| 1. |
- Akatsu, Chizuru, et al.
(författare)
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Dermatan sulfate epimerase 2 is the predominant isozyme in the formation of the chondroitin sulfate/dermatan sulfate hybrid structure in postnatal developing mouse brain
- 2011
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 21:5, s. 565-574
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Tidskriftsartikel (refereegranskat)abstract
- Chondroitin sulfate (CS) and dermatan sulfate (DS) are expressed in significant amounts in the brain and play important roles in the development of the central nervous system in mammals. CS and DS structures are often found in a single CS/DS hybrid chain. The L-iduronic acid (IdoA)-containing domain, which defines a DS-type domain, appears key to the biological functions of the CS/DS hybrid chain. In this study, to clarify the distribution of the DS-type structure in the brain during development, the expression patterns of DS epimerase 1 (DS-epi1) and DS-epi2, both of which convert D-glucuronic acid into IdoA, were investigated by in situ hybridization. DS-epi2 was ubiquitously expressed in the developing brain after birth, whereas the expression of DS-epi1 was faint and obscure at all developmental stages. Quantitative real-time polymerase chain reaction revealed the expression of DS-epi2 to be higher than that of DS-epi1 throughout development, suggesting that DS-epi2 but not DS-epi1 is mostly expressed in the brain and plays key roles in the epimerization of CS/DS during its biosynthesis. Moreover, an analysis of the disaccharides of CS/DS demonstrated significant amounts of IdoA-containing iD units [IdoA(2S)-GalNAc(6S)] and iB units [IdoA(2S)-GalNAc(4S)], where 2S, 4S and 6S stand for 2-O-, 4-O- and 6-O-sulfate, respectively, in every region of the brain examined. The proportion of these units in cerebellar CS/DS was greatly altered during postnatal development. These results suggest that the IdoA-containing structures in the developing brain are mainly produced by the actions of DS-epi2 and play crucial roles in postnatal development.
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| 2. |
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| 3. |
- Allahgholi, Leila, et al.
(författare)
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Exploring a novel β-1,3-glucanosyltransglycosylase, MlGH17B, from a marine Muricauda lutaonensis strain for modification of laminari-oligosaccharides
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Ingår i: Glycobiology. - 1460-2423.
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Tidskriftsartikel (refereegranskat)abstract
- The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4 °C and 73.2 °C, but an activity optimum at 20 °C, indicating temperature sensitive active site interactions. MlGH17B uses β-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and + 2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming β-1,3 glucan acceptor, making a β-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several β-1,6-linked branches. The modelled structure revealed an active site comprising five subsites: three glycone (-3, -2 and - 1) and two aglycone (+1 and + 2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-β-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-β-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modelled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny.
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| 4. |
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| 5. |
- Andersson, Rolf, et al.
(författare)
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Structures of two novel, serologically nonrelated core oligosaccharides of Yokenella regensburgei lipopolysaccharides differing only by a single hexose substitution
- 2010
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 20, s. 207-214
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Tidskriftsartikel (refereegranskat)abstract
- Immunochemical analysis of the Yokenella regensburgei lipopolysaccharides (LPS) indicated the presence of the core oligosaccharide-related immunotypes among the investigated strains. The structure of the core oligosaccharide segment of the Y. regensburgei LPS has been investigated using chemical methods, mass spectrometry, and (1)H, (13)C NMR spectroscopy. It was concluded that the core oligosaccharides of the strains PCM 2476 and PCM 2477 are composed of an undecasaccharide. The combined data revealed two immunotypes of the core oligosaccharide recognized by antibodies against the whole bacterial cells. The structural differences between the core oligosaccharides are limited to the outermost terminal hexopyranose residue. In the core oligosaccharide of the strain PCM 2476, it was identified as alpha-D-Glcp and in that of the strain PCM 2477 as alpha-D-Galp. This subtle difference between the glycoforms of the LPS core appeared to be essential for formation of the epitopes recognized by the specific antibodies directed against the Y. regensburgei whole bacterial cells. The oligosaccharides are not substituted by phosphate groups. Instead, the carboxyl groups of Kdo and galacturonic acid residues present in the core provide the negative charges. The undecasaccharides represent a novel core type of bacterial LPS, which is characteristic for Y. regensburgei.
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| 6. |
- Andersson Sjöland, Annika, et al.
(författare)
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Versican in inflammation and tissue remodelling: the impact on lung disorders.
- 2015
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 25:3, s. 243-251
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Forskningsöversikt (refereegranskat)abstract
- Versican is a proteoglycan that has many different roles in tissue homeostasis and inflammation. The biochemical structure is comprised of four different types of the core protein with attached glycosaminoglycans that can be sulphated to various extents and has the capacity to regulate differentiation of different cell types, migration, cell adhesion, proliferation, tissue stabilization and inflammation. Versican's regulatory properties are of importance during both homeostasis and changes that lead to disease progression. The glycosaminoglycans that are attached to the core protein are of the chondroitin sulfate/dermatan sulfate type and are known to be important in inflammation through interactions with cytokines and growth factors. For a more complex understanding of versican it is of importance to study the tissue niche, where the wound healing process in both healthy and diseased conditions take place. In previous studies our group has identified changes in the amount of the multifaceted versican in chronic lung disorders such as asthma, chronic obstructive pulmonary disease and bronchiolitis obliterans syndrome, which could be a result of pathologic, transforming growth factor β driven, on-going remodelling processes. Reversely, the context of versican in its niche is of great importance since versican has been reported to have a beneficial role in other contexts e.g. emphysema. Here we explore the vast mechanisms of versican in healthy lung and in lung disorders.
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| 7. |
- Ara, Kazi Zubaida Gulshan, et al.
(författare)
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Engineering CGTase to improve synthesis of alkyl glycosides
- 2021
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 31:5, s. 603-612
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Tidskriftsartikel (refereegranskat)abstract
- Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyse the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase CspCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype CspCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties.
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| 8. |
- Arike, Liisa, et al.
(författare)
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Intestinal Muc2 mucin O-glycosylation is affected by microbiota and regulated by differential expression of glycosyltranferases
- 2017
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 27:4, s. 318-328
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Tidskriftsartikel (refereegranskat)abstract
- Intestinal cells are covered by mucus. In the small intestine, a single unattached mucus is present whereas the colon has both an inner attached mucus layer and an outer loose mucus. The attached mucus of the colon is impenetrable to bacteria while the loose mucus acts as a habitat for commensal bacteria. In germ-free (GF) mice, small intestinal mucus is attached to the epithelium and the inner colon mucus is penetrable. O-glycosylation plays an important role in the host-microbiota interactions as the commensal bacteria use glycans as nutrient sources and attachment sites. While mucus protein composition is relatively homogenous along the intestine, its main component the Muc2 mucin shows regiospecific O-glycan patterns. We have now analyzed the glycosyltransferase relative concentrations in the epithelial cells along the intestine in GF and conventionally raised mice and compared this with the O-glycans formed. As Muc2 is the main O-glycosylated product in mucus, we made the simplified assumption that most of the glycosyltransferases found in the epithelial cells are involved in Muc2 O-glycan biosynthesis. The O-glycosyltransferase abundances along the intestine correlated well with the Muc2 O-glycan patterns. Some of the glycosyltransferases involved in the O-glycan elongation were decreased in GF mice, something that is in concordance with the observed shorter Muc2 O-glycans.
