Amongst the most commonly used markers to identify human pluripotent stem cells are the globo-series glycosphingolipids SSEA-3 and SSEA-4. However, our knowledge regarding human pluripotent stem cell glycosphingolipid expression was until recently mainly based on immunological assays of intact cells due to the very limited amounts of cell material available. In recent years the knowledge regarding glycosphingolipids in human embryonic stem cells has been extended by biochemical studies, which is the focus of this review. In addition, the distribution of the human pluripotent stem cell glycosphingolipids in human tissues, and glycosphingolipid changes during human stem cell differentiation, are discussed.
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| 3. |
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| 4. |
- Cheng, Fang, et al.
(författare)
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Suppression of glypican-1 autodegradation by NO-deprivation correlates with nuclear accumulation of amyloid beta in normal fibroblasts.
- 2015
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Ingår i: Glycoconjugate Journal. - : Springer Science and Business Media LLC. - 1573-4986 .- 0282-0080. ; 32:9, s. 675-684
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate (HS)-containing, S-nitrosylated (SNO) glypican-1 (Gpc-1) releases anhydromannose-containing HS (anMan-HS) by SNO-catalyzed autodegradation in endosomes. Transport of anMan-HS to the nucleus requires processing of the amyloid precursor protein (APP) to amyloid beta peptides (Aβ). To further examine the relationship between APP and Gpc-1 processing in normal fibroblasts we have suppressed Gpc-1 autodegradation by aminoguanidine inhibition of NO synthesis and prevented lysosomal degradation of anMan-HS by using chloroquine. Deconvolution immunofluorescence microscopy and SDS-PAGE using anMan- and APP/Aβ-specific antibodies and markers for nuclei and autophagosomes were used to identify subcellular localization of Aβ and its oligomeric state. Wild-type mouse embryonic fibroblasts (WT MEF) grown during NO-deprivation accumulated 95-98 % of Aβ as oligomers in the nucleus. WT MEF treated with chloroquine accumulated both anMan-HS and Aβ, first in the nucleus then in autophagosomes. Maximal nuclear anMan-HS and Aβ accumulation was obtained after 4 and 7 h of growth, respectively. Both yielded similar banding patterns on SDS-PAGE which were also similar to the Aβ oligomers obtained after NO-deprivation. Nuclear Aβ accumulation was marginally increased (from 54 to 58 %) by suppression of both release and degradation of anMan-HS. Nuclear exit of Aβ, accumulated during growth in aminoguanidine, was enhanced by ascorbate-induced reactivation of anMan-HS production. Transgenic Alzheimer disease mouse (Tg2576) MEF, which produces excess amount of Aβ was used for comparison. Overall, nuclear Aβ exit and lysosomal degradation was compromised by inhibition of the autophagosome-lysosome pathway in both WT and Tg2576 MEF, while only WT MEF was sensitive to suppression of Gpc-1 autodegradation.
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| 5. |
- D'Arrigo, I., et al.
(författare)
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Diverse IgG serum response to novel glycopeptide epitopes detected within immunodominant stretches of Epstein-Barr virus glycoprotein 350/220: diagnostic potential of O-glycopeptide microarrays
- 2013
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Ingår i: Glycoconjugate Journal. - : Springer Science and Business Media LLC. - 0282-0080 .- 1573-4986. ; 30:7, s. 633-640
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Tidskriftsartikel (refereegranskat)abstract
- The Epstein-Barr virus (EBV) envelope glycoprotein 350/220 (gp350/220) is the most abundant molecule on the viral surface and it is responsible for the initial viral attachment to cell surface of the host. As many other viral envelope proteins, it is highly glycosylated, not least with O-linked glycans, most of which essential for EBV life cycle. EBV gp350/220 is also a primary target for neutralizing antibodies in the human hosts and a promising candidate for an EBV vaccine. Here we showed that recombinant GalNAc transferases can glycosylate scan peptides of the EBV gp350/220 envelope protein immobilized on microarray glass slides. We also identified serum IgG antibodies to a selection of peptides and O-glycopeptides, whereas sera from EBV-IgG negative individuals remained negative. We here describe novel glycopeptide epitopes present within immunodominant stretches of EBV gp350/220 and demonstrate a remarkable variability between individual samples with respect to their reactivity patterns to peptides and glycopeptides. The study provides additional insights into the complex B-cell response towards the EBV gp350/220 envelope protein, which may have implications for diagnostic and vaccine developments.
