| 1. |
- Bax, Marieke, et al.
(författare)
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Dendritic cell maturation results in pronounced changes in glycan expression affecting recognition by siglecs and galectins
- 2007
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Ingår i: Journal of Immunology. - 1550-6606. ; 179:12, s. 8216-8224
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Tidskriftsartikel (refereegranskat)abstract
- Dendritic cells (DC) are the most potent APC in the organism. Immature dendritic cells (iDC) reside in the tissue where they capture pathogens whereas mature dendritic cells (mDC) are able to activate T cells in the lymph node. This dramatic functional change is mediated by an important genetic reprogramming. Glycosylation is the most common form of posttranslational modification of proteins and has been implicated in multiple aspects of the immune response. To investigate the involvement of glycosylation in the changes that occur during DC maturation, we have studied the differences in the glycan profile of iDC and mDC as well as their glycosylation machinery. For information relating to glycan biosynthesis, gene expression profiles of human monocyte-derived iDC and mDC were compared using a gene microarray and quantitative real-time PCR. This gene expression profiling showed a profound maturation-induced up-regulation of the glycosyltransferases involved in the expression of LacNAc, core 1 and sialylated structures and a down-regulation of genes involved in the synthesis of core 2 O-glycans. Glycosylation changes during DC maturation were corroborated by mass spectrometric analysis of N- and O-glycans and by flow cytometry using plant lectins and glycan-specific Abs. Interestingly, the binding of the LacNAc-specific lectins galectin-3 and -8 increased during maturation and up-regulation of sialic acid expression by mDC correlated with an increased binding of siglec-1, -2, and -7.
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| 2. |
- Burguière, Adeline, et al.
(författare)
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LosA, a key glycosyltransferase involved in the biosynthesis of a novel family of glycosylated acyltrehalose lipooligosaccharides from Mycobacterium marinum.
- 2005
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 280:51, s. 42124-33
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Tidskriftsartikel (refereegranskat)abstract
- Members of the genus Mycobacterium are characterized by cell envelopes rich in unusual free lipids, interacting with a covalently anchored mycolyl-arabinogalactan matrix. Previous studies have shown that Mycobacterium marinum produces large amounts of a diacylglycosylphenolphthiocerol, "phenolic" glycolipid. When cultivated on liquid Sauton medium, traces of a polar lipooligosaccharide (LOS) glycolipid antigen were also previously indicated. In this study, it was found that growth of the type strain of M. marinum on solid Sauton or Middlebrook 7H10 agar gave substantial, but different, amounts of a family of four major trehalose-based LOSs. The core pentasaccharide LOS-I was a rhamnosyl diglucosyl-acylated trehalose. The heptasaccharide, LOS-II, was derived from LOS-I by adding xylose accompanied by a novel sugar (X); repeated addition of this sugar unit X gave the octasaccharide LOS-III. LOS-IV has a decasaccharide component with two additional unusual sugar units, YZ. In a recent study (Alexander, D. C., Jones, J. R., Tan, T., Chen, J. M., and Liu, J. (2004) J. Biol. Chem. 279, 18824-18833), chromatographically similar glycolipids were assigned to the family of phosphatidylinositol mannosides (PIMs) and a "PimF" (Rv1500) glycosyltransferase implicated in the conversion of a supposed "PIM5" to a "PIM7." The present study indicates that these putative PIMs are in fact members of the phosphorus-free LOS family of glycolipids and that the protein product of Rv1500, which we have now termed LosA, is a glycosyltransferase involved in transferring sugars to LOS-III to form LOS-IV of M. marinum.
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| 3. |
- Clark, Gary F., et al.
(författare)
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Tumor Biomarker Glycoproteins in the Seminal Plasma of Healthy Human Males Are Endogenous Ligands for DC-SIGN
- 2012
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- DC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewis(x) and Lewis(y) carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewis(x) and Lewis(y) sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus.
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| 4. |
- Magalhães, Ana, et al.