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| 9. |
- Asplund, Annika, 1979, et al.
(författare)
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Hypoxic regulation of secreted proteoglycans in macrophages.
- 2010
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 20:1, s. 33-40
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Tidskriftsartikel (refereegranskat)abstract
- Macrophages are prominent in hypoxic areas of atherosclerotic lesions, and their secreted proteoglycans (PG), such as versican, can modulate the retention of lipoproteins and the activity of enzymes, cytokines, and growth factors involved in atherogenesis. In this study, we report the effects of hypoxia on PG secreted by human monocyte-derived macrophages (HMDM) and the potential regulation by the transcription factor hypoxia-inducible factor (HIF-1alpha and HIF-2alpha). We found that versican co-localized with HIF-1alpha in macrophage-rich areas in human advanced atherosclerotic lesions. Versican and perlecan mRNA expression increased after exposure to 0.5% O(2) (hypoxia) compared with 21% O(2) (control cells). Using precursors to GAG biosynthesis combined with immunoabsorption with a versican antibody an increased versican synthesis was detected at hypoxia. Furthermore, siRNA knockdown of HIF-1alpha and HIF-2alpha in THP-1 cells showed that the hypoxic induction of versican and perlecan mRNA expression involved HIF signaling. Versican expression was co-regulated by HIF-1alpha and HIF-2alpha but expression of perlecan was influenced only by HIF-1alpha and not by HIF-2alpha knockdown. The results show that oxygen concentration is an important modulator of PG expression in macrophages. This may be a novel component of the complex role of macrophages in atherosclerosis.
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| 10. |
- Axelsson, Magnus A. B., et al.
(författare)
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Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins.
- 2001
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 11:8, s. 633-44
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Tidskriftsartikel (refereegranskat)abstract
- Addition of the weak base ammonium chloride (NH4Cl) or the proton pump inhibitor bafilomycin A1 to cultured HeLa and LS 174T cells effectively neutralized the pH gradient of the secretory pathway. This resulted in relocalization of the three studied glycosyltransferases, N-acetylgalactosaminyltransferase 2, beta1,2 N-acetylglucosaminyltransferase I, and beta1,4 galactosyltransferase 1, normally localized to the Golgi stack, the medial/trans-Golgi and the trans-Golgi/TGN, respectively. Indirect immunofluorescence microscopy, immunoelectron microscopy, and subcellular fractionation of the tagged or native glycosyltransferases showed that NH4Cl caused a relocalization of the enzymes mainly to vesicles of endosomal type, whereas bafilomycin A1 gave mainly cell surface staining. The general morphology of the endoplasmic reticulum and Golgi apparatus was retained as judged from immunofluorescence and electron microscopy studies. When the O-glycans on the guanidinium chloride insoluble gel-forming mucins from the LS 174T cells were analyzed by gas chromatography-mass spectrometry after neutralization of the secretory pathway pH by NH4Cl over 10 days shorter O-glycans were observed. However, no decrease in the number of oligosaccharide chains was indicated. Together, the results suggest that pH is a contributing factor for proper steady-state distribution of glycosyltransferases over the Golgi apparatus and that altered pH may cause alterations in glycosylation possibly due to a relocalization of glycosyltransferases.
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| 11. |
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| 12. |
- Bartolini, Barbara, et al.
(författare)
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Mouse development is not obviously affected by the absence of dermatan sulfate epimerase 2 in spite of a modified brain dermatan sulfate composition.
- 2012
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 22:7, s. 1007-1016
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Tidskriftsartikel (refereegranskat)abstract
- Dermatan sulfate epimerase 2 (DS-epi2), together with its homologue DS-epi1, transform glucuronic acid into iduronic acid in dermatan sulfate polysaccharide chains. Iduronic acid gives dermatan sulfate increased chain flexibility and promotes protein binding. DS-epi2 is ubiquitously expressed and is the predominant epimerase in brain. Here we report the generation and initial characterization of DS-epi2 null mice. DS-epi2 deficient mice showed no anatomical, histological or morphological abnormalities. The body weights and lengths of mutated and wild-type littermates were indistinguishable. They were fertile and had a normal lifespan. Chondroitin/dermatan sulfate (CS/DS) isolated from newborn mutated mouse brains had a 38% reduction in iduronic acid compared to wild type littermates and compositional analysis revealed a decrease of 4-O-sulfate and an increase of 6-O-sulfate containing structures. Despite the reduction in iduronic acid, adult DS-epi2-/- brain showed normal extracellular matrix features by immunohistological stainings. We conclude that DS-epi1 compensates in vivo for the loss of DS-epi2.. These results extend previous findings of functional redundancy of brain extracellular matrix components.
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| 13. |
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| 14. |
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| 15. |
- Bleuler-Martinez, S, et al.
(författare)
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Structure-function relationship of a novel fucoside-binding fruiting body lectin from Coprinopsis cinerea exhibiting nematotoxic activity
- 2022
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 32:7, s. 600-615
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Tidskriftsartikel (refereegranskat)abstract
- Lectins are non-immunoglobulin-type proteins that bind to specific carbohydrate epitopes and play important roles in intra- and inter-organismic interactions. Here, we describe a novel fucose-specific lectin, termed CML1, which we identified from fruiting body extracts of Coprinopsis cinerea. For further characterization, the coding sequence for CML1 was cloned and heterologously expressed in Escherichia coli. Feeding of CML1-producing bacteria inhibited larval development of the bacterivorous nematode Caenorhabditis tropicalis, but not of C. elegans. The crystal structure of the recombinant protein in its apo-form and in complex with H type I or Lewis A blood group antigens was determined by X-ray crystallography. The protein folds as a sandwich of 2 antiparallel β-sheets and forms hexamers resulting from a trimer of dimers. The hexameric arrangement was confirmed by small-angle X-ray scattering (SAXS). One carbohydrate-binding site per protomer was found at the dimer interface with both protomers contributing to ligand binding, resulting in a hexavalent lectin. In terms of lectin activity of recombinant CML1, substitution of the carbohydrate-interacting residues His54, Asn55, Trp94, and Arg114 by Ala abolished carbohydrate-binding and nematotoxicity. Although no similarities to any characterized lectin were found, sequence alignments identified many non-characterized agaricomycete proteins. These results suggest that CML1 is the founding member of a novel family of fucoside-binding lectins involved in the defense of agaricomycete fruiting bodies against predation by fungivorous nematodes.