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| 6. |
- Domino, Steven E, et al.
(författare)
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Cervical mucins carry alpha(1,2)fucosylated glycans that partly protect from experimental vaginal candidiasis.
- 2009
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Ingår i: Glycoconjugate journal. - : Springer Science and Business Media LLC. - 1573-4986 .- 0282-0080. ; 26:9, s. 1125-34
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Tidskriftsartikel (refereegranskat)abstract
- Cervical mucins are glycosylated proteins that form a protective cervical mucus. To understand the role of mucin glycans in Candida albicans infection, oligosaccharides from mouse cervical mucins were analyzed by liquid chromatography-mass spectrometry. Cervical mucins carry multiple alpha(1-2)fucosylated glycans, but alpha(1,2)fucosyltransferase Fut2-null mice are devoid of these epitopes. Epithelial cells in vaginal lavages from Fut2-null mice lacked Ulex europaeus agglutinin-1 (UEA-I) staining for alpha(1-2)fucosylated glycans. Hysterectomy to remove cervical mucus eliminated UEA-I and acid mucin staining in vaginal epithelial cells from wild type mice indicating the cervix as the source of UEA-I positive epithelial cells. To assess binding of alpha(1-2) fucosylated glycans on C. albicans infection, an in vitro adhesion assay was performed with vaginal epithelial cells from wild type and Fut2-null mice. Vaginal epithelial cells from Fut2-null mice were found to bind increased numbers of C. albicans compared to vaginal epithelial cells obtained from wild type mice. Hysterectomy lessened the difference between Fut2-null and wild type mice in binding of C. ablicans in vitro and susceptibility to experimental C. albicans vaginitis in vivo. We generated a recombinant fucosylated MUC1 glycanpolymer to test whether the relative protection of wild type mice compared to Fut2-null mice could be mimicked with exogenous mucin. While a small portion of the recombinant MUC1 epitopes displayed alpha(1-2)fucosylated glycans, the predominant epitopes were sialylated due to endogenous sialyltransferases in the cultured cells. Intravaginal instillation of recombinant MUC1 glycanpolymer partially reduced experimental yeast vaginitis suggesting that a large glycanpolymer, with different glycan epitopes, may affect fungal burden.
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| 7. |
- Ekman, Bertil, et al.
(författare)
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Urine oligosaccharide pattern in patients with hyperprolactinaemia
- 2015
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Ingår i: Glycoconjugate Journal. - : Springer. - 0282-0080 .- 1573-4986. ; 32:8, s. 635-641
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Tidskriftsartikel (refereegranskat)abstract
- Free milk-type oligosaccharides are produced during pregnancy and lactation and may have an impact on several cells in the immune system. Our aim was to investigate if patients with isolated hyperprolactinaemia, not related to pregnancy, also have increased synthesis and urinary excretion of milk-type oligosaccharides and to compare the excretion pattern with that found during pregnancy. Urine samples were collected as morning sample from 18 patients with hyperprolactinaemia, 13 healthy controls with normal prolactin levels and four pregnant women. After purification, lactose and free oligosaccharides were analysed and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection. The identity of peaks was confirmed by exoglycosidase treatment and comparison with oligosaccharide standards. Prolactin was measured in serum collected between 09 and 11 a.m. by a standardized immunochemical method. Patients with hyperprolactinaemia had higher urinary excretion of lactose than normoprolactinemic controls and urinary lactose correlated positively to prolactin levels (r = 0.51, p < 0.05). Increased levels of the fucosylated oligosaccharides 2-fucosyl lactose and lacto-di-fucotetraose were found in urine from three and two patients, respectively. The acidic oligosaccharide 3-sialyl lactose was found in high amount in urine from two patients with prolactin of >10,000 mU/l. However, pregnant women in their third trimester had the highest concentration of all these oligosaccharides and excretion increased during pregnancy. This study is first to show that both lactose and certain fucosylated and sialylated milk-type oligosaccharides are increased in some patients with hyperprolactinaemia. It remains to elucidate the functional importance of these findings.