(författare)
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Fut2-null mice display an altered glycosylation profile and impaired BabA-mediated Helicobacter pylori adhesion to gastric mucosa
- 2009
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 19:12, s. 1525-1536
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Tidskriftsartikel (refereegranskat)abstract
- Glycoconjugates expressed on gastric mucosa play a crucial role in host-pathogen interactions. The FUT2 enzyme catalyzes the addition of terminal alpha(1,2)fucose residues, producing the H type 1 structure expressed on the surface of epithelial cells and in mucosal secretions of secretor individuals. Inactivating mutations in the human FUT2 gene are associated with reduced susceptibility to Helicobacter pylori infection. H. pylori infects over half the world's population and causes diverse gastric lesions, from gastritis to gastric cancer. H. pylori adhesion constitutes a crucial step in the establishment of a successful infection. The BabA adhesin binds the Le(b) and H type 1 structures expressed on gastric mucins, while SabA binds to sialylated carbohydrates mediating the adherence to inflamed gastric mucosa. In this study, we have used an animal model of nonsecretors, Fut2-null mice, to characterize the glycosylation profile and evaluate the effect of the observed glycan expression modifications in the process of H. pylori adhesion. We have demonstrated expression of terminal difucosylated glycan structures in C57Bl/6 mice gastric mucosa and that Fut2-null mice showed marked alteration in gastric mucosa glycosylation, characterized by diminished expression of alpha(1,2)fucosylated structures as indicated by lectin and antibody staining and further confirmed by mass spectrometry analysis. This altered glycosylation profile was further confirmed by the absence of Fucalpha(1,2)-dependent binding of calicivirus virus-like particles. Finally, using a panel of H. pylori strains, with different adhesin expression profiles, we have demonstated an impairment of BabA-dependent adhesion of H. pylori to Fut2-null mice gastric mucosa, whereas SabA-mediated binding was not affected.
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| 5. |
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| 6. |
- Sassi, Atfa, et al.
(författare)
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Hypomorphic homozygous mutations in phosphoglucomutase 3 (PGM3) impair immunity and increase serum IgE levels
- 2014
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Ingår i: Journal of Allergy and Clinical Immunology. - : Elsevier BV. - 0091-6749 .- 1097-6825. ; 133:5, s. 1410-U681
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Tidskriftsartikel (refereegranskat)abstract
- Background: Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in signal transducer and activator of transcription 3 (STAT3) and dedicator of cytokinesis 8 (DOCK8), which are involved in signal transduction pathways. However, glycosylation defects have not been described in patients with HIES. One crucial enzyme in the glycosylation pathway is phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of uridine diphosphate N-acetylglucosamine, which is required for the biosynthesis of N-glycans. Objective: We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. Methods: After establishing a linkage interval by means of SNPchip genotyping and homozygosity mapping in 2 families with HIES from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by means of Western blotting, and glycosylation was profiled by using mass spectrometry. Results: Mutational analysis of candidate genes in an 11.9-Mb linkage region on chromosome 6 shared by 2 multiplex families identified 2 homozygous mutations in PGM3 that segregated with disease status and followed recessive inheritance. The mutations predict amino acid changes in PGM3 (p. Glu340del and p. Leu83Ser). A third homozygous mutation (p. Asp502Tyr) and the p. Leu83Ser variant were identified in 2 other affected families, respectively. These hypomorphic mutations have an effect on the biosynthetic reactions involving uridine diphosphate N-acetylglucosamine. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-antennary and tetra-antennary N-glycans. T-cell proliferation and differentiation were impaired in patients. Most patients had developmental delay, and many had psychomotor retardation. Conclusion: Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity because biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper-IgE-like phenotype.
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| 7. |
- Struwe, Weston B, et al.
(författare)
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The minimum information required for a glycomics experiment (MIRAGE) project: sample preparation guidelines for reliable reporting of glycomics datasets.
- 2016
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 26:9, s. 907-910
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Tidskriftsartikel (refereegranskat)abstract
- Theminimum information required for a glycomics experiment (MIRAGE) project was established in 2011 to provide guidelines to aid in data reporting from all types of experiments in glycomics research including mass spectrometry (MS), liquid chromatography, glycan arrays, data handling and sample preparation. MIRAGE is a concerted effort of the wider glycomics community that considers the adaptation of reporting guidelines as an important step towards critical evaluation and dissemination of datasets as well as broadening of experimental techniques worldwide. The MIRAGE Commission published reporting guidelines for MS data and here we outline guidelines for sample preparation. The sample preparation guidelines include all aspects of sample generation, purification and modification from biological and/or synthetic carbohydrate material. The application of MIRAGE sample preparation guidelines will lead to improved recording of experimental protocols and reporting of understandable and reproducible glycomics datasets.