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| 16. |
- Breimer, Michael, 1951, et al.
(författare)
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Glycosphingolipid composition of epithelial cells isolated along the villus axis of small intestine of a single human individual
- 2012
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 22:12, s. 1721-1730
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Tidskriftsartikel (refereegranskat)abstract
- A 6-cm fresh proximal ileum surgical specimen from a blood group A(1)Le(a-b+) secretor individual was used for stepwise isolation of epithelial cells from villus tip to crypt bottom by gentle washing with ethylenediaminetetraacetic acid-containing buffer. Acid and non-acid sphingolipids were prepared from the epithelial cell fractions and the non-epithelial intestinal residue. Molecular information on the sphingolipid composition was obtained without further isolation of individual species by applying thin-layer chromatography using chemical and biological (monoclonal antibodies, cholera toxin, Escherichia coli) detection reagents, mass spectrometry and proton NMR spectroscopy of derivatized glycolipids. In this way, the structure of major and minor saccharides, ceramide components and their relative amounts were obtained. Epithelial cells and non-epithelial residue were distinctly different in their sphingolipid composition. Sphingomyelin was the major single component in both compartments. Characteristic for epithelial cells was the dominance of monoglycosylceramides, sulphatides and blood group fucolipids (mainly Le(b) hexaglycosylceramides and ALe(b) heptaglycosylceramides). The non-epithelial residue had about five times less glycolipids mainly mono-, di-, tri- and tetra-glycosylceramides and gangliosides, including the GM1 ganglioside. The ceramides were more hydroxylated (1-2 additional hydroxyls) in epithelial cell glycolipids compared with the non-epithelial residue. Combined with a separate detailed study on the glycoproteins of the same epithelial cell preparation, this human intestinal sample is the only epithelial cell preparation where both protein- and lipid-linked saccharides are characterized in detail.
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| 17. |
- Broeker, N. K., et al.
(författare)
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Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity
- 2013
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 23:1, s. 59-68
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 18. |
- Bäcker, Annika E., 1965, et al.
(författare)
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Biochemical and enzymatic characterization of blood group ABH and related histo-blood group glycosphingolipids in the epithelial cells of porcine small intestine.
- 1997
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 7:7, s. 943-53
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Tidskriftsartikel (refereegranskat)abstract
- Non-acid glycosphingolipids were isolated from small intestinal epithelial cells of a single blood group A pig. One very predominant blood group compound was obtained chemically pure upon HPLC fractionation. It was characterized by mass spectrometry and 1H NMR spectroscopy to be the type 1 chain blood group A hexaglycosylceramide. Support for the presence of minute amounts of additional A glycolipids was obtained by mass spectrometry and immunostaining of TLC plates with anti-A antibodies specific for A type 2 chain, A type 3 and 4 chain, and the ALe(b) determinant. Among precursor chains, globoside (type 4) and lactotetraosylceramide (type 1) were immunologically identified, whereas no neolactotetraosylceramide (type 2) and gangliotetraosylceramide reactivities were detected. We addressed the question whether the predominant expression of type 1 chain based A glycolipids reflects a restricted glycolipid precursor chain specificity of the alpha 1-2 fucosyl- and/or the alpha 1-3 N-acetylgalactosaminyltransferases, or if the biosynthesis of the precursor chains themselves is regulated. All precursor core saccharides, lacto- (type 1), neolacto-(type 2), and gangliotetraosylceramide as well as globopentaosylceramide (type 4), could serve as acceptors for fucose in vitro when a crude microsomal fraction obtained from mechanically released, porcine intestinal epithelial cells was used as an enzyme source. Under the same conditions an N-acetylgalactosamine residue could be transferred to the blood group H structures based on these core saccharide chains. Lactotriaosylceramide, but not gangliotriaosylceramide, could serve as an acceptor for UDP-galactose. When the product was digested with beta-galactosidase (EC 3.2.1.23) from S.pneumoniae, under conditions where it specifically cleaves Gal beta 1-4 residues, approximately 40% of the radioactivity was cleaved off, indicating that a substantial amount of neolactotetraosylceramide was made in vitro, as opposed to the predominance of lactotetraosylceramide-based structures found in vivo.
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| 19. |
- Carlsson, Susanne, et al.
(författare)
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Affinity of galectin-8 and its carbohydrate recognition domains for ligands in solution and at the cell surface.
- 2007
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 17:6, s. 663-76
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Tidskriftsartikel (refereegranskat)abstract
- Galectin-8 has two different carbohydrate recognition domains (CRDs), the N-terminal Gal-8N and the C-terminal Gal-8C linked by a peptide, and has various effects on cell adhesion and signaling. To understand the mechanism for these effects further, we compared the binding activities of galectin-8 in solution with its binding and activation of cells. We used glycan array analysis to broaden the specificity profile of the two galectin-8 CRDs, as well as intact galectin-8s (short and long linker), confirming the unique preference for sulfated and sialylated glycans of Gal-8N. Using a fluorescence anisotropy assay, we examined the solution affinities for a subset of these glycans, the highest being 50 nM for NeuAcalpha2,3Lac by Gal-8N. Thus, carbohydrate-protein interactions can be of high affinity without requiring multivalency. More importantly, using fluorescence polarization, we also gained information on how the affinity is built by multiple weak interactions between different fragments of the glycan and its carrier molecule and the galectin CRD subsites (A-E). In intact galectin-8 proteins, the two domains act independently of each other in solution, whereas at a surface they act together. Ligands with moderate or weak affinity for the isolated CRDs on the array are bound strongly by intact galectin-8s. Also galectin-8 binding and signaling at cell surfaces can be explained by combined binding of the two CRDs to low or medium affinity ligands, and their highest affinity ligands, such as sialylated galactosides, are not required.
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| 20. |
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| 21. |
- Cheng, Fang, et al.
(författare)
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Complex modulation of cytokine-induced α-synuclein aggregation by glypican-1-derived heparan sulfate in neural cells
- 2022
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 32:4, s. 333-342
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Tidskriftsartikel (refereegranskat)abstract
- In Parkinson's disease (PD), there is accumulation of α-synuclein (SYN) aggregates in neurons, which is promoted by neuroinflammation. The cytokines TNF-α, IL-1β and IL-6 induce accumulation of degradation products of the amyloid precursor protein (APP) combined with heparan sulfate (HS) chains released from glypican-1 (Gpc-1) by NO-dependent cleavage. We have investigated the effects of the cytokines and HS on SYN aggregation and secretion in dividing human neuroblastoma (SH-SY5Y) and inducible neural progenitor cells (NPC) by using immunofluorescence microscopy, vesicle isolation and slot blotting with antibodies recognizing SYN monomers and aggregates, Gpc-1, the released HS, endosomes, and autophagosomes. In SH-SY5Y cells, the capacity to release HS was fully utilized, while NPC displayed dormant capacity. TNF-α induced increased formation of SYN aggregates and clustering of HS in SH-SY5Y cells. When the supply of NO was simultaneously increased, SYN and HS accumulation disappeared. When NO formation was inhibited, SYN and HS aggregation also disappeared, but there was now a 4-fold increase in SYN secretion. In NPC, IL-6 induced increased aggregation of SYN and stimulated HS release from Gpc-1. Both SYN and HS co-localized with autophagosome marker. When HS-deficient Gpc-1 was simultaneously generated, by using a cyanobacterial neurotoxin, accumulation diminished and there was massive secretion of SYN. We suggest that the cytokines increase APP processing, which initiates NO-dependent release of HS from Gpc-1. The APP degradation products also trigger SYN aggregation. As HS can inhibit APP processing, HS-or NO-deficiency may result in autophagosomal dysfunction and both APP degradation products and SYN are secreted.