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| 8. |
- Eriksson, Christer, et al.
(författare)
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Variant size- and glycoforms of the scavenger receptor cysteine-rich protein gp-340 with differential bacterial aggregation
- 2007
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Ingår i: Glycoconjugate Journal. - : Springer Science and Business Media LLC. - 1573-4986 .- 0282-0080. ; 24:2-3, s. 131-142
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Tidskriftsartikel (refereegranskat)abstract
- Glycoprotein gp-340 aggregates bacteria in saliva as part of innate defence at mucosal surfaces. We have detected size- and glycoforms of gp-340 between human saliva samples (n=7) and lung gp-340 from a proteinosis patient using antibodies and lectins in Western blots and ELISA measurements. Western blots of saliva samples, and of gp-340 purified, from the seven donors using a gp-340 specific antibody distinguished four gp-340 size variants, designated I to IV (n=2,2,2 and 1). While saliva gp-340 variants I to III had single bands of increasing sizes, variant IV and lung gp-340 had double bands. Purified I to IV proteins all revealed a N-terminal sequence TGGWIP upon Edman degradation. Moreover, purified gp-340 from the seven donors and lung gp-340 shared N-glycans, sialylated Gal beta 1-3GalNAc and (poly)lactosamine structures. However, the larger size gp-340 grouping II/III (n=4) and smaller size grouping I/IV correlated with a secretor, Se(+), and a non secretor, Se(-), dependent glycoform of gp-340, respectively (p=0.03). The Se(+) glycoforms contained ABH, Le(b), Le(y) and polylactosamine structures, while the Se(-) glycoforms lacked ABH antigens but expressed Lea, Lex and lactosamine structures. By contrast, lung gp- 340 completely lacked ABH, Le(a/b), Le(x/y) or sLe(x) structures. Gp-340 and secretor typing of saliva from additional donors (n=29) showed gp-340 glycoforms I to IV for 6, 16, 4 and 0 donors, respectively, and 3 non-typeable donors, and verified that gp-340 glycoforms I and II/III correlate with Se(-) and Se(+) phenotypes, respectively (p < 0.0001). The glycoforms of saliva and lung gp-340 mediated differential aggregation of Le(b)-(Helicobacter pylori), sialylpolylactosamine(Streptococcus suis) or sialic acid- (Streptococcus mutans) binding bacteria. In conclusion, variant size- and glycoforms of gp-340 are expressed by different individuals and may modulate the biological properties of gp-340 pertinent to health and disease.
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| 9. |
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| 10. |
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| 11. |
- Gaunitz, Stefan, et al.
(författare)
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Avian influenza H5 hemagglutinin binds with high avidity to sialic acid on different O-linked core structures on mucin-type fusion proteins
- 2014
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Ingår i: Glycoconjugate Journal. - : Springer Science and Business Media LLC. - 0282-0080 .- 1573-4986. ; 31:2, s. 145-159
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between P-selectin glycoprotein ligand-1/mouse IgG(2b) (PSGL-1/mIgG(2b)) fusion protein carrying multiple copies of the influenza hemagglutinin receptor Sia alpha 2-3Gal on different O-glycan chains and recombinant human influenza H5N1 A/Vietnam/1203/04 hemagglutinin was investigated with a Biacore biosensor. The fusion protein was produced by stable cell lines in large scale cultures and purified with affinity- and gel filtration chromatography. The C-P55 and 293-P cell lines were established by transfecting the Chinese hamster ovary (CHO)-K1 and Human embryonic kidney (HEK)-293 cell lines with plasmids encoding the PSGL-1/mIgG(2b) fusion protein, while the C-PSLex cell line was engineered by transfecting CHO-K1 cells with the plasmids encoding the core 2 beta 1,6GnT-I and FUT-VII glycosyltransferases. Glycosylation was characterized by lectin Western blotting of the proteins and liquid chromatography - mass spectrometry of released non-derivatized O-glycans. Biacore experiments revealed that PSGL-1/mIgG(2b) is a good binding partner of H5. The binding curves displayed a slow dissociation indicating a multivalent binding. The H5 hemagglutinin binds with similar strength to PSGL-1/mIgG(2b) carrying mostly sialylated core 1 (clone C-P55), a mix of sialylated core 1 and sialylated lactosamine (clone 293-P) or mainly sialylated lactosamine (clone C-PSLex) O-glycans, indicating that this hemagglutinin is unable to discriminate between these structures. The potential use of the large, flexible PSGL-1/mIgG(2b) mucin-type fusion protein carrying Sia alpha 2-3Gal as a multivalent inhibitor of influenza virus is discussed.