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| 8. |
- Sun, Wei, 1981-, et al.
(författare)
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Further insight into the roles of the glycans attached to human blood protein C inhibitor
- 2010
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 403:2, s. 198-202
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Tidskriftsartikel (refereegranskat)abstract
- Protein C inhibitor (PCI) is a 57-kDa glycoprotein that exists in many tissues and secretions in human. As a member of the serpin superfamily of proteins it displays unusually broad protease specificity. PCI is implicated in the regulation of a wide range of processes, including blood coagulation, fertilization, prevention of tumors and pathogen defence. It has been reported that PCI isolated from human blood plasma is highly heterogeneous, and that this heterogeneity is caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of two forms that differ by the presence or absence of 6 amino acids at the N-terminus. In this study we have verified that such heterogeneity exists in PCI purified from single individuals, and that individuals of two different ethnicities possess a similar PCI pattern, verifying that the microheterogeneity is conserved among humans. Furthermore, we have provided experimental evidence that PCI is O-glycosylated on Thr20 with the O-glycan structure composition NeuAcGalGalNAc. This glycan was conserved in the two individuals and did not contribute to the size heterogeneity. Modeling suggested that the O-glycan attachment site is located in proximity to several ligand-binding sites of the inhibitor.
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| 9. |
- Sun, Wei, et al.
(författare)
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N-glycans and the N terminus of protein C inhibitor affect the cofactor-enhanced rates of thrombin inhibition
- 2008
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:27, s. 18601-11
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Tidskriftsartikel (refereegranskat)abstract
- Protein C inhibitor (PCI) is a serine protease inhibitor, displaying broad protease specificity, found in blood and other tissues. In blood, it is capable of inhibiting both procoagulant and anticoagulant proteases. Mechanisms that provide specificity to PCI remain largely unrevealed. In this study we have for the first time provided a full explanation for the marked size heterogeneity of blood-derived PCI and identified functional differences between naturally occurring PCI variants. The heterogeneity was caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of a Delta6-N-cleaved form. Bi-, tri-, and tetra-antennary complex N-glycans were identified. Fucose residues were identified both on the core GlcNAc and as parts of sialyl-Le(a/x) epitopes. Moreover, a glycan with a composition that implied a di-sialyl antenna was observed. PCI was N-glycosylated at all three potential N-glycosylation sites, Asn-230, Asn-243, and Asn-319, but a small fraction of PCI lacked the N-glycan at Asn-243. The overall removal of N-glycans affected the maximal heparin- and thrombomodulin-enhanced rates of thrombin inhibition differently in different solution conditions. In contrast, the Delta6-N-region increased both the heparin- and the thrombomodulin-enhanced rates of thrombin inhibition at all conditions examined. These results thus demonstrate that the N-linked glycans and the N-terminal region of blood-derived PCI in different ways affect the cofactor-enhanced rates of thrombin inhibition and provide information on the mechanisms by which this may be achieved. The findings are medically important, in view of the documented association of PCI with atherosclerotic plaques and the promising effect of PCI on reducing hypercoagulability states.
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| 10. |
- Sun, Wei, 1981-, et al.
(författare)
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N-glycans of Human Protein C Inhibitor : Tissue-Specific Expression and Function
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:12, s. e29011-
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Tidskriftsartikel (refereegranskat)abstract
- Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH(2)-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc(3)Hex(5)HexNAc(4), consistent with a core fucosylated bi-antennary glycan with terminal Lewis x. A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH(2)-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3 +/- 0.2 and 4.1 +/- 0.5 M(-1) s(-1) for the natural full-length PCI and a form lacking six amino acids at the NH(2)-terminus, respectively, whereas these constants were 4.8 +/- 0.1 and 29 +/- 7 M(-1) s(-1) for the corresponding PNGase F-treated forms. The 7-8-fold higher rate constants obtained when both the N-glycans and the NH(2-)terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA.
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