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| 22. |
- Cheng, Fang, et al.
(författare)
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Hypoxia induces NO-dependent release of heparan sulfate in fibroblasts from the Alzheimer mouse Tg2576 by activation of nitrite reduction.
- 2016
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 26:6, s. 623-634
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Tidskriftsartikel (refereegranskat)abstract
- There is a functional relationship between the heparan sulfate proteoglycan glypican-1 and the amyloid precursor protein of Alzheimer disease. In wild-type mouse embryonic fibroblasts, expression and processing of the amyloid precursor protein is required for endosome-to-nucleus translocation of anhydromannose-containing heparan sulfate released from S-nitrosylated glypican-1 by ascorbate-induced, nitrosothiol-catalyzed deaminative cleavage. In fibroblasts from the transgenic Alzheimer mouse Tg2576 there is increased processing of the amyloid precursor protein to amyloid-β peptides. Simultaneously, there is spontaneous formation of anhydromannose-containing heparan sulfate by an unknown mechanism. We have explored the effect of hypoxia on anhydromannose-containing heparan sulfate formation in wild-type and Tg2576 fibroblasts by deconvolution immunofluorescence microscopy and flow cytometry using an anhydromannose-specific monoclonal antibody and by (35)SO4-labeling experiments. Hypoxia prevented ascorbate-induced heparan sulfate release in wild-type fibroblasts, but induced an increased formation of anhydromannose-positive and (35)S-labeled heparan sulfate in Tg2576 fibroblasts. This appeared to be independent of glypican-1 S-nitrosylation as demonstrated by using a monoclonal antibody specific for S-nitrosylated glypican-1. In hypoxic wild-type fibroblasts, addition of nitrite to the medium restored anhydromannose-containing heparan sulfate formation. The increased release of anhydromannose-containing heparan sulfate in hypoxic Tg2576 fibroblasts did not require addition of nitrite. However, it was suppressed by inhibition of the nitrite reductase activity of xanthine oxidoreductase/aldehyde oxidase or by inhibition of p38 mitogen-activated protein kinase or by chelation of iron. We propose that normoxic Tg2576 fibroblasts maintain a high level of anhydromannose-containing heparan sulfate production by a stress-activated generation of nitric oxide from endogenous nitrite. This activation is enhanced by hypoxia.
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| 23. |
- Cheng, Fang, et al.
(författare)
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Interplay between APP and glypican-1 processing and α-synuclein aggregation in undifferentiated and differentiated human neural progenitor cells
- 2023
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 33:4, s. 325-341
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Tidskriftsartikel (refereegranskat)abstract
- In Parkinson’s disease, there is an accumulation of α-synuclein (SYN) aggregates in neurons, which is promoted by neuroinflammation. In neural cells, cytokine-induced SYN aggregation is modulated by heparan sulfate (HS) derived from glypican-1 (GPC1) by amyloid precursor protein (APP) and nitric oxide (NO)-dependent cleavage. We have explored possible interplay between APP, GPC1, and SYN in undifferentiated and differentiated neural progenitor cells (NPCs) by modulating APP and GPC1 processing. Effects were monitored by immunofluorescence microscopy and slot immunoblotting using antibodies recognizing APP degradation products, HS released from GPC1, and SYN aggregates (filamentous SYN [SYNfil]). Suppression of HS release from GPC1 by inhibition of β-secretase or by NO deprivation resulted in no or slight increase in SYNfil aggregation. Stimulation of HS release by ascorbate did not further increase SYNfil staining. Interleukin-6 (IL-6) induced increased APP and GPC1 processing and SYNfil formation, which was reduced when βsecretase was inhibited and when HS release was impeded by NO deprivation. Ascorbate restored APP and GPC1 processing but did not affect SYNfil formation. Ascorbate-dependent differentiation of NPC resulted in the expression of tyrosine hydroxylase (TH) which colocalized with SYNfil. Suppression of APP processing by inhibition of β-secretase greatly disturbed the differentiation process. IL-6 induced coclustering of APP-degradation products, TH, HS, and SYNfil, which could be reversed by stimulation of HS release from GPC1 by excess ascorbate. We suggest that continuous release of HS from GPC1 moderates SYN aggregation and supports differentiation of NPC to dopaminergic neurons.
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| 24. |
- Cheng, Fang, et al.
(författare)
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Non-conserved, S-nitrosylated cysteines in glypican-1 react with N-unsubstituted glucosamines in heparan sulfate and catalyze deaminative cleavage.
- 2012
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 22:11, s. 1480-1485
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Tidskriftsartikel (refereegranskat)abstract
- The membrane lipid-anchored glypicans (heparan sulfate proteoglycans) are present in both vertebrates and invertebrates and serve as important modulators of growth factors and morphogens during development. Their core proteins are similar and consist of a large N-terminal domain comprising 14 evolutionary conserved cysteines and a C-terminal stalk carrying the heparan sulfate side-chains and the lipid anchor. Cysteines in glypican-1 can be S-nitrosylated but their positions have not been identified. The recently determined crystal structure of the N-terminal domain of glypican-1 has revealed that all the evolutionary conserved cysteines form intramolecular disulfide bonds. However, glypican-1 contains two more, non-conserved cysteines in the C-terminal stalk, located near the heparan sulfate attachment sites. We show here that the non-conserved cysteines are free thiols as a glypican-1 core protein containing the C-terminal stalk could be biotinylated by biotin-BMCC. After S-nitrosylation by using an NO-donor and copper ions, the glypican-1 core protein was retained on an affinity matrix substituted with heparan sulfate oligosaccharides containing N-unsubstituted glucosamines. The protein was displaced with 0.2 M glucosamine but also by 2 mM ascorbate. In the latter case, the heparan sulfate of the affinity matrix was simultaneously cleaved into fragments containing anhydromannose. We propose that the S-nitrosocysteine residues interact with closely located N-unsubstituted glucosamines in the heparan sulfate side-chains of the glypican-1 proteoglycan. Addition of ascorbate induces a series of reactions that eventually releases heparan sulfate fragments with reducing terminal anhydromannose, presumably without the formation of free nitric oxide.