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| 12. |
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| 13. |
- Gustafson, Stefan, et al.
(författare)
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Accessible hyaluronan receptors identical to ICAM-1 in mouse mast-cell tumours
- 1995
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Ingår i: Glycoconjugate Journal. - 0282-0080 .- 1573-4986. ; 12:3, s. 350-355
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Tidskriftsartikel (refereegranskat)abstract
- Immunohistochemical studies of the hyaluronan (HA)-receptor (R), originally found on liver endothelial cells (LEC) and related to the intercellular adhesion molecule 1 (ICAM-1), showed that polyclonal antibodies against HARLEC (HA receptor on LEC) also stain structures in mouse mastocytomas, mainly vessels. To test if intravenously administered HA might target the tumour receptors in vivo, mice carrying an inoculated mastocytoma in one hind leg muscle were injected in the tail vein with 125I-tyrosine (T)-labelled HA and killed 75 min after injection when organs and tissues were checked for radioactivity. When doses exceeding the binding capacity of the liver were injected, a significant increase in radioactivity (up to five-fold) within the tumour tissue was found. The weight adjusted difference between control and tumour tissue was greater for smaller tumours, probably due to necrosis in the larger. HA-staining of tumours from animals receiving 125I-T-HA, showed HA in areas that also stained weakly for ICAM-1 using monoclonal antibodies. ICAM-1 staining was dramatically increased after hyaluronidase treatment of the sections, indicating that the HA is bound to these receptors and thereby blocks antibody recognition.
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| 14. |
- Gustafsson, A., et al.
(författare)
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Carbohydrate phenotyping of human and animal milk glycoproteins
- 2005
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Ingår i: Glycoconjugate journal. - : Springer Science and Business Media LLC. - 0282-0080 .- 1573-4986. ; 22:3, s. 109-18
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Tidskriftsartikel (refereegranskat)abstract
- Breast-milk has a well-known anti-microbial effect, which is in part due to the many different carbohydrate structures expressed. This renders it a position as a potential therapeutic for treatment of infection by different pathogens, thus avoiding the drawbacks of many antibiotics. The plethora of carbohydrate epitopes in breast-milk is known to differ between species, with human milk expressing the most complex one. We have investigated the expression of protein-bound carbohydrate epitopes in milk from man, cow, goat, sheep, pig, horse, dromedary and rabbit. Proteins were separated by SDS-PAGE and the presence of carbohydrate epitopes on milk proteins were analysed by Western blotting using different lectins and carbohydrate-specific antibodies. We show that ABH, Lewis (Le)x, sialyl-Lex, Lea, sialyl-Lea and Leb carbohydrate epitopes are expressed mainly on man, pig and horse milk proteins. The blood group precursor structure H type 1 is expressed in all species investigated, while only pig, dromedary and rabbit milk proteins carry H type 2 epitopes. These epitopes are receptors for Helicobacter pylori (Leb and sialyl-Lex), enteropathogenic (H type 1, Lea and Lex) and enterotoxic Escherichia coli (heat-stable toxin; H type 1 and 2), and Campylobacter jejuni (H type 2). Thus, milk from these animals or their genetically modified descendants could have a therapeutic effect by inhibiting pathogen colonization and infection.
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| 15. |
- Hallén, Ulrika, et al.
(författare)
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Glycolipid binding epitopes involved in adherence of the periodontitis-associated bacterium Porphyromonas gingivalis.