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| 25. |
- Cheng, Fang, et al.
(författare)
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Non-toxic amyloid beta formed in the presence of glypican-1 or its deaminatively generated heparan sulfate degradation products
- 2013
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 23:12, s. 1510-1519
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Tidskriftsartikel (refereegranskat)abstract
- The amyloid beta (A beta) peptides (mainly A beta 40 and A beta 42), which are derived from the amyloid precursor protein (APP), can oligomerize into antibody A11-positive, neurotoxic species, believed to be involved in Alzheimer's disease. Interestingly, APP binds strongly to the heparan sulfate (HS) proteoglycan (PG) glypican-1 (Gpc-1) in vitro and both proteins are colocalized inside cells. In endosomes, APP is proteolytically processed to yield A beta peptides. The HS chains of S-nitrosylated (SNO) Gpc-1 PG are cleaved into anhydromannose (anMan)-containing di- and oligosaccharides by an NO-dependent reaction in the same compartments. Here, we have studied the toxicity of oligomers/aggregates of A beta 40 and A beta 42, as well as A beta 40/42 mixtures that were formed in the presence of immobilized Gpc-1 PG or immobilized HS oligosaccharides. Afterwards, A beta was displaced from the matrices, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for A11 immunoreactivity, for effects on growth of mouse N2a neuroblastoma cells and for membrane leakage in rat cortical neurons. HS generally promoted and accelerated A beta multimerization into oligomers as well as larger aggregates that were mostly A11 positive and showed toxic effects. However, non-toxic A beta was formed in the presence of Gpc-1 PG or when anMan-containing HS degradation products were simultaneously generated. Both toxic and non-toxic A beta peptides were taken up by the cells but toxic forms appeared to enter the nuclei to a larger extent. The protection afforded by the presence of HS degradation products may reflect a normal intracellular function for the A beta peptides.
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| 26. |
- Cheng, Fang, et al.
(författare)
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Patterns of uronosyl epimerization and 4-/6-O-sulphation in chondroitin/dermatan sulphate from decorin and biglycan of various bovine tissues
- 1994
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Ingår i: Glycobiology. - 1460-2423. ; 4:5, s. 685-696
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Tidskriftsartikel (refereegranskat)abstract
- Dermatan sulphate is a co-polymer of two types of disaccharide repeats: D-glucuronate-N-acetylgalactosamine and L-iduronate-N-acetylgalactosamine. The former can be O-sulphated at C-4 or C-6 of the galactosamine, whereas the latter contains almost exclusively 4-O-sulphated galactosamine. A minor proportion of the L-iduronate may be O-sulphated at C-2. Chondroitin sulphate has no L-iduronate-containing repeats. We have used our recently developed methods for sequence analysis of galactosaminoglycans to investigate the structure of dermatan/chondroitin sulphates of the proteoglycans decorin and biglycan derived from various bovine tissues, like dermis, sclera, tendon, aorta, cartilage and bone. The glycan chains, radioiodinated at the reducing end, were partially cleaved with specific enzymes (chondroitin lyases), and subjected to high-resolution polyacrylamide gel electrophoresis, blotting and autoradiography to identify fragments extending from the labelled reducing end to the point of cleavage. We used chondroitin B lyase to identify the location of L-iduronate, chondroitin AC-I lyase to locate D-glucuronate and chondroitin C lyase to cleave where D-glucuronate residues were succeeded by 6-O-sulphated N-acetylgalactosamine. We could demonstrate tissue-specific, periodic and wave-like patterns of distribution for the two epimeric uronic acids, as well as specific patterns of sulphation in dermatan sulphates derived from either decorin or biglycan. For example, some dermatan sulphates contained D-glucuronate-rich domains that were always 6-sulphated (scleral decorin), others were always 4-sulphated (decorin from bovine dermis, cartilage and bone; biglycan from aorta) or 6-sulphated near the linkage region, but 4-sulphated in more distal domains (decorin from porcine dermis and bovine tendon). Decorin from bone and articular cartilage, as well as biglycan from articular and nasal cartilage, carried largely chondroitin sulphate chains, but also some dermatan sulphate, whereas galactosaminoglycan chains derived from aggrecan of nasal cartilage were free of L-iduronate. Decorin and biglycan from the same tissue (articular cartilage or sclera) had similar glycan chains. The two side chains in a biglycan molecule are probably also similar to one another. The portion of the glycan chains nearest to the core protein was substituted with charged groups to a variable degree, which may correlate with the structural features of the main chain.
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| 27. |
- Cheng, Fang, et al.
(författare)
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Proinflammatory cytokines induce accumulation of glypican-1-derived heparan sulfate and the C-terminal fragment of β-cleaved APP in autophagosomes of dividing neuronal cells
- 2020
-
Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 30:8, s. 539-549
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Tidskriftsartikel (refereegranskat)abstract
- Proinflammatory cytokines stimulate expression of β-secretase, which increases processing of amyloid precursor protein (APP), ultimately leading to the deposition of amyloid beta (Aβ). The N-terminal domain of β-cleaved APP supports Cu/NO-dependent release of heparan sulfate (HS) from the glypican-1 (Gpc-1) proteoglycan. HS is an inhibitor of β-secretase, thereby constituting a regulatory, negative feedback loop. Here, we have investigated the effect of the proinflammatory cytokines TNF-α, IL-1β and IL-6 on the interplay between APP processing and release of HS from Gpc-1 in neuronal cells. We have used deconvolution immunofluorescence microscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a panel of monoclonal/polyclonal antibodies recognizing the released HS, the N-terminus of Aβ, Aβ, the C-terminus of APP and the autophagosome marker LC3 as well as the chemical lysosome marker LysoTrackerRed (LTR). We repeatedly found that N2a neuroblastoma cells and human neural stem cells grown in the presence of the cytokines developed large cytoplasmic clusters, which stained positive for HS, the N-terminus of Aβ, Aβ, the C-terminus of APP, LC3 and LTR, indicating accumulation of HS and APP/APP degradation products in enlarged autophagosomes/lysosomes. The SDS-PAGE of immunoisolates obtained from TNF-α-treated N2a cells by using anti-C-terminus of APP revealed the presence of SDS-stable complexes between HS and the C-terminal fragment of β-cleaved APP (βCTF) migrating in the range 10-18 kDa. Clustered accumulation of βCTF disappeared when HS release was prevented and slightly enhanced when HS release was increased. Hence, when proinflammatory cytokines induce increased processing of APP, inhibition of β-secretase by HS is insufficient, which may lead to the impaired autophagosomal degradation.
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| 28. |
- Cheng, Fang, et al.
(författare)
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Rapid nuclear transit and impaired degradation of amyloid beta and glypican-1-derived heparan sulfate in Tg2576 mouse fibroblasts.
- 2015
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 25:5, s. 548-556
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Tidskriftsartikel (refereegranskat)abstract
- Anhydromannose (anMan)-containing heparan sulfate (HS) derived from S-nitrosylated glypican-1 is generated in endosomes by an endogenously or ascorbate induced S-nitrosothiol-catalyzed reaction. Expression and processing of amyloid precursor protein (APP) is required to initiate formation and endosome-to-nucleus translocation of anMan-containing HS in wild-type mouse embryonic fibroblasts (WT MEF). HS is then transported to autophagosomes and finally degraded in lysosomes. To investigate how APP-derived amyloid beta peptide (Aβ) affects intracellular trafficking of HS we have studied nuclear transit as well as autophagosome/lysosome targeting and degradation in Tg2576 MEF which produce increased amounts of Aβ. Deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody showed anMan-staining in the nuclei of Tg2576 MEF after 5 min of ascorbate treatment and after 15 min in WT MEF. There was also greater nuclear accumulation of HS in Tg2576 MEF as determined by (35)S-sulfate labeling experiments. Tg2576 MEF was less sensitive to inhibition of NO production and copper-chelation than WT MEF. By using APP- and Aβ-recognizing antibodies we observed nuclear translocation of Aβ peptide in Tg2576 MEF but not in WT MEF. HS remained in the nucleus of WT MEF for at least 8 h and was then transported to autophagosomes. By 8 h HS had disappeared from the nuclei of Tg2576 MEF but colocalized poorly with the autophagosome marker LC3. Aβ also disappeared rapidly from the nuclei of Tg2576 MEF. Initially it appeared in acidic vesicles and later it accumulated extracellularly. Thus, in Tg2576 MEF there is nuclear accumulation as well as secretion of Aβ and impaired degradation of HS.
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| 29. |
- Cheng, Fang, et al.
(författare)
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Reversal of apolipoprotein E4-dependent or chemical-induced accumulation of APP degradation products by vitamin C-induced release of heparan sulfate from glypican-1
- 2021
-
Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 31:7, s. 800-811
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Tidskriftsartikel (refereegranskat)abstract
- The Apolipoprotein E4 (ApoE4) genotype is the most influential risk factor for sporadic Alzheimer's disease. It appears to be associated with retarded endosome-to-autophagosome trafficking. The amyloid precursor protein (APP) and the heparan sulfate (HS)-containing proteoglycan glypican-1 (Gpc-1) are both processed in endosomes, and mutually regulated by the APP degradation products and the released HS. We have investigated APP and Gpc-1 processing in ApoE3 and ApoE4 expressing human fibroblasts, in human neural stem cells (NSC) exposed to the cholesterol transport inhibitor U18666A and in induced neurons obtained by reprogramming of ApoE fibroblasts (ApoE-iN). We have used immunofluorescence microscopy, flow cytometry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis western blotting with antibodies recognizing the released HS, APP, amyloid β(Aβ), late endosomes (Rab7), autophagosomes (LC3) and neurons (Tuj1). We found that the capacity to release HS was not fully utilized in ApoE4 expressing fibroblasts and that HS-Aβ complexes accumulated in the nuclei. In ApoE3 fibroblasts, the β-cleaved APP C-terminal fragment (β-CTF) and Aβ were primarily present in late endosomes and autophagosomes. When HS release from Gpc-1 was enhanced by ascorbate in ApoE4/4 fibroblasts, there was efficient transfer of Aβ and HS from the nuclei to autophagosomes. In U18666A-treated NSC as well as in ApoE4/4-iN we repeatedly found accumulation of APP degradation products (β-CTF/Aβ). This was reversed by subsequent exposure to ascorbate or dehydroascorbic acid.
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| 30. |
- Cherian, Reeja Maria, et al.
(författare)
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Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins
- 2014
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 24:1, s. 26-38
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Tidskriftsartikel (refereegranskat)abstract
- The binding of Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) to a mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG(2b) (PSGL-1/mIgG(2b)), carrying multiple copies of the blood group P1 determinant on O-glycans was investigated with western blot and the biosensor Biacore. Chinese hamster ovary K-1 (CHO-K1) cells were stably transfected with linearized plasmids encoding the PSGL-1/mIgG(2b) fusion protein, the pigeon alpha 1,4-galactosyltransferase (alpha 4Gal-T) and the core 2 beta 1,6-N-acetylglucosaminyltransferase (C2GnT-I). Western blot analyses of purified PSGL-1/mIgG(2b) and liquid chromatography-mass spectrometry (LC-MS) of released O-glycans confirmed the presence of the P1 determinant. Western blot analysis indicated strong binding of Stx1, but not Stx2, to PSGL-1/mIgG(2b). In a Biacore assay, Stx1 and Stx2 were immobilized on a dextran chip and the binding of purified PSGL-1/mIgG(2b) and a P-k-albumin neoglycoprotein was analyzed. Stx1 and Stx2 bound with high avidity to both PSGL-1/mIgG(2b) and P-k-albumin, while the Stx1 binding was the strongest. In summary, we have shown that the pigeon alpha 4Gal-T can be aberrantly expressed in CHO cells together with the core 2 enzyme to generate multiple, O-linked P1 determinants on a simultaneously expressed mucin-type fusion protein. P1-decorated PSGL-1/mIgG(2b) bound with high avidity to both Stx1 and Stx2, and as such constitutes a potential therapeutic inhibitor of these toxins.
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| 31. |
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| 32. |
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| 33. |
- Conze, Tim, 1977-, et al.
(författare)
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MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas
- 2010
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 20:2, s. 199-206
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 34. |
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| 35. |
- Cui, Hao, et al.
(författare)
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Glucuronyl C5-epimerase is crucial for epithelial cell maturation during embryonic lung development
- 2021
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Ingår i: Glycobiology. - : Oxford University Press. - 0959-6658 .- 1460-2423. ; 31:3, s. 223-230
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Tidskriftsartikel (refereegranskat)abstract
- Glucuronyl C5-epimerase (Hsepi) is a key enzyme in the biosynthesis of heparan sulfate that is a sulfated polysaccharide expressed on the cell surface and in the extracellular matrix of alveolar walls and blood vessels. Targeted interruption of the Hsepi gene, Glce, in mice resulted in neonatal lethality, which is most likely due to lung atelectasis. In this study, we examined the potential mechanisms behind the defect in lung development. Histological analysis of the lungs from embryos revealed no difference in the morphology between wild-type and mutant animals up to E16.5. This suggests that the initial events leading to formation of the lung primordium and branching morphogenesis are not disturbed. However, the distal lung of E17.5-18.5 mutants is still populated by epithelial tubules, lacking the typical saccular structural characteristic of a normal E17.5 lung. Immunostaining revealed strong signals of surfactant protein-C, but a weaker signal of T1 alpha in the mutant lungs in comparison to WT littermates, suggesting differentiation of type I alveolar epithelial cells (AT1) is impaired. One of the parameters contributed to the failure of AT1 maturation is reduced vascularization in the developing lungs.
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| 36. |
- Cui, Hao, et al.