- 2008
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Ingår i: Glycoconjugate journal. - : Springer Science and Business Media LLC. - 0282-0080 .- 1573-4986. ; 25:6, s. 561-572
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Tidskriftsartikel (refereegranskat)abstract
- The ability of the periodontal pathogen Porphyromonas gingivalis to use different glycolipid structures as receptors has previously been demonstrated. The bacterium adhered to acid and nonacid glycolipids originating from human organs and to nonacid glycolipids of porcine origin. The aim of the present study was to analyze these binding epitopes by structural characterization. Glycolipid fractions with positive bacterial binding from e.g. human and porcine origin, were purified by the high performance liquid chromatography technique and thereafter used in bacterial overlay assays with (35)S-labeled P. gingivalis. Purified fractions with positive binding were structurally characterized by proton nuclear magnetic resonance spectroscopy. Complementing thin-layer chromatograms and bacterial overlay assays with pure reference glycolipid fractions and competition experiments with lactose were performed to define potential receptors. The P. gingivalis binding epitopes, including cerebrosides with nonhydroxy fatty acids, lactosylceramide with hydroxy fatty acids, sulfatides, lacto-, neolacto- and gangliotetraosylceramides, are in several instances similar to those found for other bacteria, e.g. H. pylori, H. influenzae and N. meningitidis. In addition P. gingivalis also bound to the Galalpha4Gal epitope of the globo series of glycolipids. In the future these results may be valuable for development of new treatment strategies, such as anti-adhesion therapies and vaccines specifically directed against P. gingivalis infection.
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| 16. |
- Harrison, Amanda L., et al.
(författare)
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A synthetic globotriaosylceramide analogue inhibits HIV-1 infection in vitro by two mechanisms
- 2010
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Ingår i: Glycoconjugate Journal. - : Springer Science and Business Media LLC. - 1573-4986 .- 0282-0080. ; 27:5, s. 515-524
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Tidskriftsartikel (refereegranskat)abstract
- Previously, it was shown that the cell-membrane-expressed glycosphingolipid, globotriaosylceramide (Gb(3)/P-k/CD77), protects against HIV-1 infection and may be a newly described natural resistance factor against HIV infection. We have now investigated the potential of a novel, water soluble, non-toxic and completely synthetic analogue of Gb(3)/P-k (FSL-Gb(3)) to inhibit HIV-1 infection in vitro. A uniquely designed analogue, FSL-Gb(3), of the natural Gb(3)/P-k molecule was synthesized. HIV-1(IIIB) (X4 virus) and HIV-1(Ba-L) (R5 virus) infection of PHA/interleukin-2-activated, peripheral blood mononuclear cells (PBMCs) and Jurkat T cells in vitro was assessed, as well as infection of U87.CD4.CCR5 by various clinical R5 tropic viruses after treatment with FSL-Gb(3). We monitored Gb(3), CD4 and CXCR4 expression by fluorescent antibody cell sorting and viral replication by p24 (gag) ELISA. Total cellular Gb(3) was examined by glycosphingolipid extraction and thin layer chromatography. In vivo toxicity was monitored in mice by histological assessment of vital organs and lymphoid tissue. FSL-Gb(3) blocked X4 and R5 of both lab and clinical viral strains in activated PBMCs or the U87.CD4.CCR5 cell line with a 50% inhibitory concentration (IC50) of approximately 200-250 mu M. FACS and TLC overlay showed that FSL-Gb(3) can insert itself into cellular plasma membranes and that cellular membrane-absorbed FSL-Gb(3) is able to inhibit subsequent HIV-1 infection. There was no effect of FSL-Gb(3) on cell surface levels of CD4 or CXCR4. Thus, FSL-Gb(3) can inhibit HIV-1 by two mechanisms: direct inhibition of virus and inhibition of viral entry. Infusion of FSL-Gb(3) into laboratory mice at doses well in excess of theoretical therapeutic doses was tolerated with no untoward reactions. Our results demonstrate the potential utility of using a completely synthetic, water soluble globotriaosylceramide analogue, FSL-Gb(3), having low toxicity, for possible future use as a novel therapeutic approach for the systemic treatment of HIV/AIDS.
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| 17. |
- Hoja-Łukowicz, Dorota, et al.