(författare)
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Re-expression of glucuronyl C5-epimerase in the mutant MEF cells increases heparan sulfate epimerization but has no influence on the Golgi localization and enzymatic activity of 2-O-sulfotransferase
- 2021
-
Ingår i: Glycobiology. - : Oxford University Press. - 0959-6658 .- 1460-2423. ; 31:8, s. 1018-1025
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate (HS) is a linear and complex polysaccharide that modulates the biological activities through protein recognition and interaction. Evidence indicates that protein-binding properties of HS are largely dependent on distinctive sulfation and epimerization patterns that are modified by a series of Golgi-localized enzymes. In particular, the glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) residues to L-iduronic acid (IdoA) and 2-O-sulfotransferase (2OST) catalyzes sulfation at C2 position of IdoA and rarely GlcA residues. Mice lacking both Hsepi and 2OST display multiple development defects, indicating the importance of IdoA in HS. Here, to gain greater insights of HS structure-function relationships, as well as a better understanding of the regulatory mechanisms of Hsepi and 2OST, the fine structure and cellular signaling functions of HS were investigated after restoration of Hsepi in the mutant mouse embryonic fibroblast (MEF) cells. Introduction of Hsepi into the Hsepi mutant MEF cells led to robustly increased proportion of IdoA residues, which rescued the cell signaling in response to fibroblast growth factor 2. However, we found that Hsepi knockout had no influence on either cellular transport or enzymatic activity of 2OST in the MEF cells, which is not in accord with the findings suggesting that the enzymatic activity and cellular transport of 2OST and Hsepi might be differently regulated.
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| 37. |
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| 38. |
- Derba-Maceluch, Marta, et al.
(författare)
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O-Acetylation of glucuronoxylan in Arabidopsis thaliana wild type and its change in xylan biosynthesis mutants
- 2014
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 24, s. 494-506
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Tidskriftsartikel (refereegranskat)abstract
- O-Acetylglucuronoxylans (AcGX) in Arabidopsis thaliana carry acetyl residues on the 2-O and/or 3-O positions of the xylopyranosyl (Xylp) units, but the distribution of different O-acetylated Xylp units is partly unclear. We studied a possible correlation of xylan acetylation and the activities of different glycosyltransferases involved in xylan biosynthesis by analyzing the distribution of O-acetyl substituents on AcGX from Arabidopsis wild-type and mutants irx7, irx9-1, irx10, irx14 and gux1gux2. The relative contents of the Xylp structural units were determined with quantitative two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. In the wild type, the degree of acetylation (DA) was 60%. Mono- and diacetylated Xylp units constituted 44 and 6% of the AcGX backbone, respectively; while (4-O-methyl)-glucopyranosyluronic acid (1 -> 2)-linked Xylp units, most of which also carry 3-O-acetylation, represented 13%. The DA was decreased in irx7, irx9-1 and irx14 due to the decrease in monoacetylation (2-O and 3-O), indicating a relationship between acetylation and other AcGX biosynthetic processes. The possible interactions that could lead to such changes have been discussed. No change in DA was observed in irx10 and gux1gux2, but monoacetylation was nonetheless elevated in gux1gux2. This indicates that acetylation occurs after addition of GlcpA to the xylan backbone. Mass fragmentation analysis suggests that the prevalent acetylation pattern is the acetyl group added on every other Xylp unit.
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| 39. |
- Dick, Gunnar, et al.
(författare)
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PAPST1 regulates sulfation of heparan sulfate proteoglycans in epithelial MDCK II cells.
- 2015
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 25:1, s. 30-41
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycan (PG) sulfation depends on activated nucleotide sulfate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Transporters in the Golgi membrane translocate PAPS from the cytoplasm into the organelle lumen where PG sulfation occurs. Silencing of PAPS transporter (PAPST) 1 in epithelial MDCK cells reduced PAPS uptake into Golgi vesicles. Surprisingly, at the same time sulfation of heparan sulfate (HS) was stimulated. The effect was pathway specific in polarized epithelial cells. Basolaterally secreted PGs displayed an altered HS sulfation pattern and increased growth factor binding capacity. In contrast, the sulfation pattern of apically secreted PGs was unchanged while the secretion was reduced. Regulation of PAPST1 allows epithelial cells to prioritize between PG sulfation in the apical and basolateral secretory routes at the level of the Golgi apparatus. This provides sulfation patterns that ensure PG functions at the extracellular level, such as growth factor binding.
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| 40. |
- Diskin, Shiri, et al.
(författare)
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The role of integrin glycosylation in galectin-8-mediated trabecular meshwork cell adhesion and spreading
- 2009
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 19:1, s. 29-37
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Tidskriftsartikel (refereegranskat)abstract
- Primary open angle glaucoma (POAG) is a major blindness-causing disease, characterized by elevated intraocular pressure due to an insufficient outflow of aqueous humor. The trabecular meshwork (TM) lining the aqueous outflow pathway modulates the aqueous outflow facility. TM cell adhesion, cell-matrix interactions, and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. In a recent study, we demonstrated that galectin-8 (Gal8) modulates the adhesion and cytoskeletal arrangement of TM cells and that it does so through binding to beta(1) integrins and inducing Rho signaling. The current study is aimed at the characterization of the mechanism by which Gal8 mediates TM cell adhesion and spreading. We demonstrate here that TM cells adhere to and spread on Gal8-coated wells but not on galectin-1 (Gal1)- or galectin-3 (Gal3)-coated wells. The adhesion of TM cells to Gal8-coated wells was abolished by a competing sugar, beta-lactose, but not by a noncompeting sugar, sucrose. Also, a trisaccharide, NeuAc alpha 2-3Gal beta 1-4GlcNAc, which binds specifically to the N-CRD of Gal8, inhibited the spreading of TM cells to Gal8-coated wells. In contrast, NeuAc alpha 2-6Gal beta 1-4GlcNAc which lacks affinity for Gal8 had no effect. Affinity chromatography of cell extracts on a Gal8-affinity column and binding experiments with plant lectins, Maakia Amurensis and Sambucus Nigra, revealed that alpha(3)beta(1), alpha(5)beta(1), and alpha(v)beta(1) integrins are major counterreceptors of Gal8 in TM cells and that TM cell beta(1) integrins carry predominantly alpha 2-3-sialylated glycans, which are high-affinity ligands for Gal8 but not for Gal1 or Gal3. These data lead us to propose that Gal8 modulates TM cell adhesion and spreading, at least in part, by interacting with alpha 2-3-sialylated glycans on beta(1) integrins.
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| 41. |
- Dong, Chuqiao, et al.
(författare)
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Structure, Dynamics, and Interactions of GPI-Anchored Human Glypican-1 with Heparan Sulfates in a Membrane
- 2021
-
Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 31:5, s. 593-602
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Tidskriftsartikel (refereegranskat)abstract
- Glypican-1 and its heparan sulfate (HS) chains play important roles in modulating many biological processes including growth factor signaling. Glypican-1 is bound to a membrane surface via a glycosylphosphatidylinositol (GPI)-anchor. In this study, we used all-atom molecular modeling and simulation to explore the structure, dynamics, and interactions of GPI-anchored glypican-1, three HS chains, membranes, and ions. The folded glypican-1 core structure is stable, but has substantial degrees of freedom in terms of movement and orientation with respect to the membrane due to the long unstructured C-terminal region linking the core to the GPI-anchor. With unique structural features depending on the extent of sulfation, high flexibility of HS chains can promote multi-site interactions with surrounding molecules near and above the membrane. This study is a first step toward all-atom molecular modeling and simulation of the glycocalyx, as well as its modulation of interactions between growth factors and their receptors.
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| 42. |
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| 43. |
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| 44. |
- Fagerberg, David, et al.
(författare)
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Novel Leb-like Helicobacter pylori-binding glycosphingolipid created by the expression of human alpha-1,3/4-fucosyltransferase in FVB/N mouse stomach.
- 2009
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 19:2, s. 182-91
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 45. |
- Fairweather, J. K., et al.
(författare)
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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)
- 2004
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 14:9, s. 775-781
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 46. |
- Falck, Peter, et al.
(författare)
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Characterization of a family 43 β-xylosidase from the xylooligosaccharide utilizing putative probiotic Weissella sp. strain 92.
- 2015
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 26:2, s. 193-202
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Tidskriftsartikel (refereegranskat)abstract
- In this work we present the first XOS degrading glycoside hydrolase from Weissella, WXyn43, a two-domain enzyme from GH43. The gene was amplified from genomic DNA of the XOS utilizing Weissella strain 92, classified under the species pair Weissella cibaria/W.confusa, and expressed in Escherichia coli. The enzyme is lacking a putative signal peptide and is, from a homology model, shown to be composed of an N-terminal 5-fold ß-propeller catalytic domain and a C-terminal ß-sandwich domain of unknown function. WXyn43 hydrolysed short (1-4)-β-D-xylooligosaccharides, with similar kcat/KM for Xylobiose (X2) and xylotriose (X3) and clearly lower efficiency in xylotetraose (X4) conversion. WXyn43 displays the highest reported kcat for conversion of X3 (900 s(-1) at 37°C) and X4 (770 s(-1)), and kcat for hydrolysis of X2 (907 s(-1)) is comparable to or greater than the highest previously reported. The purified enzyme adopted a homotetrameric state in solution, while a truncated form with isolated N-terminal catalytic domain adopted a mixture of oligomeric states and lacked detectable activity. The homology model shows that residues from both domains are involved in monomer-monomer hydrogen bonds, while the bonds creating dimer-dimer interactions only involved residues from the N-terminal domain. Docking of X2 and X3 in the active site show interactions corresponding to sub-sites -1 and +1, while presence of a third subsite is unclear, but interactions between a loop and the reducing-end xylose of X3 may be present.
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| 47. |
- Fontana, Carolina, et al.
(författare)
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Rapid structural elucidation of polysaccharides employing predicted functions of glycosyltransferases and NMR data : Application to the O-antigen of Escherichia coli O59
- 2014
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 24:5, s. 450-457
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 48. |
- Fontana, Carolina, 1980-, et al.
(författare)
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Structural studies of the O-antigen polysaccharide from Escherichia coli O115 and biosynthetic aspects thereof
- 2013
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 23:3, s. 354-362
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 49. |
- Forsman, Huamei, et al.
(författare)
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The beta-galactoside binding immunomodulatory lectin galectin-3 reverses the desensitized state induced in neutrophils by the chemotactic peptide f-Met-Leu-Phe: role of reactive oxygen species generated by the NADPH-oxidase and inactivation of the agonist
- 2008
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 18:11, s. 905-12
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Tidskriftsartikel (refereegranskat)abstract
- Neutrophils interacting with a chemoattractant gradually become nonresponsive to further stimulation by the same agonist, a process known as desensitization. Receptor desensitization is a highly regulated process that involves different mechanisms depending on which receptor-ligand pair that is studied. Galectin-3, a member of a large family of beta-galactoside-binding lectins, has been suggested to be a regulator of the inflammatory process, augmenting or directly triggering the neutrophil functional repertoire. We show here that the desensitized state of neutrophils interacting with the chemotactic peptide fMLF is broken by galectin-3 and that this is achieved through an oxygen radical-mediated inactivation of the chemoattractant. The effect was inhibited by the competitor lactose and required the affinity of galectin-3 for N-acetyllactosamine, a saccharide typically found on cell surface glycoproteins. The latter was shown using a galectin-3 mutant that lacked N-acetyllactosamine binding activity, and this protein was not active. The mechanism behind the inactivation of the chemoattractant was found to depend on the ability of galectin-3 to induce a neutrophil generation/secretion of reactive oxygen species which in combined action with myeloperoxidase inactivated the peptides.
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| 50. |
- Fransson, Lars-Åke, et al.
(författare)
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Recycling of a glycosylphosphatidylinositol-anchored heparan sulphate proteoglycan (glypican) in skin fibroblasts
- 1995
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Ingår i: Glycobiology. - 1460-2423. ; 5:4, s. 407-415
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Tidskriftsartikel (refereegranskat)abstract
- We have used suramin and brefeldin A to investigate the nature of a heparan sulphate proteoglycan that appears to recycle from the cell surface to intracellular compartments which synthesize new heparan sulphate chains. Suramin, which would block internalization and deglycanation of a putative recycling cell surface proteoglycan, markedly increases the yield of a membrane-bound proteoglycan with a core protein of 60-70 kDa and unusually long heparan sulphate side chains. When transport of newly made core proteins to their Golgi sites for glycosaminoglycan assembly is blocked, by using brefeldin A, [3H]glucosamine and [35S]sulphate incorporation into cell surface-bound heparan sulphate proteoglycan can still take place. After chemical biotinylation of cell surface proteins in brefeldin A-treated cells, followed by metabolic [35S]sulphation in the presence of the same drug, biotin-tagged [35S]proteoglycan can be demonstrated, indicating the presence of recycling proteoglycan species. By pre-labelling cells with [3H]leucine or [3H]inositol in the presence of suramin, followed by chase labelling with [35S]sulphate in the presence of brefeldin A, a 3H- and 35S-labelled, hydrophobic heparan sulphate proteoglycan with a core protein of 60-65 kDa is obtained. The proteoglycan loses its hydrophobicity when glucosamine-inositol bonds are cleaved, indicating that it is membrane bound via a glycosylphosphatidylinositol anchor. However, treatment with phosphatidylinositol-specific phospholipase C has no effect, suggesting that the inositol moiety may be acylated. We propose that a portion of the lipid-anchored proteoglycan glypican is internalized, recycled via the Golgi, where heparan sulphate chains are added, and finally re-deposited at the cell surface.
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