(författare)
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L1CAM from human melanoma carries a novel type of N-glycan with Galβ1-4Galβ1- motif : Involvement of N-linked glycans in migratory and invasive behaviour of melanoma cells
- 2013
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Ingår i: Glycoconjugate Journal. - : Springer Netherlands. - 0282-0080 .- 1573-4986. ; 30:3, s. 205-225
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Tidskriftsartikel (refereegranskat)abstract
- Dramatic changes in glycan biosynthesis during oncogenic transformation result in the emergence of marker glycans on the cell surface. We analysed the N- linked glycans of L1CAM from different stages of melanoma progression, using high-performance liquid chromatography combined with exoglycosidase sequencing, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and lectin probes. L1CAM oligosaccharides are heavily sialylated, mainly digalactosylated, biantennary complex-type structures with galactose β1-4/3-linked to GlcNAc and with or without fucose α1-3/6-linked to GlcNAc. Hybrid, bisected hybrid, bisected triantennary and tetraantennary complex oligosaccharides, and β1-6-branched complex-type glycans with or without lactosamine extensions are expresses at lower abundance. We found that metastatic L1CAM possesses only α2-6-linked sialic acid and the loss of α2-3-linked sialic acid in L1CAM is a phenomenon observed during the transition of melanoma cells from VGP to a metastatic stage. Unexpectedly, we found a novel monoantennary complex-type oligosaccharide with a Galβ1-4Galβ1- epitope capped with sialic acid residues A1[3]G(4)2S 2-3 . To our knowledge this is the first report documenting the presence of this oligosaccharide in human cancer. The novel and unique N- glycan should be recognised as a new class of human melanoma marker. In functional tests we demonstrated that the presence of cell surface α2-3-linked sialic acid facilitates the migratory behaviour and increases the invasiveness of primary melanoma cells, and it enhances the motility of metastatic cells. The presence of cell surface α2-6-linked sialic acid enhances the invasive potential of both primary and metastatic melanoma cells. Complex-type oligosaccharides in L1CAM enhance the invasiveness of metastatic melanoma cells.
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| 18. |
- Ito, H., et al.
(författare)
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Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines
- 2016
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Ingår i: Glycoconjugate Journal. - : Springer Science and Business Media LLC. - 0282-0080 .- 1573-4986. ; 33:3, s. 405-415
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Tidskriftsartikel (refereegranskat)abstract
- The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.
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| 19. |
- Jansson, Lena, 1979, et al.
(författare)
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No direct binding of the heat-labile enterotoxin of Escherichia coli to E. coli lipopolysaccharides.
- 2009
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Ingår i: Glycoconjugate journal. - : Springer Science and Business Media LLC. - 1573-4986 .- 0282-0080.
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Tidskriftsartikel (refereegranskat)abstract
- A novel carbohydrate binding site recognizing blood group A and B determinants in a hybrid of cholera toxin and Escherichia coli heat-labile enterotoxin B-subunits (termed LCTBK) has previously been described, and also the native heat-labile enterotoxin bind to some extent to blood group A/B terminated glycoconjugates. The blood group antigen binding site is located at the interface of the B-subunits. Interestingly, the same area of the B-subunits has been proposed to be involved in binding of the heat-labile enterotoxin to lipopolysaccharides on the bacterial cell surface. Binding of the toxin to lipopolysaccharides does not affect the GM1 binding capacity. The present study aimed at characterizing the relationship between the blood group A/B antigen binding site and the lipopolysaccharide binding site. However, no binding of the B-subunits to E. coli lipopolysaccharides in microtiter wells or on thin-layer chromatograms was obtained. Incubation with lipopolysaccharides did not affect the binding of the B-subunits of heat-labile enterotoxin of human isolates to blood group A-carrying glycosphingolipids, indicating that the blood group antigen site is not involved in LPS binding. However, the saccharide competition experiments showed that GM1 binding reduced the affinity for blood group A determinants and vice versa, suggesting that a concurrent occupancy of the two binding sites does not occur. The latter finding is related to a connection between the blood group antigen binding site and the GM1 binding site through residues interacting with both ligands.
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| 20. |
- Jin, Chunsheng, et al.
(författare)
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Identification by mass spectrometry and immunoblotting of xenogeneic antigens in theN- andO-glycomes of porcine, bovine and equine heart tissues
- 2020
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Ingår i: Glycoconjugate Journal. - : Springer Science and Business Media LLC. - 0282-0080 .- 1573-4986. ; 37, s. 485-498
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Tidskriftsartikel (refereegranskat)abstract
- Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which alpha-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins.N-glycans were released by PNGase F digestion andO-glycans by beta-elimination. Released oligosaccharides were analyzed by liquid chromatography - tandem mass spectrometry. In total, 102N-glycans and 40O-glycans were identified in animal heart tissue lysates. TheN- andO-glycan patterns were different between species. alpha-Gal and Neu5Gc were identified on bothN- andO-linked glycans,N,N '-diacetyllactosamine (LacdiNAc) onN-glycans only and sulfatedO-glycans. The relative amounts of alpha-Gal-containingN-glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying alpha-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylatedO-glycans, and bovine pericardium the highest level of Neu5Gc-sialylatedN-glycans. The identifiedN-andO-linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.
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| 21. |
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| 22. |
- Lahmann, Martina, 1963, et al.
(författare)
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Synthesis of the Lewis b hexasaccharide and HSA-conjugates thereof.
- 2004
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Ingår i: Glycoconjugate journal. - 0282-0080 .- 1573-4986. ; 21:5, s. 251-6
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Tidskriftsartikel (refereegranskat)abstract
- An efficient and short route has been elaborated for the aminopropyl spacer equipped Leb hexasaccharide. For the preparation of HSA-conjugates of this oligosaccharide, the use of disuccinimidyl suberate (DSS) and disuccinimidyl glutarate (DSG) as cross-linker reagents has been evaluated. This conjugation method emerged as being faster and easier to monitor by standard MALDI-TOF spectrometry than squarate ester based conjugations of similar efficiency if DSS is used as cross-linker.
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| 23. |
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| 24. |
- Landström, Jens, et al.
(författare)
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Small molecules containing hetero-bicyclic ring systems compete with UDP-Glc for binding to WaaG glycosyltransferase
- 2012
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Ingår i: Glycoconjugate Journal. - : Springer. - 0282-0080 .- 1573-4986. ; 29:7, s. 491-502
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Tidskriftsartikel (refereegranskat)abstract
- The α-1,3-glucosyltransferase WaaG is involved in the synthesis of the core region of lipopolysaccharides in E. coli. A fragment-based screening for inhibitors of the WaaG glycosyltrasferase donor site has been performed using NMR spectroscopy. Docking simulations were performed for three of the compounds of the fragment library that had shown binding activity towards WaaG and yielded 3D models for the respective complexes. The three ligands share a hetero-bicyclic ring system as a common structural motif and they compete with UDP-Glc for binding. Interestingly, one of the compounds promoted binding of uridine to WaaG, as seen from STD NMR titrations, suggesting a different binding mode for this ligand. We propose these compounds as scaffolds for the design of selective high-affinity inhibitors of WaaG. Binding of natural substrates, enzymatic activity and donor substrate selectivity were also investigated by NMR spectroscopy. Molecular dynamics simulations of WaaG were carried out with and without bound UDP and revealed structural changes compared to the crystal structure and also variations in flexibility for some amino acid residues between the two WaaG systems studied.
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| 25. |
- Lidholt, Kerstin, et al.
(författare)
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Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma.
- 1997
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Ingår i: Glycoconjugate Journal. - 0282-0080 .- 1573-4986. ; 14:6, s. 737-742
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Tidskriftsartikel (refereegranskat)abstract
- Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and GlcA beta 1-3Gal(4-O-sulfate)beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, were tested as hexosamine accepters, using UDP-[H-3]GlcNAc and UDP-[H-3]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with alpha-N-acetylgalactosaminidase and beta-N-acetylhexosaminidase revealed that the [H-3]GalNAc unit was alpha-linked, as in the product previously synthesized using serum enzymes, and not beta-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [H-3]GlcNAc from UDP-[H-3]GlcNAc could be detected. By contrast, transfer of a [H-3]GlcNAc unit to a [GlcA beta 1-4GlcNAca1-4](2)-GlcA beta 1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as accepters for the first HexNAc transfer reactions involved in the formation of these polysaccharides.
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